free full text journal articles: microbiology and immunology
(skip the 30 most recent)


Advertisement


 

Google
 
Web www.neurotransmitter.net

Recent Articles in Microbiology and Molecular Biology Reviews

Kane PM
The where, when, and how of organelle acidification by the yeast vacuolar H+-ATPase.
Microbiol Mol Biol Rev. 2006 Mar;70(1):177-91.
All eukaryotic cells contain multiple acidic organelles, and V-ATPases are central players in organelle acidification. Not only is the structure of V-ATPases highly conserved among eukaryotes, but there are also many regulatory mechanisms that are similar between fungi and higher eukaryotes. These mechanisms allow cells both to regulate the pHs of different compartments and to respond to changing extracellular conditions. The Saccharomyces cerevisiae V-ATPase has emerged as an important model for V-ATPase structure and function in all eukaryotic cells. This review discusses current knowledge of the structure, function, and regulation of the V-ATPase in S. cerevisiae and also examines the relationship between biosynthesis and transport of V-ATPase and compartment-specific regulation of acidification. [Abstract/Link to Full Text]

van Hijum SA, Kralj S, Ozimek LK, Dijkhuizen L, van Geel-Schutten IG
Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria.
Microbiol Mol Biol Rev. 2006 Mar;70(1):157-76.
Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and alpha-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with alpha-amylase enzymes (family GH13), with a predicted permuted (beta/alpha)(8) barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of alpha-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize beta-fructan polymers with either beta-(2-->6) (inulin) or beta-(2-->1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed beta-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either beta-(2-->6) or beta-(2-->1) linkages, degree and type of branching, and fructan molecular mass remain to be identified. [Abstract/Link to Full Text]

Ilouze M, Dishon A, Kotler M
Characterization of a novel virus causing a lethal disease in carp and koi.
Microbiol Mol Biol Rev. 2006 Mar;70(1):147-56.
Since 1998 a lethal disease of carp and ornamental koi (Cyprinus carpio) has afflicted fisheries in North America, Europe, and Asia, causing severe economic losses to the fish farming industry. This review summarizes the isolation and identification of the disease-causing agent and describes the currently known molecular characteristics of this newly isolated virus, distinguishing it from other known large DNA viruses. In addition, we summarize the clinical and histopathological manifestations of the disease. Providing information on the immune response to this virus and evaluating the available means of diagnosis and protection should help to reduce the damage induced by this disease. This review does not discuss the economic aspects of the disease or the debate on whether the disease should be registered; both of these issues were recently reviewed in detail (O. L. M. Haenen, K. Way, S. M. Bergmann, and E. Ariel, Bull. Eur. Assoc. Fish Pathol. 24:293-307, 2004; D. Pokorova, T. Vesely, V. Piackova, S. Reschova, and J. Hulova, Vet. Med. Czech. 50:139-147, 2005). [Abstract/Link to Full Text]

Grünewald J, Marahiel MA
Chemoenzymatic and template-directed synthesis of bioactive macrocyclic peptides.
Microbiol Mol Biol Rev. 2006 Mar;70(1):121-46.
Non-ribosomally synthesized peptides have compelling biological activities ranging from antimicrobial to immunosuppressive and from cytostatic to antitumor. The broad spectrum of applications in modern medicine is reflected in the great structural diversity of these natural products. They contain unique building blocks, such as d-amino acids, fatty acids, sugar moieties, and heterocyclic elements, as well as halogenated, methylated, and formylated residues. In the past decades, significant progress has been made toward the understanding of the biosynthesis of these secondary metabolites by nonribosomal peptide synthetases (NRPSs) and their associated tailoring enzymes. Guided by this knowledge, researchers genetically redesigned the NRPS template to synthesize new peptide products. Moreover, chemoenzymatic strategies were developed to rationally engineer nonribosomal peptides products in order to increase or alter their bioactivities. Specifically, chemical synthesis combined with peptide cyclization mediated by nonribosomal thioesterase domains enabled the synthesis of glycosylated cyclopeptides, inhibitors of integrin receptors, peptide/polyketide hybrids, lipopeptide antibiotics, and streptogramin B antibiotics. In addition to the synthetic potential of these cyclization catalysts, which is the main focus of this review, different enzymes for tailoring of peptide scaffolds as well as the manipulation of carrier proteins with reporter-labeled coenzyme A analogs are discussed. [Abstract/Link to Full Text]

Ren G, Vajjhala P, Lee JS, Winsor B, Munn AL
The BAR domain proteins: molding membranes in fission, fusion, and phagy.
Microbiol Mol Biol Rev. 2006 Mar;70(1):37-120.
The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt alpha-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes. [Abstract/Link to Full Text]

Trinh V, Langelier MF, Archambault J, Coulombe B
Structural perspective on mutations affecting the function of multisubunit RNA polymerases.
Microbiol Mol Biol Rev. 2006 Mar;70(1):12-36.
High-resolution crystallographic structures of multisubunit RNA polymerases (RNAPs) have increased our understanding of transcriptional mechanisms. Based on a thorough review of the literature, we have compiled the mutations affecting the function of multisubunit RNA polymerases, many of which having been generated and studied prior to the publication of the first high-resolution structure, and highlighted the positions of the altered amino acids in the structures of both the prokaryotic and eukaryotic enzymes. The observations support many previous hypotheses on the transcriptional process, including the implication of the bridge helix and the trigger loop in the processivity of RNAP, the importance of contacts between the RNAP jaw-lobe module and the downstream DNA in the establishment of a transcription bubble and selection of the transcription start site, the destabilizing effects of ppGpp on the open promoter complex, and the link between RNAP processivity and termination. This study also revealed novel, remarkable features of the RNA polymerase catalytic mechanisms that will require additional investigation, including the putative roles of fork loop 2 in the establishment of a transcription bubble, the trigger loop in start site selection, and the uncharacterized funnel domain in RNAP processivity. [Abstract/Link to Full Text]

Sobczak I, Lolkema JS
The 2-hydroxycarboxylate transporter family: physiology, structure, and mechanism.
Microbiol Mol Biol Rev. 2005 Dec;69(4):665-95.
The 2-hydroxycarboxylate transporter family is a family of secondary transporters found exclusively in the bacterial kingdom. They function in the metabolism of the di- and tricarboxylates malate and citrate, mostly in fermentative pathways involving decarboxylation of malate or oxaloacetate. These pathways are found in the class Bacillales of the low-CG gram-positive bacteria and in the gamma subdivision of the Proteobacteria. The pathways have evolved into a remarkable diversity in terms of the combinations of enzymes and transporters that built the pathways and of energy conservation mechanisms. The transporter family includes H+ and Na+ symporters and precursor/product exchangers. The proteins consist of a bundle of 11 transmembrane helices formed from two homologous domains containing five transmembrane segments each, plus one additional segment at the N terminus. The two domains have opposite orientations in the membrane and contain a pore-loop or reentrant loop structure between the fourth and fifth transmembrane segments. The two pore-loops enter the membrane from opposite sides and are believed to be part of the translocation site. The binding site is located asymmetrically in the membrane, close to the interface of membrane and cytoplasm. The binding site in the translocation pore is believed to be alternatively exposed to the internal and external media. The proposed structure of the 2HCT transporters is different from any known structure of a membrane protein and represents a new structural class of secondary transporters. [Abstract/Link to Full Text]

Weiss SR, Navas-Martin S
Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus.
Microbiol Mol Biol Rev. 2005 Dec;69(4):635-64.
Coronaviruses are a family of enveloped, single-stranded, positive-strand RNA viruses classified within the Nidovirales order. This coronavirus family consists of pathogens of many animal species and of humans, including the recently isolated severe acute respiratory syndrome coronavirus (SARS-CoV). This review is divided into two main parts; the first concerns the animal coronaviruses and their pathogenesis, with an emphasis on the functions of individual viral genes, and the second discusses the newly described human emerging pathogen, SARS-CoV. The coronavirus part covers (i) a description of a group of coronaviruses and the diseases they cause, including the prototype coronavirus, murine hepatitis virus, which is one of the recognized animal models for multiple sclerosis, as well as viruses of veterinary importance that infect the pig, chicken, and cat and a summary of the human viruses; (ii) a short summary of the replication cycle of coronaviruses in cell culture; (iii) the development and application of reverse genetics systems; and (iv) the roles of individual coronavirus proteins in replication and pathogenesis. The SARS-CoV part covers the pathogenesis of SARS, the developing animal models for infection, and the progress in vaccine development and antiviral therapies. The data gathered on the animal coronaviruses continue to be helpful in understanding SARS-CoV. [Abstract/Link to Full Text]

Barabote RD, Saier MH
Comparative genomic analyses of the bacterial phosphotransferase system.
Microbiol Mol Biol Rev. 2005 Dec;69(4):608-34.
We report analyses of 202 fully sequenced genomes for homologues of known protein constituents of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). These included 174 bacterial, 19 archaeal, and 9 eukaryotic genomes. Homologues of PTS proteins were not identified in archaea or eukaryotes, showing that the horizontal transfer of genes encoding PTS proteins has not occurred between the three domains of life. Of the 174 bacterial genomes (136 bacterial species) analyzed, 30 diverse species have no PTS homologues, and 29 species have cytoplasmic PTS phosphoryl transfer protein homologues but lack recognizable PTS permeases. These soluble homologues presumably function in regulation. The remaining 77 species possess all PTS proteins required for the transport and phosphorylation of at least one sugar via the PTS. Up to 3.2% of the genes in a bacterium encode PTS proteins. These homologues were analyzed for family association, range of protein types, domain organization, and organismal distribution. Different strains of a single bacterial species often possess strikingly different complements of PTS proteins. Types of PTS protein domain fusions were analyzed, showing that certain types of domain fusions are common, while others are rare or prohibited. Select PTS proteins were analyzed from different phylogenetic standpoints, showing that PTS protein phylogeny often differs from organismal phylogeny. The results document the frequent gain and loss of PTS protein-encoding genes and suggest that the lateral transfer of these genes within the bacterial domain has played an important role in bacterial evolution. Our studies provide insight into the development of complex multicomponent enzyme systems and lead to predictions regarding the types of protein-protein interactions that promote efficient PTS-mediated phosphoryl transfer. [Abstract/Link to Full Text]

Scheffers DJ, Pinho MG
Bacterial cell wall synthesis: new insights from localization studies.
Microbiol Mol Biol Rev. 2005 Dec;69(4):585-607.
In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization. [Abstract/Link to Full Text]

Neiman AM
Ascospore formation in the yeast Saccharomyces cerevisiae.
Microbiol Mol Biol Rev. 2005 Dec;69(4):565-84.
Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore. [Abstract/Link to Full Text]

Harold FM
Molecules into cells: specifying spatial architecture.
Microbiol Mol Biol Rev. 2005 Dec;69(4):544-64.
A living cell is not an aggregate of molecules but an organized pattern, structured in space and in time. This article addresses some conceptual issues in the genesis of spatial architecture, including how molecules find their proper location in cell space, the origins of supramolecular order, the role of the genes, cell morphology, the continuity of cells, and the inheritance of order. The discussion is framed around a hierarchy of physiological processes that bridge the gap between nanometer-sized molecules and cells three to six orders of magnitude larger. Stepping stones include molecular self-organization, directional physiology, spatial markers, gradients, fields, and physical forces. The knowledge at hand leads to an unconventional interpretation of biological order. I have come to think of cells as self-organized systems composed of genetically specified elements plus heritable structures. The smallest self that can be fairly said to organize itself is the whole cell. If structure, form, and function are ever to be computed from data at a lower level, the starting point will be not the genome, but a spatially organized system of molecules. This conclusion invites us to reconsider our understanding of what genes do, what organisms are, and how living systems could have arisen on the early Earth. [Abstract/Link to Full Text]

Kazmierczak MJ, Wiedmann M, Boor KJ
Alternative sigma factors and their roles in bacterial virulence.
Microbiol Mol Biol Rev. 2005 Dec;69(4):527-43.
Sigma factors provide promoter recognition specificity to RNA polymerase holoenzyme, contribute to DNA strand separation, and then dissociate from the core enzyme following transcription initiation. As the regulon of a single sigma factor can be composed of hundreds of genes, sigma factors can provide effective mechanisms for simultaneously regulating expression of large numbers of prokaryotic genes. One newly emerging field is identification of the specific roles of alternative sigma factors in regulating expression of virulence genes and virulence-associated genes in bacterial pathogens. Virulence genes encode proteins whose functions are essential for the bacterium to effectively establish an infection in a host organism. In contrast, virulence-associated genes can contribute to bacterial survival in the environment and therefore may enhance the capacity of the bacterium to spread to new individuals or to survive passage through a host organism. As alternative sigma factors have been shown to regulate expression of both virulence and virulence-associated genes, these proteins can contribute both directly and indirectly to bacterial virulence. Sigma factors are classified into two structurally unrelated families, the sigma70 and the sigma54 families. The sigma70 family includes primary sigma factors (e.g., Bacillus subtilis sigma(A)) as well as related alternative sigma factors; sigma54 forms a distinct subfamily of sigma factors referred to as sigma(N) in almost all species for which these proteins have been characterized to date. We present several examples of alternative sigma factors that have been shown to contribute to virulence in at least one organism. For each sigma factor, when applicable, examples are drawn from multiple species. [Abstract/Link to Full Text]

Neylon C, Kralicek AV, Hill TM, Dixon NE
Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex.
Microbiol Mol Biol Rev. 2005 Sep;69(3):501-26.
The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process. [Abstract/Link to Full Text]

Pomeranz LE, Reynolds AE, Hengartner CJ
Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine.
Microbiol Mol Biol Rev. 2005 Sep;69(3):462-500.
Pseudorabies virus (PRV) is a herpesvirus of swine, a member of the Alphaherpesvirinae subfamily, and the etiological agent of Aujeszky's disease. This review describes the contributions of PRV research to herpesvirus biology, neurobiology, and viral pathogenesis by focusing on (i) the molecular biology of PRV, (ii) model systems to study PRV pathogenesis and neurovirulence, (iii) PRV transsynaptic tracing of neuronal circuits, and (iv) veterinary aspects of pseudorabies disease. The structure of the enveloped infectious particle, the content of the viral DNA genome, and a step-by-step overview of the viral replication cycle are presented. PRV infection is initiated by binding to cellular receptors to allow penetration into the cell. After reaching the nucleus, the viral genome directs a regulated gene expression cascade that culminates with viral DNA replication and production of new virion constituents. Finally, progeny virions self-assemble and exit the host cells. Animal models and neuronal culture systems developed for the study of PRV pathogenesis and neurovirulence are discussed. PRV serves asa self-perpetuating transsynaptic tracer of neuronal circuitry, and we detail the original studies of PRV circuitry mapping, the biology underlying this application, and the development of the next generation of tracer viruses. The basic veterinary aspects of pseudorabies management and disease in swine are discussed. PRV infection progresses from acute infection of the respiratory epithelium to latent infection in the peripheral nervous system. Sporadic reactivation from latency can transmit PRV to new hosts. The successful management of PRV disease has relied on vaccination, prevention, and testing. [Abstract/Link to Full Text]

Pernthaler J, Amann R
Fate of heterotrophic microbes in pelagic habitats: focus on populations.
Microbiol Mol Biol Rev. 2005 Sep;69(3):440-61.
Major biogeochemical processes in the water columns of lakes and oceans are related to the activities of heterotrophic microbes, e.g., the mineralization of organic carbon from photosynthesis and allochthonous influx or its transport to the higher trophic levels. During the last 15 years, cultivation-independent molecular techniques have substantially contributed to our understanding of the diversity of the microbial communities in different aquatic systems. In parallel, the complexity of aquatic habitats at a microscale has inspired research on the ecophysiological properties of uncultured microorganisms that thrive in a continuum of dissolved to particulate organic matter. One possibility to link these two aspects is to adopt a"Gleasonian" perspective, i.e., to study aquatic microbial assemblages in situ at the population level rather than looking at microbial community structure, diversity, or function as a whole. This review compiles current knowledge about the role and fate of different populations of heterotrophic picoplankton in marine and inland waters. Specifically, we focus on a growing suite of techniques that link the analysis of bacterial identity with growth, morphology, and various physiological activities at the level of single cells. An overview is given of the potential and limitations of methodological approaches, and factors that might control the population sizes of different microbes in pelagic habitats are discussed. [Abstract/Link to Full Text]

Cordes SP
N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express.
Microbiol Mol Biol Rev. 2005 Sep;69(3):426-39.
In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated. [Abstract/Link to Full Text]

Eichler J, Adams MW
Posttranslational protein modification in Archaea.
Microbiol Mol Biol Rev. 2005 Sep;69(3):393-425.
One of the first hurdles to be negotiated in the postgenomic era involves the description of the entire protein content of the cell, the proteome. Such efforts are presently complicated by the various posttranslational modifications that proteins can experience, including glycosylation, lipid attachment, phosphorylation, methylation, disulfide bond formation, and proteolytic cleavage. Whereas these and other posttranslational protein modifications have been well characterized in Eucarya and Bacteria, posttranslational modification in Archaea has received far less attention. Although archaeal proteins can undergo posttranslational modifications reminiscent of what their eucaryal and bacterial counterparts experience, examination of archaeal posttranslational modification often reveals aspects not previously observed in the other two domains of life. In some cases, posttranslational modification allows a protein to survive the extreme conditions often encountered by Archaea. The various posttranslational modifications experienced by archaeal proteins, the molecular steps leading to these modifications, and the role played by posttranslational modification in Archaea form the focus of this review. [Abstract/Link to Full Text]

Yuan L, Kurek I, English J, Keenan R
Laboratory-directed protein evolution.
Microbiol Mol Biol Rev. 2005 Sep;69(3):373-92.
Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to "evolve" in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences. [Abstract/Link to Full Text]

Fong TT, Lipp EK
Enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools.
Microbiol Mol Biol Rev. 2005 Jun;69(2):357-71.
Waterborne enteric viruses threaten both human and animal health. These pathogens are host specific and cause a wide range of diseases and symptoms in humans or other animals. While considerable research has documented the risk of enteric viruses to human health from contact with contaminated water, the current bacterial indicator-based methods for evaluation of water quality are often ineffectual proxies for pathogenic viruses. Additionally, relatively little work has specifically investigated the risk of waterborne viruses to animal health, and this risk currently is not addressed by routine water quality assessments. Nonetheless, because of their host specificity, enteric viruses can fulfill a unique role both for assessing health risks and as measures of contamination source in a watershed, yet the use of animal, as well as human, host-specific viruses in determining sources of fecal pollution has received little attention. With improved molecular detection assays, viruses from key host groups can be targeted directly using PCR amplification or hybridization with a high level of sensitivity and specificity. A multispecies viral analysis would provide needed information for controlling pollution by source, determining human health risks based on assessments of human virus loading and exposure, and determining potential risks to production animal health and could indicate the potential for the presence of other zoonotic pathogens. While there is a need to better understand the prevalence and environmental distribution of nonhuman enteric viruses, the development of improved methods for specific and sensitive detection will facilitate the use of these microbes for library-independent source tracking and water quality assessment tools. [Abstract/Link to Full Text]

Ramos JL, Martínez-Bueno M, Molina-Henares AJ, Terán W, Watanabe K, Zhang X, Gallegos MT, Brennan R, Tobes R
The TetR family of transcriptional repressors.
Microbiol Mol Biol Rev. 2005 Jun;69(2):326-56.
We have developed a general profile for the proteins of the TetR family of repressors. The stretch that best defines the profile of this family is made up of 47 amino acid residues that correspond to the helix-turn-helix DNA binding motif and adjacent regions in the three-dimensional structures of TetR, QacR, CprB, and EthR, four family members for which the function and three-dimensional structure are known. We have detected a set of 2,353 nonredundant proteins belonging to this family by screening genome and protein databases with the TetR profile. Proteins of the TetR family have been found in 115 genera of gram-positive, alpha-, beta-, and gamma-proteobacteria, cyanobacteria, and archaea. The set of genes they regulate is known for 85 out of the 2,353 members of the family. These proteins are involved in the transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity. The regulatory network in which the family member is involved can be simple, as in TetR (i.e., TetR bound to the target operator represses tetA transcription and is released in the presence of tetracycline), or more complex, involving a series of regulatory cascades in which either the expression of the TetR family member is modulated by another regulator or the TetR family member triggers a cell response to react to environmental insults. Based on what has been learned from the cocrystals of TetR and QacR with their target operators and from their three-dimensional structures in the absence and in the presence of ligands, and based on multialignment analyses of the conserved stretch of 47 amino acids in the 2,353 TetR family members, two groups of residues have been identified. One group includes highly conserved positions involved in the proper orientation of the helix-turn-helix motif and hence seems to play a structural role. The other set of less conserved residues are involved in establishing contacts with the phosphate backbone and target bases in the operator. Information related to the TetR family of regulators has been updated in a database that can be accessed at www.bactregulators.org. [Abstract/Link to Full Text]

Khalikova E, Susi P, Korpela T
Microbial dextran-hydrolyzing enzymes: fundamentals and applications.
Microbiol Mol Biol Rev. 2005 Jun;69(2):306-25.
Dextran is a chemically and physically complex polymer, breakdown of which is carried out by a variety of endo- and exodextranases. Enzymes in many groups can be classified as dextranases according to function: such enzymes include dextranhydrolases, glucodextranases, exoisomaltohydrolases, exoisomaltotriohydrases, and branched-dextran exo-1,2-alpha-glucosidases. Cycloisomalto-oligosaccharide glucanotransferase does not formally belong to the dextranases even though its side reaction produces hydrolyzed dextrans. A new classification system for glycosylhydrolases and glycosyltransferases, which is based on amino acid sequence similarities, divides the dextranases into five families. However, this classification is still incomplete since sequence information is missing for many of the enzymes that have been biochemically characterized as dextranases. Dextran-degrading enzymes have been isolated from a wide range of microorganisms. The major characteristics of these enzymes, the methods for analyzing their activities and biological roles, analysis of primary sequence data, and three-dimensional structures of dextranases have been dealt with in this review. Dextranases are promising for future use in various scientific and biotechnological applications. [Abstract/Link to Full Text]

Sapp J
The prokaryote-eukaryote dichotomy: meanings and mythology.
Microbiol Mol Biol Rev. 2005 Jun;69(2):292-305.
Drawing on documents both published and archival, this paper explains how the prokaryote-eukaryote dichotomy of the 1960s was constructed, the purposes it served, and what it implied in terms of classification and phylogeny. In doing so, I first show how the concept was attributed to Edouard Chatton and the context in which he introduced the terms. Following, I examine the context in which the terms were reintroduced into biology in 1962 by Roger Stanier and C. B. van Niel. I study the discourse over the subsequent decade to understand how the organizational dichotomy took on the form of a natural classification as the kingdom Monera or superkingdom Procaryotae. Stanier and van Niel admitted that, in regard to constructing a natural classification of bacteria, structural characteristics were no more useful than physiological properties. They repeatedly denied that bacterial phylogenetics was possible. I thus examine the great historical irony that the "prokaryote," in both its organizational and phylogenetic senses, was defined (negatively) on the basis of structure. Finally, we see how phylogenetic research based on 16S rRNA led by Carl Woese and his collaborators confronted the prokaryote concept while moving microbiology to the center of evolutionary biology. [Abstract/Link to Full Text]

Levin DE
Cell wall integrity signaling in Saccharomyces cerevisiae.
Microbiol Mol Biol Rev. 2005 Jun;69(2):262-91.
The yeast cell wall is a highly dynamic structure that is responsible for protecting the cell from rapid changes in external osmotic potential. The wall is also critical for cell expansion during growth and morphogenesis. This review discusses recent advances in understanding the various signal transduction pathways that allow cells to monitor the state of the cell wall and respond to environmental challenges to this structure. The cell wall integrity signaling pathway controlled by the small G-protein Rho1 is principally responsible for orchestrating changes to the cell wall periodically through the cell cycle and in response to various forms of cell wall stress. This signaling pathway acts through direct control of wall biosynthetic enzymes, transcriptional regulation of cell wall-related genes, and polarization of the actin cytoskeleton. However, additional signaling pathways interface both with the cell wall integrity signaling pathway and with the actin cytoskeleton to coordinate polarized secretion with cell wall expansion. These include Ca(2+) signaling, phosphatidylinositide signaling at the plasma membrane, sphingoid base signaling through the Pkh1 and -2 protein kinases, Tor kinase signaling, and pathways controlled by the Rho3, Rho4, and Cdc42 G-proteins. [Abstract/Link to Full Text]

Rediers H, Rainey PB, Vanderleyden J, De Mot R
Unraveling the secret lives of bacteria: use of in vivo expression technology and differential fluorescence induction promoter traps as tools for exploring niche-specific gene expression.
Microbiol Mol Biol Rev. 2005 Jun;69(2):217-61.
A major challenge for microbiologists is to elucidate the strategies deployed by microorganisms to adapt to and thrive in highly complex and dynamic environments. In vitro studies, including those monitoring genomewide changes, have proven their value, but they can, at best, mimic only a subset of the ensemble of abiotic and biotic stimuli that microorganisms experience in their natural habitats. The widely used gene-to-phenotype approach involves the identification of altered niche-related phenotypes on the basis of gene inactivation. However, many traits contributing to ecological performance that, upon inactivation, result in only subtle or difficult to score phenotypic changes are likely to be overlooked by this otherwise powerful approach. Based on the premise that many, if not most, of the corresponding genes will be induced or upregulated in the environment under study, ecologically significant genes can alternatively be traced using the promoter trap techniques differential fluorescence induction and in vivo expression technology (IVET). The potential and limitations are discussed for the different IVET selection strategies and system-specific variants thereof. Based on a compendium of genes that have emerged from these promoter-trapping studies, several functional groups have been distinguished, and their physiological relevance is illustrated with follow-up studies of selected genes. In addition to confirming results from largely complementary approaches such as signature-tagged mutagenesis, some unexpected parallels as well as distinguishing features of microbial phenotypic acclimation in diverse environmental niches have surfaced. On the other hand, by the identification of a large proportion of genes with unknown function, these promoter-trapping studies underscore how little we know about the secret lives of bacteria and other microorganisms. [Abstract/Link to Full Text]

Vemuri GN, Aristidou AA
Metabolic engineering in the -omics era: elucidating and modulating regulatory networks.
Microbiol Mol Biol Rev. 2005 Jun;69(2):197-216.
The importance of regulatory control in metabolic processes is widely acknowledged, and several enquiries (both local and global) are being made in understanding regulation at various levels of the metabolic hierarchy. The wealth of biological information has enabled identifying the individual components (genes, proteins, and metabolites) of a biological system, and we are now in a position to understand the interactions between these components. Since phenotype is the net result of these interactions, it is immensely important to elucidate them not only for an integrated understanding of physiology, but also for practical applications of using biological systems as cell factories. We present some of the recent "-omics" approaches that have expanded our understanding of regulation at the gene, protein, and metabolite level, followed by analysis of the impact of this progress on the advancement of metabolic engineering. Although this review is by no means exhaustive, we attempt to convey our ideology that combining global information from various levels of metabolic hierarchy is absolutely essential in understanding and subsequently predicting the relationship between changes in gene expression and the resulting phenotype. The ultimate aim of this review is to provide metabolic engineers with an overview of recent advances in complementary aspects of regulation at the gene, protein, and metabolite level and those involved in fundamental research with potential hurdles in the path to implementing their discoveries in practical applications. [Abstract/Link to Full Text]

Brencic A, Winans SC
Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria.
Microbiol Mol Biol Rev. 2005 Mar;69(1):155-94.
Diverse interactions between hosts and microbes are initiated by the detection of host-released chemical signals. Detection of these signals leads to altered patterns of gene expression that culminate in specific and adaptive changes in bacterial physiology that are required for these associations. This concept was first demonstrated for the members of the family Rhizobiaceae and was later found to apply to many other plant-associated bacteria as well as to microbes that colonize human and animal hosts. The family Rhizobiaceae includes various genera of rhizobia as well as species of Agrobacterium. Rhizobia are symbionts of legumes, which fix nitrogen within root nodules, while Agrobacterium tumefaciens is a pathogen that causes crown gall tumors on a wide variety of plants. The plant-released signals that are recognized by these bacteria are low-molecular-weight, diffusible molecules and are detected by the bacteria through specific receptor proteins. Similar phenomena are observed with other plant pathogens, including Pseudomonas syringae, Ralstonia solanacearum, and Erwinia spp., although here the signals and signal receptors are not as well defined. In some cases, nutritional conditions such as iron limitation or the lack of nitrogen sources seem to provide a significant cue. While much has been learned about the process of host detection over the past 20 years, our knowledge is far from being complete. The complex nature of the plant-microbe interactions makes it extremely challenging to gain a comprehensive picture of host detection in natural environments, and thus many signals and signal recognition systems remain to be described. [Abstract/Link to Full Text]

Demain AL, Newcomb M, Wu JH
Cellulase, clostridia, and ethanol.
Microbiol Mol Biol Rev. 2005 Mar;69(1):124-54.
Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield. [Abstract/Link to Full Text]

Laursen BS, Sřrensen HP, Mortensen KK, Sperling-Petersen HU
Initiation of protein synthesis in bacteria.
Microbiol Mol Biol Rev. 2005 Mar;69(1):101-23.
Valuable information on translation initiation is available from biochemical data and recently solved structures. We present a detailed description of current knowledge about the structure, function, and interactions of the individual components involved in bacterial translation initiation. The first section describes the ribosomal features relevant to the initiation process. Subsequent sections describe the structure, function, and interactions of the mRNA, the initiator tRNA, and the initiation factors IF1, IF2, and IF3. Finally, we provide an overview of mechanisms of regulation of the translation initiation event. Translation occurs on ribonucleoprotein complexes called ribosomes. The ribosome is composed of a large subunit and a small subunit that hold the activities of peptidyltransfer and decode the triplet code of the mRNA, respectively. Translation initiation is promoted by IF1, IF2, and IF3, which mediate base pairing of the initiator tRNA anticodon to the mRNA initiation codon located in the ribosomal P-site. The mechanism of translation initiation differs for canonical and leaderless mRNAs, since the latter is dependent on the relative level of the initiation factors. Regulation of translation occurs primarily in the initiation phase. Secondary structures at the mRNA ribosomal binding site (RBS) inhibit translation initiation. The accessibility of the RBS is regulated by temperature and binding of small metabolites, proteins, or antisense RNAs. The future challenge is to obtain atomic-resolution structures of complete initiation complexes in order to understand the mechanism of translation initiation in molecular detail. [Abstract/Link to Full Text]

Inoki K, Ouyang H, Li Y, Guan KL
Signaling by target of rapamycin proteins in cell growth control.
Microbiol Mol Biol Rev. 2005 Mar;69(1):79-100.
Target of rapamycin (TOR) proteins are members of the phosphatidylinositol kinase-related kinase (PIKK) family and are highly conserved from yeast to mammals. TOR proteins integrate signals from growth factors, nutrients, stress, and cellular energy levels to control cell growth. The ribosomal S6 kinase 1 (S6K) and eukaryotic initiation factor 4E binding protein 1(4EBP1) are two cellular targets of TOR kinase activity and are known to mediate TOR function in translational control in mammalian cells. However, the precise molecular mechanism of TOR regulation is not completely understood. One of the recent breakthrough studies in TOR signaling resulted in the identification of the tuberous sclerosis complex gene products, TSC1 and TSC2, as negative regulators for TOR signaling. Furthermore, the discovery that the small GTPase Rheb is a direct downstream target of TSC1-TSC2 and a positive regulator of the TOR function has significantly advanced our understanding of the molecular mechanism of TOR activation. Here we review the current understanding of the regulation of TOR signaling and discuss its function as a signaling nexus to control cell growth during normal development and tumorigenesis. [Abstract/Link to Full Text]


Recent Articles in Applied and Environmental Microbiology

Tinsley E, Khan SA
A Bacillus anthracis-based in vitro system supports replication of plasmid pXO2 as well as rolling-circle-replicating plasmids.
Appl Environ Microbiol. 2007 Aug;73(15):5005-10.
Capsule-encoding virulence plasmid pXO2 of Bacillus anthracis is predicted to replicate by a unidirectional theta-type mechanism. To gain a better understanding of the mechanism of replication of pXO2 and other plasmids in B. anthracis and related organisms, we have developed a cell-free system based on B. anthracis that can faithfully replicate plasmid DNA in vitro. The newly developed system was shown to support the in vitro replication of plasmid pT181, which replicates by the rolling-circle mechanism. We also demonstrate that this system supports the replication of plasmid pXO2 of B. anthracis. Replication of pXO2 required directional transcription through the plasmid origin of replication, and increased transcription through the origin resulted in an increase in plasmid replication. [Abstract/Link to Full Text]

Jiang H, Yang C, Qu H, Liu Z, Fu QS, Qiao C
Cloning of a novel aldo-keto reductase gene from Klebsiella sp. strain F51-1-2 and its functional expression in Escherichia coli.
Appl Environ Microbiol. 2007 Aug;73(15):4959-65.
A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His(6)-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the K(m) and k(cat) values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 +/- 47.0 microM and 25.7 +/- 1.7 min(-1), respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling. [Abstract/Link to Full Text]

Trosvik P, Skĺnseng B, Jakobsen KS, Stenseth NC, Naes T, Rudi K
Multivariate analysis of complex DNA sequence electropherograms for high-throughput quantitative analysis of mixed microbial populations.
Appl Environ Microbiol. 2007 Aug;73(15):4975-83.
High-throughput quantification of genetically coherent units (GCUs) is essential for deciphering population dynamics and species interactions within a community of microbes. Current techniques for microbial community analyses are, however, not suitable for this kind of high-throughput application. Here, we demonstrate the use of multivariate statistical analysis of complex DNA sequence electropherograms for the effective and accurate estimation of relative genotype abundance in cell samples from mixed microbial populations. The procedure is no more labor-intensive than standard automated DNA sequencing and provides a very effective means of quantitative data acquisition from experimental microbial communities. We present results with the Campylobacter jejuni strain-specific marker gene gltA, as well as the 16S rRNA gene, which is a universal marker across bacterial assemblages. The statistical models computed for these genes are applied to genetic data from two different experimental settings, namely, a chicken infection model and a multispecies anaerobic fermentation model, demonstrating collection of time series data from model bacterial communities. The method presented here is, however, applicable to any experimental scenario where the interest is quantification of GCUs in genetically heterogeneous DNA samples. [Abstract/Link to Full Text]

Baldridge GD, Burkhardt NY, Felsheim RF, Kurtti TJ, Munderloh UG
Transposon insertion reveals pRM, a plasmid of Rickettsia monacensis.
Appl Environ Microbiol. 2007 Aug;73(15):4984-95.
Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector. [Abstract/Link to Full Text]

Aucina A, Rudawska M, Leski T, Skridaila A, Riepsas E, Iwanski M
Growth and mycorrhizal community structure of Pinus sylvestris seedlings following the addition of forest litter.
Appl Environ Microbiol. 2007 Aug;73(15):4867-73.
We report the effects of pine and oak litter on species composition and diversity of mycorrhizal fungi colonizing 2-year-old Pinus sylvestris L. seedlings grown in a bare-root nursery in Lithuania. A layer of pine or oak litter was placed on the surface of the nursery bed soil to mimic natural litter cover. Oak litter amendment appeared to be most favorable for seedling survival, with a 73% survival rate, in contrast to the untreated mineral bed soil (44%). The concentrations of total N, P, K, Ca, and Mg were higher in oak growth medium than in pine growth medium. Relative to the control (pH 6.1), the pH was lower in pine growth medium (5.8) and higher in oak growth medium (6.3). There were also twofold and threefold increases in the C content of growth medium with the addition of pine and oak litter, respectively. Among seven mycorrhizal morphotypes, eight different mycorrhizal taxa were identified: Suillus luteus, Suillus variegatus, Wilcoxina mikolae, a Tuber sp., a Tomentella sp., Cenococcum geophilum, Amphinema byssoides, and one unidentified ectomycorrhizal symbiont. Forest litter addition affected the relative abundance of mycorrhizal symbionts more than their overall representation. This was more pronounced for pine litter than for oak litter, with 40% and 25% increases in the abundance of suilloid mycorrhizae, respectively. Our findings provide preliminary evidence that changes in the supply of organic matter through litter manipulation may have far-reaching effects on the chemistry of soil, thus influencing the growth and survival of Scots pine seedlings and their mycorrhizal communities. [Abstract/Link to Full Text]

Dillon JG, Fishbain S, Miller SR, Bebout BM, Habicht KS, Webb SM, Stahl DA
High rates of sulfate reduction in a low-sulfate hot spring microbial mat are driven by a low level of diversity of sulfate-respiring microorganisms.
Appl Environ Microbiol. 2007 Aug;73(16):5218-26.
The importance of sulfate respiration in the microbial mat found in the low-sulfate thermal outflow of Mushroom Spring in Yellowstone National Park was evaluated using a combination of molecular, microelectrode, and radiotracer studies. Despite very low sulfate concentrations, this mat community was shown to sustain a highly active sulfur cycle. The highest rates of sulfate respiration were measured close to the surface of the mat late in the day when photosynthetic oxygen production ceased and were associated with a Thermodesulfovibrio-like population. Reduced activity at greater depths was correlated with novel populations of sulfate-reducing microorganisms, unrelated to characterized species, and most likely due to both sulfate and carbon limitation. [Abstract/Link to Full Text]

Fall S, Hamelin J, Ndiaye F, Assigbetse K, Aragno M, Chotte JL, Brauman A
Differences between bacterial communities in the gut of a soil-feeding termite (Cubitermes niokoloensis) and its mounds.
Appl Environ Microbiol. 2007 Aug;73(16):5199-208.
In tropical ecosystems, termite mound soils constitute an important soil compartment covering around 10% of African soils. Previous studies have shown (S. Fall, S. Nazaret, J. L. Chotte, and A. Brauman, Microb. Ecol. 28:191-199, 2004) that the bacterial genetic structure of the mounds of soil-feeding termites (Cubitermes niokoloensis) is different from that of their surrounding soil. The aim of this study was to characterize the specificity of bacterial communities within mounds with respect to the digestive and soil origins of the mound. We have compared the bacterial community structures of a termite mound, termite gut sections, and surrounding soil using PCR-denaturing gradient gel electrophoresis (DGGE) analysis and cloning and sequencing of PCR-amplified 16S rRNA gene fragments. DGGE analysis revealed a drastic difference between the genetic structures of the bacterial communities of the termite gut and the mound. Analysis of 266 clones, including 54 from excised bands, revealed a high level of diversity in each biota investigated. The soil-feeding termite mound was dominated by the Actinobacteria phylum, whereas the Firmicutes and Proteobacteria phyla dominate the gut sections of termites and the surrounding soil, respectively. Phylogenetic analyses revealed a distinct clustering of Actinobacteria phylotypes between the mound and the surrounding soil. The Actinobacteria clones of the termite mound were diverse, distributed among 10 distinct families, and like those in the termite gut environment lightly dominated by the Nocardioidaceae family. Our findings confirmed that the soil-feeding termite mound (C. niokoloensis) represents a specific bacterial habitat in the tropics. [Abstract/Link to Full Text]

Huang TP, Wong AC
A cyclic AMP receptor protein-regulated cell-cell communication system mediates expression of a FecA homologue in Stenotrophomonas maltophilia.
Appl Environ Microbiol. 2007 Aug;73(15):5034-40.
Stenotrophomonas maltophilia WR-C possesses an rpf/diffusible signal factor (DSF) cell-cell communication system. It produces cis-Delta2-11-methyl-dodecenoic acid, a DSF, and seven structural derivatives, which require rpfF and rpfB for synthesis. Acquisition of iron from the environment is important for bacterial growth as well as the expression of virulence genes. We identified a gene homologous to fecA, which encodes a ferric citrate receptor that transports exogenous siderophore ferric citrate from the environment into the bacterial periplasm. Western blot analysis with anti-FecA-His(6) antibody showed that the FecA homologue was induced in the iron-depleted medium supplemented with a low concentration of ferric citrate. Deletion of rpfF or rpfB resulted in reduced FecA expression compared to the wild type. Synthetic DSF restored FecA expression by the DeltarpfF mutant to the wild-type level. Reverse transcription-PCR showed that the fecA transcript was decreased in the DeltarpfF mutant compared to the wild type. These data suggest that DSF affected the level of fecA mRNA. Transposon inactivation of crp, which encodes cyclic AMP (cAMP) receptor protein (CRP) resulted in reduced FecA expression and rpfF transcript level. Putative CRP binding sites were located upstream of the rpfF promoter, indicating that the effect of CRP on FecA is through the rpf/DSF pathway and by directly controlling rpfF. We propose that CRP may serve as a checkpoint for iron uptake, protease activity, and hemolysis in response to environmental changes such as changes in concentrations of glucose, cAMP, iron, or DSF. [Abstract/Link to Full Text]

Singh BK, Tate KR, Kolipaka G, Hedley CB, Macdonald CA, Millard P, Murrell JC
Effect of afforestation and reforestation of pastures on the activity and population dynamics of methanotrophic bacteria.
Appl Environ Microbiol. 2007 Aug;73(16):5153-61.
We investigated the effect of afforestation and reforestation of pastures on methane oxidation and the methanotrophic communities in soils from three different New Zealand sites. Methane oxidation was measured in soils from two pine (Pinus radiata) forests and one shrubland (mainly Kunzea ericoides var. ericoides) and three adjacent permanent pastures. The methane oxidation rate was consistently higher in the pine forest or shrubland soils than in the adjacent pasture soils. A combination of phospholipid fatty acid (PLFA) and stable isotope probing (SIP) analyses of these soils revealed that different methanotrophic communities were active in soils under the different vegetations. The C18 PLFAs (signature of type II methanotrophs) predominated under pine and shrublands, and C16 PLFAs (type I methanotrophs) predominated under pastures. Analysis of the methanotrophs by molecular methods revealed further differences in methanotrophic community structure under the different vegetation types. Cloning and sequencing and terminal-restriction fragment length polymorphism analysis of the particulate methane oxygenase gene (pmoA) from different samples confirmed the PLFA-SIP results that methanotrophic bacteria related to type II methanotrophs were dominant in pine forest and shrubland, and type I methanotrophs (related to Methylococcus capsulatus) were dominant in all pasture soils. We report that afforestation and reforestation of pastures caused changes in methane oxidation by altering the community structure of methanotrophic bacteria in these soils. [Abstract/Link to Full Text]

Sunnotel O, Snelling WJ, McDonough N, Browne L, Moore JE, Dooley JS, Lowery CJ
Effectiveness of standard UV depuration at inactivating Cryptosporidium parvum recovered from spiked Pacific oysters (Crassostrea gigas).
Appl Environ Microbiol. 2007 Aug;73(16):5083-7.
When filter-feeding shellfish are consumed raw, because of their ability to concentrate and store waterborne pathogens, they are being increasingly associated with human gastroenteritis and have become recognized as important pathogen vectors. In the shellfish industry, UV depuration procedures are mandatory to reduce pathogen levels prior to human consumption. However, these guidelines are based around more susceptible fecal coliforms and Salmonella spp. and do not consider Cryptosporidium spp., which have significant resistance to environmental stresses. Thus, there is an urgent need to evaluate the efficiency of standard UV depuration against the survival of Cryptosporidium recovered from shellfish. Our study found that in industrial-scale shellfish depuration treatment tanks, standard UV treatment resulted in a 13-fold inactivation of recovered, viable C. parvum oocysts from spiked (1 x 10(6) oocysts liter (-1)) Pacific oysters. Depuration at half power also significantly reduced (P < 0.05; ninefold) the number of viable oocysts recovered from oysters. While UV treatment resulted in significant reductions of recovered viable oocysts, low numbers of viable oocysts were still recovered from oysters after depuration, making their consumption when raw a public health risk. Our study highlights the need for increased periodic monitoring programs for shellfish harvesting sites, improved depuration procedures, and revised microbial quality control parameters, including Cryptosporidium assessment, to minimize the risk of cryptosporidiosis. [Abstract/Link to Full Text]

de Kerchove AJ, Elimelech M
Impact of alginate conditioning film on deposition kinetics of motile and nonmotile Pseudomonas aeruginosa strains.
Appl Environ Microbiol. 2007 Aug;73(16):5227-34.
The initial deposition of bacteria in most aquatic systems is affected by the presence of a conditioning film adsorbed at the liquid-solid interface. Due to the inherent complexity of such films, their impact on bacterial deposition remains poorly defined. The aim of this study was to gain a better understanding of the effect of a conditioning film on the deposition of motile and nonmotile Pseudomonas aeruginosa cells in a radial stagnation point flow system. A well-defined alginate film was used as a model conditioning film because of its polysaccharide and polyelectrolyte nature. Deposition experiments under favorable (nonrepulsive) conditions demonstrated the importance of swimming motility for cell transport towards the substrate. The impact of the flagella of motile cells on deposition is dependent on the presence of the conditioning film. We showed that on a clean substrate surface, electrostatic repulsion governs bacterial deposition and the presence of flagella increases cell deposition. However, our results suggest that steric interactions between flagella and extended polyelectrolytes of the conditioning film hinder cell deposition. At a high ionic strength (100 mM), active swimming motility and changes in alginate film structure suppressed the steric barrier and allowed conditions favorable for deposition. We demonstrated that bacterial deposition is highly influenced by cell motility and the structure of the conditioning film, which are both dependent on ionic strength. [Abstract/Link to Full Text]

Fox JT, Renter DG, Sanderson MW, Thomson DU, Lechtenberg KF, Nagaraja TG
Evaluation of culture methods to identify bovine feces with high concentrations of Escherichia coli O157.
Appl Environ Microbiol. 2007 Aug;73(16):5253-60.
Our objective was to evaluate methods for identifying cattle with high concentrations of Escherichia coli O157 in their feces. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid (Nal)-resistant E. coli O157, and direct plating of diluted feces on sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) containing Nal was considered the gold standard (GS) method. In experiment 1, methods evaluated were preenrichment direct streak, immunomagnetic separation with most probable number (MPN), and postenrichment direct streak with MPN, all using CT-SMAC. The mean concentration of Nal-resistant E. coli O157 in samples (n = 59) by use of the GS was 3.6 log10 CFU/g. The preenrichment streak detected >3.0 log10 CFU/g samples with a 74.4% sensitivity and 68.8% specificity. Postenrichment direct streak-MPN and immunomagnetic separation-MPN concentrations were correlated significantly with GS concentrations (r = 0.53 and r = 0.39, respectively). In experiment 2 (480 samples), pre- and postenrichment direct streaking performed in triplicate and spiral plating on CT-SMAC were evaluated. For preenrichment streaks, sensitivity was 79.7% and specificity was 96.7% for detecting >3.0 log10 CFU/g when the criterion was positive cultures on at least two plates. For spiral plating at that concentration, sensitivity and specificity were 83.9% and 56.3%, respectively. Postenrichment streaking performed relatively poorly. Triplicate preenrichment streaks of 1:10-diluted feces on CT-SMAC may be useful for identifying cattle shedding high concentrations of E. coli O157. Estimates of sensitivity and specificity enable appropriate application of methods and interpretation of results and may enhance applied research, surveillance, and risk assessments. [Abstract/Link to Full Text]

Richter H, Lanthier M, Nevin KP, Lovley DR
Lack of electricity production by Pelobacter carbinolicus indicates that the capacity for Fe(III) oxide reduction does not necessarily confer electron transfer ability to fuel cell anodes.
Appl Environ Microbiol. 2007 Aug;73(16):5347-53.
The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different. [Abstract/Link to Full Text]

Quesada A, Guijo MI, Merchán F, Blázquez B, Igeńo MI, Blasco R
Essential role of cytochrome bd-related oxidase in cyanide resistance of Pseudomonas pseudoalcaligenes CECT5344.
Appl Environ Microbiol. 2007 Aug;73(16):5118-24.
Pseudomonas pseudoalcaligenes CECT5344 grows in minimal medium containing cyanide as the sole nitrogen source. Under these conditions, an O2-dependent respiration highly resistant to cyanide was detected in cell extracts. The structural genes for the cyanide-resistant terminal oxidase, cioA and cioB, are clustered and encode the integral membrane proteins that correspond to subunits I and II of classical cytochrome bd, although the presence of heme d in the membrane could not be detected by difference spectra. The cio operon from P. pseudoalcaligenes presents a singular organization, starting upstream of cioAB by the coding sequence of a putative ferredoxin-dependent sulfite or nitrite reductase and spanning downstream two additional open reading frames that encode uncharacterized gene products. PCR amplifications of RNA (reverse transcription-PCR) indicated the cyanide-dependent up-regulation and cotranscription along the operon. The targeted disruption of cioA eliminates both the expression of the cyanide-stimulated respiratory activity and the growth with cyanide as the nitrogen source, which suggests a critical role of this cytochrome bd-related oxidase in the metabolism of cyanide by P. pseudoalcaligenes CECT5344. [Abstract/Link to Full Text]

Besemer K, Singer G, Limberger R, Chlup AK, Hochedlinger G, Hödl I, Baranyi C, Battin TJ
Biophysical controls on community succession in stream biofilms.
Appl Environ Microbiol. 2007 Aug;73(15):4966-74.
Biofilm formation is controlled by an array of coupled physical, chemical, and biotic processes. Despite the ecological relevance of microbial biofilms, their community formation and succession remain poorly understood. We investigated the effect of flow velocity, as the major physical force in stream ecosystems, on biofilm community succession (as continuous shifts in community composition) in microcosms under laminar, intermediate, and turbulent flow. Flow clearly shaped the development of biofilm architecture and community composition, as revealed by microscopic investigation, denaturing gradient gel electrophoresis (DGGE) analysis, and sequencing. While biofilm growth patterns were undirected under laminar flow, they were clearly directed into ridges and conspicuous streamers under turbulent flow. A total of 51 biofilm DGGE bands were detected; the average number ranged from 13 to 16. Successional trajectories diverged from an initial community that was common in all flow treatments and increasingly converged as biofilms matured. We suggest that this developmental pattern was primarily driven by algae, which, as "ecosystem engineers," modulate their microenvironment to create similar architectures and flow conditions in all treatments and thereby reduce the physical effect of flow on biofilms. Our results thus suggest a shift from a predominantly physical control to coupled biophysical controls on bacterial community succession in stream biofilms. [Abstract/Link to Full Text]

Akeboshi H, Chiba Y, Kasahara Y, Takashiba M, Takaoka Y, Ohsawa M, Tajima Y, Kawashima I, Tsuji D, Itoh K, Sakuraba H, Jigami Y
Production of recombinant beta-hexosaminidase A, a potential enzyme for replacement therapy for Tay-Sachs and Sandhoff diseases, in the methylotrophic yeast Ogataea minuta.
Appl Environ Microbiol. 2007 Aug;73(15):4805-12.
Human beta-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of alpha- and beta-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using alpha-1,6-mannosyltransferase-deficient (och1Delta) yeast as the host. Genes encoding the alpha- and beta-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (alphaalpha) and HexB (betabeta). A total of 57 mg of beta-hexosaminidase isozymes, of which 13 mg was HexA (alphabeta), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the beta-subunit. The purified HexA was treated with alpha-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 +/- 0.1 and 1.7 +/- 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the beta-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed. [Abstract/Link to Full Text]

Santos R, Fernandes J, Fernandes N, Oliveira F, Cadete M
Mycobacterium parascrofulaceum in acidic hot springs in Yellowstone National Park.
Appl Environ Microbiol. 2007 Aug;73(15):5071-3.
Mycobacterium parascrofulaceum was found in Norris Geyser Basin, Yellowstone National Park, in a system composed of two acidic (pH 3.0) springs with temperatures between 56 degrees C at the source and 40 degrees C at the confluence of both springs. Growth and survival assays at 56 degrees C for 60 days were performed, confirming the origin of the strain. [Abstract/Link to Full Text]

Koutsoumanis K, Angelidis AS
Probabilistic modeling approach for evaluating the compliance of ready-to-eat foods with new European Union safety criteria for Listeria monocytogenes.
Appl Environ Microbiol. 2007 Aug;73(15):4996-5004.
Among the new microbiological criteria that have been incorporated in EU Regulation 2073/2005, of particular interest are those concerning Listeria monocytogenes in ready-to eat (RTE) foods, because for certain food categories, they no longer require zero tolerance but rather specify a maximum allowable concentration of 100 CFU/g or ml. This study presents a probabilistic modeling approach for evaluating the compliance of RTE sliced meat products with the new safety criteria for L. monocytogenes. The approach was based on the combined use of (i) growth/no growth boundary models, (ii) kinetic growth models, (iii) product characteristics data (pH, a(w), shelf life) collected from 160 meat products from the Hellenic retail market, and (iv) storage temperature data recorded from 50 retail stores in Greece. This study shows that probabilistic analysis of the above components using Monte Carlo simulation, which takes into account the variability of factors affecting microbial growth, can lead to a realistic estimation of the behavior of L. monocytogenes throughout the food supply chain, and the quantitative output generated can be further used by food managers as a decision-making tool regarding the design or modification of a product's formulation or its "use-by" date in order to ensure its compliance with the new safety criteria. The study also argues that compliance of RTE foods with the new safety criteria should not be considered a parameter with a discrete and binary outcome because it depends on factors such as product characteristics, storage temperature, and initial contamination level, which display considerable variability even among different packages of the same RTE product. Rather, compliance should be expressed and therefore regulated in a more probabilistic fashion. [Abstract/Link to Full Text]

Bibbal D, Dupouy V, Ferré JP, Toutain PL, Fayet O, Prčre MF, Bousquet-Mélou A
Impact of three ampicillin dosage regimens on selection of ampicillin resistance in Enterobacteriaceae and excretion of blaTEM genes in swine feces.
Appl Environ Microbiol. 2007 Aug;73(15):4785-90.
The aim of this study was to assess the impact of three ampicillin dosage regimens on ampicillin resistance among Enterobacteriaceae recovered from swine feces by use of phenotypic and genotypic approaches. Phenotypically, ampicillin resistance was determined from the percentage of resistant Enterobacteriaceae and MICs of Escherichia coli isolates. The pool of ampicillin resistance genes was also monitored by quantification of bla(TEM) genes, which code for the most frequently produced beta-lactamases in gram-negative bacteria, using a newly developed real-time PCR assay. Ampicillin was administered intramuscularly and orally to fed or fasted pigs for 7 days at 20 mg/kg of body weight. The average percentage of resistant Enterobacteriaceae before treatment was between 2.5% and 12%, and bla(TEM) gene quantities were below 10(7) copies/g of feces. By days 4 and 7, the percentage of resistant Enterobacteriaceae exceeded 50% in all treated groups, with some highly resistant strains (MIC of >256 microg/ml). In the control group, bla(TEM) gene quantities fluctuated between 10(4) and 10(6) copies/g of feces, whereas they fluctuated between 10(6) to 10(8) and 10(7) to 10(9) copies/g of feces for the intramuscular and oral routes, respectively. Whereas phenotypic evaluations did not discriminate among the three ampicillin dosage regimens, bla(TEM) gene quantification was able to differentiate between the effects of two routes of ampicillin administration. Our results suggest that fecal bla(TEM) gene quantification provides a sensitive tool to evaluate the impact of ampicillin administration on the selection of ampicillin resistance in the digestive microflora and its dissemination in the environment. [Abstract/Link to Full Text]

Miller SR, Castenholz RW, Pedersen D
Phylogeography of the thermophilic cyanobacterium Mastigocladus laminosus.
Appl Environ Microbiol. 2007 Aug;73(15):4751-9.
We have taken a phylogeographic approach to investigate the demographic and evolutionary processes that have shaped the geographic patterns of genetic diversity for a sample of isolates of the cosmopolitan thermophilic cyanobacterial Mastigocladus laminosus morphotype collected from throughout most of its range. Although M. laminosus is found in thermal areas throughout the world, our observation that populations are typically genetically differentiated on local geographic scales suggests the existence of dispersal barriers, a conclusion corroborated by evidence for genetic isolation by distance. Genealogies inferred using nitrogen metabolism gene sequence data suggest that a significant amount of the extant global diversity of M. laminosus can be traced back to a common ancestor associated with the western North American hot spot currently located below Yellowstone National Park. Estimated intragenic recombination rates are comparable to those of pathogenic bacteria known for their capacity to exchange DNA, indicating that genetic exchange has played an important role in generating novel variation during M. laminosus diversification. Selection has constrained protein changes at loci involved in the assimilation of both dinitrogen and nitrate, suggesting the historic use of both nitrogen sources in this heterocystous cyanobacterium. Lineage-specific differences in thermal performance were also observed. [Abstract/Link to Full Text]

Devescovi G, Bigirimana J, Degrassi G, Cabrio L, LiPuma JJ, Kim J, Hwang I, Venturi V
Involvement of a quorum-sensing-regulated lipase secreted by a clinical isolate of Burkholderia glumae in severe disease symptoms in rice.
Appl Environ Microbiol. 2007 Aug;73(15):4950-8.
Burkholderia glumae is an emerging rice pathogen in several areas around the world. Closely related Burkholderia species are important opportunistic human pathogens for specific groups of patients, such as patients with cystic fibrosis and patients with chronic granulomatous disease. Here we report that the first clinical isolate of B. glumae, strain AU6208, has retained its capability to be very pathogenic to rice. As previously reported for rice isolate B. glumae BGR1 (and also for the clinical isolate AU6208), TofI or TofR acyl homoserine lactone (AHL) quorum sensing played a pivotal role in rice virulence. We report that AHL quorum sensing in B. glumae AU6208 regulates secreted LipA lipase and toxoflavin, the phytotoxin produced by B. glumae. B. glumae AU6208 lipA mutants were no longer pathogenic to rice, indicating that the lipase is an important virulence factor. It was also established that type strain B. glumae ATCC 33617 did not produce toxoflavin and lipase and was nonpathogenic to rice. It was determined that in strain ATCC 33617 the LuxR family quorum-sensing sensor/regulator TofR was inactive. Introducing the tofR gene of B. glumae AU6208 in strain ATCC 33617 restored its ability to produce toxoflavin and the LipA lipase. This study extends the role of AHL quorum sensing in rice pathogenicity through the regulation of a lipase which was demonstrated to be a virulence factor. It is the first report of a clinical B. glumae isolate retaining strong rice pathogenicity and finally determined that B. glumae can undergo phenotypic conversion through a spontaneous mutation in the tofR regulator. [Abstract/Link to Full Text]

Paranjpye RN, Johnson AB, Baxter AE, Strom MS
Role of type IV pilins in persistence of Vibrio vulnificus in Crassostrea virginica oysters.
Appl Environ Microbiol. 2007 Aug;73(15):5041-4.
Vibrio vulnificus is part of the natural estuarine microflora and accumulates in shellfish through filter feeding. It is responsible for the majority of seafood-associated fatalities in the United States mainly through consumption of raw oysters. Previously we have shown that a V. vulnificus mutant unable to express PilD, the type IV prepilin peptidase, does not express pili on the surface of the bacterium and is defective in adherence to human epithelial cells (R. N. Paranjpye, J. C. Lara, J. C. Pepe, C. M. Pepe, and M. S. Strom, Infect. Immun. 66:5659-5668, 1998). A mutant unable to express one of the type IV pilins, PilA, is also defective in adherence to epithelial cells as well as biofilm formation on abiotic surfaces (R. N. Paranjpye and M. S. Strom, Infect. Immun. 73:1411-1422, 2005). In this study we report that the loss of PilD or PilA significantly reduces the ability of V. vulnificus to persist in Crassostrea virginica over a 66-h interval, strongly suggesting that pili expressed by this bacterium play a role in colonization or persistence in oysters. [Abstract/Link to Full Text]

Van der Meulen R, Scheirlinck I, Van Schoor A, Huys G, Vancanneyt M, Vandamme P, De Vuyst L
Population dynamics and metabolite target analysis of lactic acid bacteria during laboratory fermentations of wheat and spelt sourdoughs.
Appl Environ Microbiol. 2007 Aug;73(15):4741-50.
Four laboratory sourdough fermentations, initiated with wheat or spelt flour and without the addition of a starter culture, were prepared over a period of 10 days with daily back-slopping. Samples taken at all refreshment steps were used for determination of the present microbiota. Furthermore, an extensive metabolite target analysis of more than 100 different compounds was performed through a combination of various chromatographic methods including liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. The establishment of a stable microbial ecosystem occurred through a three-phase evolution within a week, as revealed by both microbiological and metabolite analyses. Strains of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus rossiae, Lactobacillus brevis, and Lactobacillus paraplantarum were dominating some of the sourdough ecosystems. Although the heterofermentative L. fermentum was dominating one of the wheat sourdoughs, all other sourdoughs were dominated by a combination of obligate and facultative heterofermentative taxa. Strains of homofermentative species were not retrieved in the stable sourdough ecosystems. Concentrations of sugar and amino acid metabolites hardly changed during the last days of fermentation. Besides lactic acid, ethanol, and mannitol, the production of succinic acid, erythritol, and various amino acid metabolites, such as phenyllactic acid, hydroxyphenyllactic acid, and indolelactic acid, was shown during fermentation. Physiologically, they contributed to the equilibration of the redox balance. The biphasic approach of the present study allowed us to map some of the interactions taking place during sourdough fermentation and helped us to understand the fine-tuned metabolism of lactic acid bacteria, which allows them to dominate a food ecosystem. [Abstract/Link to Full Text]

Montero CI, Johnson MR, Chou CJ, Conners SB, Geouge SG, Tachdjian S, Nichols JD, Kelly RM
Responses of wild-type and resistant strains of the hyperthermophilic bacterium Thermotoga maritima to chloramphenicol challenge.
Appl Environ Microbiol. 2007 Aug;73(15):5058-65.
Transcriptomes and growth physiologies of the hyperthermophile Thermotoga maritima and an antibiotic-resistant spontaneous mutant were compared prior to and following exposure to chloramphenicol. While the wild-type response was similar to that of mesophilic bacteria, reduced susceptibility of the mutant was attributed to five mutations in 23S rRNA and phenotypic preconditioning to chloramphenicol. [Abstract/Link to Full Text]

Cordaux R, Paces-Fessy M, Raimond M, Michel-Salzat A, Zimmer M, Bouchon D
Molecular characterization and evolution of arthropod-pathogenic Rickettsiella bacteria.
Appl Environ Microbiol. 2007 Aug;73(15):5045-7.
We determined the 16S rRNA gene sequences of three crustacean "Rickettsiella armadillidii" strains. Rickettsiella bacteria overall appear to form a monophyletic group that diverged from Coxiella bacteria approximately 350 million years ago. Therefore, the genus Rickettsiella as a whole (not just Rickettsiella grylli) should be classified among the Gammaproteobacteria instead of the Alphaproteobacteria. [Abstract/Link to Full Text]

Gourmelon M, Caprais MP, Ségura R, Le Mennec C, Lozach S, Piriou JY, Rincé A
Evaluation of two library-independent microbial source tracking methods to identify sources of fecal contamination in French estuaries.
Appl Environ Microbiol. 2007 Aug;73(15):4857-66.
In order to identify the origin of the fecal contamination observed in French estuaries, two library-independent microbial source tracking (MST) methods were selected: (i) Bacteroidales host-specific 16S rRNA gene markers and (ii) F-specific RNA bacteriophage genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, and bird) feces. Two human-specific markers (HF183 and HF134), one ruminant-specific marker (CF193'), and one pig-specific marker (PF163) showed a high level of specificity (>90%). However, the data suggest that the proposed ruminant-specific CF128 marker would be better described as an animal marker, as it was observed in all bovine and sheep feces and 96% of pig feces. F RNA bacteriophages were detected in only 21% of individual fecal samples tested, in 60% of pig slurries, but in all sewage samples. Most detected F RNA bacteriophages were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird feces and from pig slurries. Both MST methods were applied to 28 water samples collected from three watersheds at different times. Classification of water samples as subject to human, animal, or mixed fecal contamination was more frequent when using Bacteroidales markers (82.1% of water samples) than by bacteriophage genotyping (50%). The ability to classify a water sample increased with increasing Escherichia coli or enterococcus concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers. [Abstract/Link to Full Text]

Adsul M, Khire J, Bastawde K, Gokhale D
Production of lactic acid from cellobiose and cellotriose by Lactobacillus delbrueckii mutant Uc-3.
Appl Environ Microbiol. 2007 Aug;73(15):5055-7.
Lactobacillus delbrueckii mutant Uc-3 utilizes both cellobiose and cellotriose efficiently, converting it into L(+) lactic acid. The enzyme activities of cellobiose and cellotriose utilization were determined for cell extracts, whole cells, and disrupted cells. Aryl-beta-glucosidase activity was detected only for whole cells and disrupted cells, suggesting that these activities are cell bound. The mutant produced 90 g/liter of lactic acid from 100 g/liter of cellobiose with 2.25 g/liter/h productivity. [Abstract/Link to Full Text]

Steyert SR, Pineiro SA
Development of a novel genetic system to create markerless deletion mutants of Bdellovibrio bacteriovorus.
Appl Environ Microbiol. 2007 Aug;73(15):4717-24.
Bdellovibrio bacteriovorus is a species of unique obligate predatory bacteria that utilize gram-negative bacteria as prey. Their life cycle alternates between a motile extracellular phase and a growth phase within the prey cell periplasm. The mechanism of prey cell invasion and the genetic networks and regulation during the life cycle have not been elucidated. The obligate predatory nature of the B. bacteriovorus life cycle suggests the use of this bacterium in potential applications involving pathogen control but adds complexity to the development of practical genetic systems that can be used to determine gene function. This work reports the development of a genetic technique for allelic exchange or gene inactivation by construction of in-frame markerless deletion mutants including the use of a counterselectable marker in B. bacteriovorus. A suicide plasmid carrying the sacB gene for counterselection was used to inactivate the strB gene in B. bacteriovorus HD100 by an in-frame deletion. Despite the inactivation of the strB gene, B. bacteriovorus was found to retain resistance to high concentrations of streptomycin. The stability of a plasmid for use in complementation experiments was also investigated, and it was determined that pMMB206 replicates autonomously in B. bacteriovorus. Development of this practical genetic system now facilitates the study of B. bacteriovorus at the molecular level and will aid in understanding the regulatory networks and gene function in this fascinating predatory bacterium. [Abstract/Link to Full Text]

Platero R, de Lorenzo V, Garat B, Fabiano E
Sinorhizobium meliloti fur-like (Mur) protein binds a fur box-like sequence present in the mntA promoter in a manganese-responsive manner.
Appl Environ Microbiol. 2007 Aug;73(15):4832-8.
In Sinorhizobium meliloti, the Mur(Sm) protein, a homologue of the ferric uptake regulator (Fur), mediates manganese-dependent regulation of the MntABCD manganese uptake system. In this study, we analyzed Mur(Sm) binding to the promoter region of the S. meliloti mntA gene. We demonstrated that Mur(Sm) protein binds with high affinity to the promoter region of mntA gene in a manganese-responsive manner. Moreover, the results presented here indicate that two monomers, or one dimer, of Mur(Sm) binds the DNA. The binding region was identified by DNase I footprinting analysis and covers a region of about 30 bp long that contains a palindromic sequence. The Mur(Sm) binding site, present in the mntA promoter region, is similar to a Fur box; however, manganese-activated Mur(Sm) binds a canonical Fur box with very low affinity. Furthermore, the data obtained indicate that Mur(Sm) responds to physiological concentrations of manganese. [Abstract/Link to Full Text]

Panadero J, Hernández-López MJ, Prieto JA, Randez-Gil F
Overexpression of the calcineurin target CRZ1 provides freeze tolerance and enhances the fermentative capacity of baker's yeast.
Appl Environ Microbiol. 2007 Aug;73(15):4824-31.
Recent years have shown a huge growth in the market of industrial baker's yeasts (Saccharomyces cerevisiae), with the need for strains affording better performance in prefrozen dough. Evidence suggests that during the freezing process, cells can suffer biochemical damage caused by osmotic stress. Nevertheless, the involvement of ion-responsive transcriptional factors and pathways in conferring freeze resistance has not yet been examined. Here, we have investigated the role of the salt-responsive calcineurin-Crz1p pathway in mediating tolerance to freezing by industrial baker's yeast. Overexpression of CRZ1 in the industrial HS13 strain increased both salt and freeze tolerance and improved the leavening ability of baker's yeast in high-sugar dough. Moreover, engineered cells were able to produce more gas during fermentation of prefrozen dough than the parental strain. Similar effects were observed for overexpression of TdCRZ1, the homologue to CRZ1 in Torulaspora delbrueckii, suggesting that expression of calcineurin-Crz1p target genes can alleviate the harmful effects of ionic stress during freezing. However, overexpression of STZ and FTZ, two unrelated Arabidopsis thaliana genes encoding Cys(2)/His(2)-type zinc finger proteins, also conferred freeze resistance in yeast. Furthermore, experiments with Deltacnb1 and Deltacrz1 mutants failed to show a freeze-sensitive phenotype, even in cells pretreated with NaCl. Overall, our results demonstrate that overexpression of CRZ1 has the potential to be a useful tool for increasing freeze tolerance and fermentative capacity in industrial strains. However, these effects do not appear to be mediated through activation of known salt-responding pathways. [Abstract/Link to Full Text]

Coutard F, Lozach S, Pommepuy M, Hervio-Heath D
Real-time reverse transcription-PCR for transcriptional expression analysis of virulence and housekeeping genes in viable but nonculturable Vibrio parahaemolyticus after recovery of culturability.
Appl Environ Microbiol. 2007 Aug;73(16):5183-9.
A real-time reverse transcription-PCR method was developed to determine whether the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus induced the expression of virulence genes coding for the thermostable direct hemolysin and for type III secretion system 2 (TTSS2). The culturability of clinical strain Vp5 of V. parahaemolyticus in artificial seawater at 4 degrees C was monitored, and the VBNC state was obtained. One day after entry into the VBNC state, temperature upshifts to 20 and 37 degrees C allowed the recovery of culturability. Standard curves for the relative quantification of expression of the housekeeping genes rpoS, pvsA, fur, and pvuA; the tdh2 gene; and the TTSS2 genes (VPA1354, VPA1346, and VPA1342) were established. The levels of expression of the pvsA and pvuA genes were stable and were used to normalize the levels of expression of the other genes. No transcriptional induction of the selected virulence genes under the temperature conditions used to recover the culturability of the VBNC bacteria was observed. The study results demonstrate that the recovery of culturability of VBNC cells of pathogenic V. parahaemolyticus is restricted to regrowth, without correlation with the induction of virulence gene expression. Disease induction would depend mainly on host-pathogen interactions that allow the expression of the virulence genes. This is the first time that the use of mRNA to detect viable cells was evaluated by computing the half-lives of multiple mRNA species under conditions inducing the VBNC state. [Abstract/Link to Full Text]


Recent Articles in Applied Microbiology

Anellis A, Shattuck E, Rowley DB, Ross EW, Whaley DN, Dowell VR
Low-temperature irradiation of beef and methods of evaluation of radappertization process.
Appl Microbiol. 1975 Nov;30(5):811-20.
An inoculated, irradiated beef pack (1,240 cans) was conducted for the determination of microbiological safety for unrestricted human consumption. Each can contained a mixture of 10(6) spores of each of 10 strains of Clostridium botulinum (5 type A and 5 type B), or a total of 10(7) spores/can. The cans were irradiated to various doses (100 cans/dose) with 60Co gamma rays at -30 +/- 10 C, incubated at 30 +/- 2 C for 6 months, and examined for swelling, toxicity, and recoverable botulinal cells. The minimal experimental sterilizing dose based on nonswollen, nontoxic sterile cans were 2.2 less than experimental sterilizing dose based on nonswollen, nontoxic sterile cans was 2.2 less than experimental sterilizing dose less than or equal to 2.6 Mrad. Using recoverable cells as the most stringent criterion of spoilage, and assuming the conventional simple exponential (without an initial shoulder) rate of spore kill, the "12D" dose was 3.7 Mrad when estimated on the basis of mixture of 10 strains totaling 10(7) spores/can, and 4.3 Mrad if it is assumed that each can of beef contained 10(6) spores of a single most resistant strain and all of these spores were of identical resistances. However, an analysis of the data by extreme value statistics indicated with 90% confidence that the spore death rate was not a simple exponential but might be a shifted exponential (with an initial shoulder), Weibull, lognormal, or normal, with a "12D" equivalent of about 3.0 Mrad regardless of the initial spore density per can. There was an apparent antagonism between the irradiated type A and B strains in the cans. Some of the cans contained type B toxin but did not include type B viable cells. Other cans had a mixture of type A and B toxins, but a large number of these cans did not yield recoverable type B cells. However, type A viable cells could always be demonstrated in those cans containing type A toxin. [Abstract/Link to Full Text]

Songer JR, Braymen DT, Mathis RG
Safe, Convenient Pipetting Station.
Appl Microbiol. 1975 Nov;30(5):887-888.
A simple convenient pipetting station is described that eliminates the need for mouth pipetting. Necessary components, fabrication procedures, and operating techniques are given. [Abstract/Link to Full Text]

Eisler WJ, Lebuis DA
Dual Channel Temperature Recorder.
Appl Microbiol. 1975 Nov;30(5):746-749.
A dual channel temperature recorder is described which can measure temperatures linearly over a range of 0 to 80 C. The sensitivity can be adjusted so that any temperature span, as small as 3 degrees , will cover recorder full scale, and temperatures can be read to 0.1 degrees . The recorder provides two independent, permanent records of temperature variations which can be related directly to the time of day. [Abstract/Link to Full Text]

Ohmiya K, Sato Y
Purification and Properties of Intracellular Proteinase from Streptococcus cremoris.
Appl Microbiol. 1975 Nov;30(5):738-745.
Proteolytic activity in the extract from the cells of Streptococcus cremoris increased in the presence of casein, lactose, glucose, and CaCl(2) in the media but was negligibly detectable in the extract of the cells harvested from the culture containing succinate or citrate. The intracellular proteinase from S. cremoris harvested from tomato medium was purified 150-fold in this experiment. The enzyme had a molecular weight of 140,000, optimum pH at 6.5 to 7.0, and maximum activity at 30 C. The proteinase was activated by Ca and inhibited by Zn, Cu, Hg, Fe, ethylenediaminetetraacetate, and sodium lauryl sulfate. The K(m) value of the enzyme towards each casein fraction was almost the same, and the V(max) of the enzyme towards alpha(s)-casein was smaller than those towards the other casein fractions. [Abstract/Link to Full Text]

Stott WT, Bullerman LB
Influence of carbohydrate and nitrogen source on patulin production by Penicillium patulum.
Appl Microbiol. 1975 Nov;30(5):850-4.
A strain of Penicillium patulum, isolated from cheddar cheese, produced patulin when grown on liquid media containing lactose and milk nitrogen sources. Patulin production was affected by the temperature of incubation, the type and amount of carbohydrate, and the type of nitrogen source present. Patulin levels generally were depressed by incubation at 5 C and low carbohydrate levels. Patulin was produced at low levels in the absence of sugars at 5 C when the mold was grown on milk nitrogen sources. No patulin was detected in cultures grown on 25% casein slurries or cheddar cheese, even though growth of the mold was extensive. [Abstract/Link to Full Text]

Kaneshiro T, Kelson BF, Sloneker JH
Fibrous material in feedlot waste fermented by Trichoderma viride.
Appl Microbiol. 1975 Nov;30(5):876-8.
Trichoderma viride QM9123 fermented fiber isolated from feedlot waste at concentrations up to 16.7% solids. The fermented fiber solids decreased by 32%, and carbohydrate decreased by 60%. Cellulotyic enzyme production was better with fiber substrates that had been alkali pretreated and had a lower hemicellulose-to-cellulose ratio. [Abstract/Link to Full Text]

Hang YD, Splittstoesser DF, Woodams EE
Utilization of brewery spent grain liquor by Aspergillus niger.
Appl Microbiol. 1975 Nov;30(5):879-80.
Aspergillus niger was found capable of rapidly converting about 97% of the sugar from brewery spent grain liquor to fungal mass. The yield of dry mycelium, based on the sugar consumed, was approximately 57%. This fungus produced 1.10% titratable acid calculated as citric acid and reduced the biochemical oxygen demand by 96%. [Abstract/Link to Full Text]

Wyatt RD, Hamilton PB
Interaction between aflatoxicosis and a natural infection of chickens with Salmonella.
Appl Microbiol. 1975 Nov;30(5):870-2.
Broiler chicks with a natural congenital infection of Salmonella worthington required a lower concentration of dietary aflatoxin (0.625 mug/g) to depress growth than uninfected chicks (2.50 mug/g). [Abstract/Link to Full Text]

Ashton DH, Wilson AA, Evancho GM
Identification of a component of crystalline egg albumin bactericidal for thermophilic aerobic sporeformers.
Appl Microbiol. 1975 Nov;30(5):821-4.
During an investigation of the effect of basic and acidic proteins on the growth of thermophilic aerobic sporeformers, crystalline egg albumin was found to be strongly bactericidal. This finding was uncharacteristic of acidic proteins. The bactericidal fraction was heat sensitive and separated from the non-bactericidal albumin fraction during gel filtration on Sephadex G-75. Cells of Micrococcus lysodeikticus and Bacillus stearothermophilus were lysed rapidly by the bactericidal component, leading to its tentative identification as lysozyme. The bactericidal substance possessed an electrophoretic mobility on polyacrylamide gel containing sodium dodecyl sulfate identical to that of crystalline egg white lysozyme. Users of crystalline egg albumin are cautioned that commerical preparations may be contaminated with lysozyme. Destruction of the thermophilic aerobes by lysozyme should be considered when performing counts on egg products. [Abstract/Link to Full Text]

Gerber NN, Stahly DP
Prodiginine (prodigiosin-like) pigments from Streptoverticillium rubrireticuli, an organism that causes pink staining of polyvinyl chloride.
Appl Microbiol. 1975 Nov;30(5):807-10.
Red pigments were extracted from Streptoverticillium rubrireticuli strain 100-19, an organism frequently incriminated in pink staining of polyvinyl chloride. These pigments were identified as undecylprodiginine and butylcycloheptylprodiginine. [Abstract/Link to Full Text]

Siebeling RJ, Neal PM, Granberry WD
Treatment of Salmonella-Arizona-infected turtle eggs with terramycin and chloromycetin by the temperature-differential egg dip method.
Appl Microbiol. 1975 Nov;30(5):791-9.
Attempts to eliminate Salmonella and Arizona infection from newly hatched turtles were made by dipping fresh eggs in cold solutions of Terramycin and Chloromycetin at 1,000, 1,200, 1,500 and 2,000 mug per ml for either 10, 20, or 30 min. Control groups consisted of hatchings produced from nondipped eggs or eggs dipped in chilled water. In two of the four experiments 5 to 10 eggs were blended on days 15, 30, and 45 post antibiotic dip treatment. Twenty-five to 60 hatchlings from each control or experimental dip groups were held in containers and the water was tested (excretion method) for Salmonella and Arizona every 15 or 30 days for 180 to 210 days after hatching. Representative turtles were homogenized (blending method) to determine if systemic infections were present. All specimens tested were enriched in tetrathionate and selenite cystine broth. Nondipped eggs and water-dipped eggs routinely showed Salmonella and Arizona present in egg homogenate and hatchlings emerging from these eggs excreted these pathogens. Terramycin- and Chloromycetin-dipped eggs were uniformly negative for these pathogens, only if fresh eggs were dipped. Bacteriological assay of container water and whole turtle homogenate from hatchlings were negative for Salmonella and Arizona if eggs were dipped in 1,000 mug of Terramycin early in the egg laying season or if eggs were dipped in 1,500 or 2,000 mug of Terramycin per ml late in the egg laying season. The results of temperature-differential egg dip studies suggest that this is a feasible and promising method by which to eradicate Salmonella and Arizona from the turtle. [Abstract/Link to Full Text]

Sanchez S, Quinto CM
D-Glucose isomerase: constitutive and catabolite repression-resistant mutants of Streptomyces phaeochromogenes.
Appl Microbiol. 1975 Nov;30(5):750-4.
As in other Streptomyces species, the enzymatic conversion of D-glucose to D-fructose is carried out in Streptomyces phaeochromogenes NRRL B-3559 by the inducible enzyme, D-xylose keto isomerase (EC 5.3.1.5). Mutants of this microorganism were selected for their ability to grow on D-lyxose (2-epimer of D-xlose). As a result of the mutational event, the microorganism constitutively produced D-xylose isomerase. As in the parent strain, the constitutive formation of the isomerase was repressed by D-glucose. The fact that this mutant was unable to grow in low D-xylose concentrations in the presence of the D-glucose analogue, 3-O-methylglucose, permitted the isolation of D-xylose isomerase constitutive mutants which were insensitive to D-glucose repression. [Abstract/Link to Full Text]

Updegraff DM, Grant WD
Microbial utilization of Pinus radiata bark.
Appl Microbiol. 1975 Nov;30(5):722-6.
A screening program using suspensions of ground bark in mineral salts media, or extracts prepared from ground bark by treating with hot water, sulfuric acid, ammonium hydroxide, or sodium hydroxide, yielded more than 200 pure cultures of fungi, yeasts, and bacteria. Only 38 of these have good growth on liquid bark media. All were filamentous fungi, although many bacteria and yeasts were among the cultures that failed to give appreciable growth. Species of Penicillium, Scopulariopsis, Aspergillus, Trichoderma, Cladosporium, and Fusarium were among the most actively growing cultures. Cell biomass yields, as measured by cell nitrogen determination, were too low for economic production of single cell protein. [Abstract/Link to Full Text]

Hedberg M, Connor DA
Evaluation of coli-count samplers for possible use in standard couting of total and fecal coliforms in recreational waters.
Appl Microbiol. 1975 Nov;30(5):881-3.
Millipore Coli-Count Samplers were used to enumerate colonies of laboratory cultureunts than standard membrane0filter procedures for both total and fecal coliforms. Althought the samplers are useful for semiquantitative analysis as indicated by the manufacturer, they are not suitable examinations of recreational waters. [Abstract/Link to Full Text]

Wilkins TD, Walker CB, Moore WE
Micromethod for identification of anaerobic bacteria: design and operation of apparatus.
Appl Microbiol. 1975 Nov;30(5):831-7.
A replicator is described for transferring 48 bacterial cultures into separate wells of microtiter plates. The device was designed for determination of carbohydrate fermentation patterns of anaerobic bacteria but should be useful for other applications. A simple device for filling microtiter wells with media is also described. [Abstract/Link to Full Text]

Wilkins TD, Walker CB
Development of a micromethod for identification of anaerobic bacteria.
Appl Microbiol. 1975 Nov;30(5):825-30.
A microprocedure was described for determining the carbohydrate fermentation patterns of 48 anaerobic bacteria at one time in microtiter plates. The cultures were transferred into agar-filled wells of microtiter plates with a replicator inside an anaerobic glove box. Fermentation was measured both with a colorimetric indicator and with a small pH electrode. The method was approximately 97% accurate. It would be most useful for laboratories that need to identify large numbers of anaerobes at one time. [Abstract/Link to Full Text]

Faust MA, Aotaky AE, Hargadon MT
Effect of physical parameters on the in situ survival of Escherichia coli MC-6 in an estuarine environment.
Appl Microbiol. 1975 Nov;30(5):800-6.
Survival of Escherichia coli MC-6 of fecal origin in an estuarine environment as affected by time, water temperature, dissolved oxygen, salinity, and montmorillonite in diffusion chambers has been elucidated. Several in situ physical parameters were recorded simultaneously, and viable cell numbers were estimated. The survival of the bacteria varied seasonally. Montmorillonite addition extended the time needed for a 50% reduction of the viable cell population (t1/2) of cells by 40% over the t1/2 of cells in Rhode River water alone. The effect of this clay was not significantly greater between 50- to 1,000 mug/ml montmorillonite concentrations. In all experiments, the relationships among pairs of variables were studied by regression and correlation analysis. The slope between viable cell numbers and water temperatures increased about 50% for each 10 C increment in temperature and gave a correlation coefficient r = 0.617, significant at 95% confidence level. A similar correlation coefficient, r = 0.670, was obtained between water temperature and t1/2 of the initial cell population. In all experiments regressions were performed considering all variables after bacteria had been in the Rhode River environment for 3 days. Coefficient of multiple determination was estimated as R2 = 0.756. Approximately 75.6% of the variance of viable cell numbers can be explained by variation in water temperature, dissolved oxygen, and salinity. Simple correlation coefficients within the regression steps were also computed. Survival of bacteria was closely and negatively correlated with increasing water temperature (r = -0.717). It is suggested that water temperature is the most important factor in predicting fecal coliform survival from point and nonpoint sources in assessing water quality in an estuarine ecosystem. [Abstract/Link to Full Text]

Puleo JR, Favero MS, Oxborrow GS, Herring CM
Method for collecting naturally occurring airborne bacterial spores for determining their thermal resistance.
Appl Microbiol. 1975 Nov;30(5):786-90.
The ability to determine the thermal resistance of naturally occurring airborne bacterial spores associated with spacecraft and their assembly areas has been hindered by lack of an effective collecting system. Efforts to collect and concentrate spores with air samplers or from air filters have not been successful. A fallout method was developed for this purpose and tested. Sterile Teflon ribbons (7.6 by 183 cm) were exposed in pertinent spacecraft assembly areas and subsequently treated with dry heat. Thermal inactivation experiments were conducted at 125 and 113 C. Heating intervals ranged from 1 to 12 h at 125 C and 6, 12, 18, and 24 h at 113 C. Eight hours was the longest heating time yielding survivors at 125 C, whereas survivors were recovered at all of the heating intervals at 113 C. D125C values were calculated using the fractional-replicate-unit-negative technique of Pflug and Schmidt (1968) and ranged from 25 to 126 min. This variation indicated that the most probable number of survivors at each heating interval did not fall on a straight line passing through the initial spore population. However, the most-probable-number values taken alone formed a straight line suggesting logarithmic thermal destruction of a subpopulation of spores with a D125C value of 6.3 h. [Abstract/Link to Full Text]

Huhtanen CN, Wasserman AE
Effect of added iron on the formation of clostridial inhibitors.
Appl Microbiol. 1975 Nov;30(5):768-70.
Inhibition of Clostridium botulinum by nitrite was potentiated by the addition of Fe(II) or Fe(III) to the culture medium. The effect of iron was more pronounced when nitrite was added after autoclaving. [Abstract/Link to Full Text]

Thomason BM, Biddle JW, Cherry WB
Dection of salmonellae in the environment.
Appl Microbiol. 1975 Nov;30(5):764-7.
The incidence of salmonellae in contrasting environments was compared in this study. Samples collected from or near surface waters in a lush hardwood forest yielded four salmonellae serotypes from six culturally positive samples. A total of 76 samples collected from the top of a granite outcropping over a 3-month period yielded 10 positive samples. Only two salmonellae serotypes were isolated, and one of these was isolated only once. The nature of the sample material had no significant effect on the detection of salmonellae from the two sampling sites. However, the presence or absence of visible moisture in the sample significantly affected the recovery of salmonellae. The results showed that even a harsh environment such as that found on top of Stone Moutain may serve as an ecological niche for the survival and transmission of salmonellae. [Abstract/Link to Full Text]

Brodsky MH, Schiemann DA
Influence of coliform source on evaluation of membrane filters.
Appl Microbiol. 1975 Nov;30(5):727-30.
Four brands of membrane filters were examined for total and fecal coliform recovery performance by two experimental approaches. Using diluted EC broth cultures of water samples, Johns-Manville filters were superior to Sartorius filters for fecal coliform but equivalent for total coliform recovery. Using river water samples, Johns-Manville filters were superior to Sartorius filters for total coliform but equivalent for fecal coliform recovery. No differences were observed between Johns-Manville and Millipore or Millipore and Sartorius filters for total or fecal coliform recoveries using either approach, nor was any difference observed between Millipore and Gelman filters for fecal coliform recovery from river water samples. These results indicate that the source of the coliform bacteria has an important influence on the conclusions of membrane filter evaluation studies. [Abstract/Link to Full Text]

Hansen JN, Levin RA
Effect of some inhibitors derived from nitrite on marcomolecular synthesis in Bacillus cereus.
Appl Microbiol. 1975 Nov;30(5):862-9.
The effect of several inhibitors derived from nitrite on incorporation of [14C]uracil into ribonucleic acid of Bacillus cereus during outgrowth and vegetative growth has been determined. A heat-induced inhibitor presumably of the Perigo type was compared with the nitrosothiols of thioglycolate and beta-mercaptoethanol. All were found to effectively inhibit uracil incorporation at all stages tested. Phase-contrast microscopy revealed that inhibition of morphological events occurred either before germination or during early outgrowth, depending on inhibitor concentration. It was also found that a precursor to the heat-induced inhibitor could be isolated from dialyzable tryptone (a pancreatic digest of casein), which chromatographed as a single species on diethylaminoethyl-cellulose and Sephadex G-25, with an apparent molecular weight of about 2,000. [Abstract/Link to Full Text]

Carney JF, Carty CE, Colwell RR
Seasonal occurrence and distribution of microbial indicators and pathogens in the Rhode River of Chesapeake Bay.
Appl Microbiol. 1975 Nov;30(5):771-80.
The seasonal incidence and occurrence of indicator organisms and pathogens were studied at four sites in the Rhode River, a subestuary of Chesapeake Bay. The highest frequency of occurrence of total and fecal coliforms and fecal streptococci was in Muddy Creek, a marsh area receiving pasture land runoff. Second highest frequency of occurrence of these bacteria was in Cadle Creek, a populated area. Lowest measurements of these parameters were obtained at stations in the central portion of the Rhode River. No Salmonella spp. were detected by the methods employed in this study. However, it is concluded that if these organisms are present, the concentrations are less than or equal to 1 organism per liter. The presence of Clostridium botulinum was detected in 12% of the samples tested. [Abstract/Link to Full Text]

Lundin A, Thore A
Comparison of methods for extraction of bacterial adenine nucleotides determined by firefly assay.
Appl Microbiol. 1975 Nov;30(5):713-21.
Adenine nucleotides in Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa were extracted using 10 different methods. Extracts were assayed for adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) by the firefly method using an improved procedure. Analytical interference by bacterial enzymes not inactivated during the extraction was found to be a major problem. However, these enzymes were inactivated to a considerable extent by the inclusion of ethylenediaminetetraacetate in the extraction reagent. The 10 extraction methods were compared with respect to yield of adenine nucleotides, interference with the enzymic assay, reproducibility of the method, and stability of the extracts. Results indicated that extraction with trichloroacetic acid was the method most closely reflecting actual levels of ATP, ADP and AMP in intact bacterial cells. However, for the extraction of ATP in some bacterial strains several other methods may be used and may be advantageous from a practical point of view. [Abstract/Link to Full Text]

Barach JT, Flowers RS, Adams DM
Repair of heat-injured Clostridium perfringens spores during outgrowth.
Appl Microbiol. 1975 Nov;30(5):873-5.
Clostridium perfringens strain NCTC 8798 spores were injured by ultrahigh temperature treatment and were unable to outgrow in the presence of antibiotics used in selective enumeration media. Injured spores underwent repair in a nonselective laboratory medium and in foods. [Abstract/Link to Full Text]

Moran DM, Tannenbaum SR, Archer MC
Inhibitor of Clostridium perfringens formed by heating sodium nitrite in a chemically defined medium.
Appl Microbiol. 1975 Nov;30(5):838-43.
An inhibitor of Clostridium perfringens formed when low levels of nitrite were autoclaved with a defined chemical medium. A systematic study of the medium revealed that only amino acids and mineral salts were involved in the production of this inhibitor, which was proven to be a toxic compound formed from cysteine, ferrous sulfate, and sodium nitrite. The inhibitor was compared to several known compounds. S-nitrosocysteine inhibited the test organism, but would not form in the test system in amounts large enough to explain the observed inhibition. Roussin red salt was unstable in the test system and therefore was not the inhibitor. Roussin black salt, which was also inhibitory, could form in sufficient amounts to explain the inhibition. A complex of cysteine, iron, and nitric oxide was detected in the autoclaved solution of cysteine, ferrous sulfate, and sodium nitrite; this cysteine complex did not appear to be inhibitory, however, at levels which could form in the autoclaved medium. The observed inhibition may have been due to the combined effects of sublethal concentrations of each compound. [Abstract/Link to Full Text]

Walden WC, Hentges DJ
Differential effects of oxygen and oxidation-reduction potential on the multiplication of three species of anaerobic intestinal bacteria.
Appl Microbiol. 1975 Nov;30(5):781-5.
The sensitivity of three strains of anaerobic intestinal bacteria, Clostridium perfringens, Bacteroides fragilis, and Peptococcus magnus, to the differential effects of oxygen and adverse oxidation-reduction potential was measured. The multiplication of the three organisms was inhibited in the presence of oxygen whether the medium was at a negative oxidation-reduction potential (Eh of -50 mV), poised by the intermittent addition of dithiothreitol, or at a positive oxidation-reduction potential (Eh of near +500 mV). However, when these organisms were cultured in the presence of oxygen, no inhibition was observed, even when the oxidation-reduction potential was maintained at an average Eh of +325 mV by the addition of potassium ferricyanide. When the cultures were aerated, the growth patterns of the three organisms demonstrated different sensitivities to oxygen. P. magnus was found to be the most sensitive. After 2 h of aerobic incubation, no viable organisms could be detected. B. fragilis was intermediately sensitive to oxygen with no viable organisms detected after 5 h of aerobic incubation. C. perfringens was the least sensitive. Under conditions of aerobic incubation, viable organisms survived for 10 h. During the experiments with Clostridium, no spores were observed by spore staining. [Abstract/Link to Full Text]

Arthur LO, Nakamura LK, Julian G, Bulla LA
Carbohydrate catabolism of selected strains in the genus Agrobacterium.
Appl Microbiol. 1975 Nov;30(5):731-7.
Radiorespirometric and enzyme analyses were used to reveal the glucose-catabolizing mechanisms functioning in single strains of seven presumed Agrobacterium species. The Entner-Doudoroff and pentose cycle pathways functioned in A. radiobacter, A. tumefaciens, A. rubi, and A. rhizogenes. Whereas both catabolic pathways were utilized to an almost equal degree in the A. radiobacter and A. tumefaciens strains, use of the Entner-Doudoroff pathway predominated in the A. rubi and A. rhizogenes strains. A stellulatum catabolized glucose almost solely through the Entner-Doudoroff pathway. In A. pseudotsugae and A. gypsophilae, glucose was metabolized mainly through the Emden-Meyerhof-Parnas pathway; the pentose phosphate pathway was also utilized. [Abstract/Link to Full Text]

Myers PS, Millar WN
Nonautotrophic Thiobacillus in acid mine water.
Appl Microbiol. 1975 Nov;30(5):884-6.
Nonautotrophic thiobacilli were isolated from the acidic water of a coal mine. Based on their mixotrophic physiology, the isolates are regarded as strains of Thiobacillus perometabolis. [Abstract/Link to Full Text]

Simonson LG, Liberta AE, Richardson A
Characterization of an extracellular dextranase from Fusarium moniliforme.
Appl Microbiol. 1975 Nov;30(5):855-61.
An extracellular dextranase (EC 3.2.1.11) was purified approximately 75-fold from cell-free culture filtrates of Fusarium moniliforme. The purified dextranase was of the endo type, and isomaltose was identified as the primary end product of dextran hydrolysis. The molecular weight of the dextranase was determined to be 39,000 by gel permeation chromatography. The enzyme was most active at pH 5.5, and the temperature optimum was near 55 C. Activity was not inhibited by either ethylenediaminetetraacetic acid or iodoacetate. The Km for dextran with an average molecular weight of 10,000 was estimated to be 1.1 X 10(-4) M. The electrophoretic mobility of the dextranase was distinctly different from that of a Penicillium-derived commercial dextranase. The F. moniliforme dextranase was also found to differ from the commercial preparation by its greater relative activity against glucans isolated from Streptococcus mutans. [Abstract/Link to Full Text]


Recent Articles in Bacteriological Reviews

Piggot PJ, Coote JG
Genetic aspects of bacterial endospore formation.
Bacteriol Rev. 1976 Dec;40(4):908-62. [Abstract/Link to Full Text]

Patterson MJ, El Batool Hafeez A
Group B streptococci in human disease.
Bacteriol Rev. 1976 Sep;40(3):774-92. [Abstract/Link to Full Text]

Wickner RB
Killer of Saccharomyces cerevisiae: a double-stranded ribonucleic acid plasmid.
Bacteriol Rev. 1976 Sep;40(3):757-73. [Abstract/Link to Full Text]

Tagg JR, Dajani AS, Wannamaker LW
Bacteriocins of gram-positive bacteria.
Bacteriol Rev. 1976 Sep;40(3):722-56. [Abstract/Link to Full Text]

Fitzgerald JW
Sulfate ester formation and hydrolysis: a potentially important yet often ignored aspect of the sulfur cycle of aerobic soils.
Bacteriol Rev. 1976 Sep;40(3):698-721. [Abstract/Link to Full Text]

Omura S
The antibiotic cerulenin, a novel tool for biochemistry as an inhibitor of fatty acid synthesis.
Bacteriol Rev. 1976 Sep;40(3):681-97. [Abstract/Link to Full Text]

Knowles CJ
Microorganisms and cyanide.
Bacteriol Rev. 1976 Sep;40(3):652-80. [Abstract/Link to Full Text]

Reanney D
Extrachromosomal elements as possible agents of adaptation and development.
Bacteriol Rev. 1976 Sep;40(3):552-90. [Abstract/Link to Full Text]

Pastan I, Adhya S
Cyclic adenosine 5'-monophosphate in Escherichia coli.
Bacteriol Rev. 1976 Sep;40(3):527-51. [Abstract/Link to Full Text]

Mäkelä PH, Mayer H
Enterobacterial common antigen.
Bacteriol Rev. 1976 Sep;40(3):591-632. [Abstract/Link to Full Text]

Bibel DJ, Chen TH
Diagnosis of plaque: an analysis of the Yersin-Kitasato controversy.
Bacteriol Rev. 1976 Sep;40(3):633-51. [Abstract/Link to Full Text]

Kalakoutskii LV, Agre NS
Comparative aspects of development and differentiation in actinomycetes.
Bacteriol Rev. 1976 Jun;40(2):469-524. [Abstract/Link to Full Text]

Vogels GD, Van der Drift C
Degradation of purines and pyrimidines by microorganisms.
Bacteriol Rev. 1976 Jun;40(2):403-68. [Abstract/Link to Full Text]

Aronson AI, Fitz-James P
Structure and morphogenesis of the bacterial spore coat.
Bacteriol Rev. 1976 Jun;40(2):360-402. [Abstract/Link to Full Text]

Cummings DJ, Bolin RW
Head length control in T4 bacteriophage morphogenesis: effect of canavanine on assembly.
Bacteriol Rev. 1976 Jun;40(2):314-59. [Abstract/Link to Full Text]

Campbell PA
Immunocompetent cells in resistance to bacterial infections.
Bacteriol Rev. 1976 Jun;40(2):284-313. [Abstract/Link to Full Text]

Penn M, Dworkin M
Robert Koch and two visions of microbiology.
Bacteriol Rev. 1976 Jun;40(2):276-83. [Abstract/Link to Full Text]

Doetsch RN
Lazzaro Spallanzani's Opuscoli of 1776.
Bacteriol Rev. 1976 Jun;40(2):270-5. [Abstract/Link to Full Text]

Porter JR
Antony van Leeuwenhoek: tercentenary of his discovery of bacteria.
Bacteriol Rev. 1976 Jun;40(2):260-9. [Abstract/Link to Full Text]

Doetsch RN, Murray RG
Editorial: Microbiological centennials: years to remember in 1976.
Bacteriol Rev. 1976 Jun;40(2):259. [Abstract/Link to Full Text]

Novick RP, Clowes RC, Cohen SN, Curtiss R, Datta N, Falkow S
Uniform nomenclature for bacterial plasmids: a proposal.
Bacteriol Rev. 1976 Mar;40(1):168-89. [Abstract/Link to Full Text]

Krieg NR
Biology of the chemoheterotrophic spirilla.
Bacteriol Rev. 1976 Mar;40(1):55-115. [Abstract/Link to Full Text]

Massey LK, Sokatch JR, Conrad RS
Branched-chain amino acid catabolism in bacteria.
Bacteriol Rev. 1976 Mar;40(1):42-54. [Abstract/Link to Full Text]

Lechevalier H
Louis Joblot and his microscopes.
Bacteriol Rev. 1976 Mar;40(1):241-58. [Abstract/Link to Full Text]

Heywood P, Magee PT
Meiosis in protists. Some structural and physiological aspects of meiosis in algae, fungi, and protozoa.
Bacteriol Rev. 1976 Mar;40(1):190-240. [Abstract/Link to Full Text]

Bachmann BJ, Low KB, Taylor AL
Recalibrated linkage map of Escherichia coli K-12.
Bacteriol Rev. 1976 Mar;40(1):116-67. [Abstract/Link to Full Text]

Schmit JC, Brody S
Biochemical genetics of Neurospora crassa conidial germination.
Bacteriol Rev. 1976 Mar;40(1):1-41. [Abstract/Link to Full Text]


Bacteriophages of Bacillus subtilis.
Bacteriol Rev. 1975 Dec;39(4):540.
[This corrects the article on p. 294 in vol. 39.][This corrects the article on p. 295 in vol. 39.][This corrects the article on p. 298 in vol. 39.]. [Abstract/Link to Full Text]

Sadoff HL
Encystment and germination in Azotobacter vinelandii.
Bacteriol Rev. 1975 Dec;39(4):516-39. [Abstract/Link to Full Text]

Hardy KG
Colicinogeny and related phenomena.
Bacteriol Rev. 1975 Dec;39(4):464-515. [Abstract/Link to Full Text]


Recent Articles in BMC Immunology

Sharif O, Bolshakov VN, Raines S, Newham P, Perkins ND
Transcriptional profiling of the LPS induced NF-kappaB response in macrophages.
BMC Immunol. 2007;81.
BACKGROUND: Exposure of macrophages to bacterial products such as lipopolysaccharide (LPS) results in activation of the NF-kappaB transcription factor, which orchestrates a gene expression programme that underpins the macrophage-dependent immune response. These changes include the induction or repression of a wide range of genes that regulate inflammation, cell proliferation, migration and cell survival. This process is tightly regulated and loss of control is associated with conditions such as septic shock, inflammatory diseases and cancer. To study this response, it is important to have in vitro model systems that reflect the behaviour of cells in vivo. In addition, it is necessary to understand the natural differences that can occur between individuals. In this report, we have investigated and compared the LPS response in macrophage derived cell lines and peripheral blood mononuclear cell (PBMC) derived macrophages. RESULTS: Gene expression profiles were determined following LPS treatment of THP-1 cells for 1 and 4 hours. LPS significantly induced or repressed 72 out of 465 genes selected as being known or putative NF-kappaB target genes, which exhibited 4 temporal patterns of expression. Results for 34 of these genes, including several genes not previously identified as LPS target genes, were validated using real time PCR. A high correlation between microarray and real time PCR data was found. Significantly, the LPS induced expression profile of THP-1 cells, as determined using real time PCR, was found to be very similar to that of human PBMC derived macrophages. Interestingly, some differences were observed in the LPS response between the two donor PBMC macrophage populations. Surprisingly, we found that the LPS response in U937 cells was dramatically different to both THP-1 and PBMC derived macrophages. CONCLUSION: This study revealed a dynamic and diverse transcriptional response to LPS in macrophages, involving both the induction and repression of gene expression in a time dependent manner. Moreover, we demonstrated that the LPS induced transcriptional response in the THP-1 cell line is very similar to primary PBMC derived macrophages. Therefore, THP-1 cells represent a good model system for studying the mechanisms of LPS and NF-kappaB dependent gene expression. [Abstract/Link to Full Text]

Shreffler WG, Visness CM, Burger M, Cruikshank WW, Lederman HM, de la Morena M, Grindle K, Calatroni A, Sampson HA, Gern JE
Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study.
BMC Immunol. 2006;729.
BACKGROUND: Cryopreservation of peripheral blood mononuclear cells has been used to preserve and standardize immunologic measurements for multicenter studies, however, effects of cryopreservation on cytokine responses are incompletely understood. In designing immunologic studies for a new multicenter birth cohort study of childhood asthma, we performed a series of experiments to determine the effects of two different methods of cryopreservation on the cytokine responses of cord and peripheral blood mononuclear cells. RESULTS: Paired samples of PBMC were processed freshly, or after cryopreservation in a Nalgene container (NC) or a controlled-rate freezer (CRF). Although there were some differences between the methods, cryopreservation inhibited PHA-induced IL-10 secretion and Der f 1-induced IL-2 secretion, and augmented PHA-induced IL-2 secretion and spontaneous secretion of TNF-alpha. In separate experiments, NC cryopreservation inhibited secretion of several cytokines (IL-13, IL-10, IFN-gamma, TNF-alpha) by PHA-stimulated cord blood mononuclear cells. With the exception of PHA-induced IL-13, results from fresh and cryopreserved cord blood samples were not significantly correlated. Finally, in reproducibility studies involving processing of identical cell samples in up to 4 separate laboratories, variances in cytokine responses of fresh cells stimulated at separate sites did not exceed those in cryopreserved cells stimulated at a central site. CONCLUSION: Collectively, these studies indicate that cryopreservation can affect mononuclear cell cytokine response profiles, and that IL-10 secretion and antigen-induced responses may be especially vulnerable. These studies also demonstrate that mononuclear cell responses can be standardized for performance in a small number of laboratories for multicenter studies, and underscore the importance of measuring reproducibility and of testing whether cryopreservation techniques alter specific immunologic outcomes. [Abstract/Link to Full Text]

Johnson JL, Ellis BA, Munafo DB, Brzezinska AA, Catz SD
Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes.
BMC Immunol. 2006;728.
BACKGROUND: Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection. RESULTS: Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX) domain of p47phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47phox, is translocated to the membrane of mature phagosomes. CONCLUSION: We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils. [Abstract/Link to Full Text]

Dawson HD, Collins G, Pyle R, Key M, Weeraratna A, Deep-Dixit V, Nadal CN, Taub DD
Direct and indirect effects of retinoic acid on human Th2 cytokine and chemokine expression by human T lymphocytes.
BMC Immunol. 2006;727.
BACKGROUND: Vitamin A (VA) deficiency induces a type 1 cytokine response and exogenously provided retinoids can induce a type 2 cytokine response both in vitro and in vivo. The precise mechanism(s) involved in this phenotypic switch are inconsistent and have been poorly characterized in humans. In an effort to determine if retinoids are capable of inducing Th2 cytokine responses in human T cell cultures, we stimulated human PBMCs with immobilized anti-CD3 mAb in the presence or absence of all-trans retinoic acid (ATRA) or 9-cis-RA. RESULTS: Stimulation of human PBMCs and purified T cells with ATRA and 9-cis-RA increased mRNA and protein levels of IL-4, IL-5, and IL-13 and decreased levels of IFN-gamma, IL-2, IL-12p70 and TNF-alpha upon activation with anti-CD3 and/or anti-CD28 mAbs. These effects were dose-dependent and evident as early as 12 hr post stimulation. Real time RT-PCR analysis revealed a dampened expression of the Th1-associated gene, T-bet, and a time-dependent increase in the mRNA for the Th2-associated genes, GATA-3, c-MAF and STAT6, upon treatment with ATRA. Besides Th1 and Th2 cytokines, a number of additional proinflammatory and regulatory cytokines including several chemokines were also differentially regulated by ATRA treatment. CONCLUSION: These data provide strong evidence for multiple inductive roles for retinoids in the development of human type-2 cytokine responses. [Abstract/Link to Full Text]

Khanna AK, Mehra MR
Targeted in vitro and in vivo gene transfer into T lymphocytes: potential of direct inhibition of allo-immune activation.
BMC Immunol. 2006;726.
BACKGROUND: Successful inhibition of alloimmune activation in organ transplantation remains one of the key events in achieving a long-term graft survival. Since T lymphocytes are largely responsible for alloimmune activation, targeted gene transfer of gene of cyclin kinase inhibitor p21 into T cells might inhibit their aberrant proliferation. A number of strategies using either adenoviral or lentiviral vectors linked to mono or bispecific antibodies directed against T cell surface markers/cytokines did not yield the desired results. Therefore, this study was designed to test if a CD3promoter-p21 chimeric construct would in vitro and in vivo transfer p21 gene to T lymphocytes and result in inhibition of proliferation. CD3 promoter-p21 chimeric constructs were prepared with p21 in the sense and antisense orientation. For in vitro studies EL4-IL-2 thyoma cells were used and for in vivo studies CD3p21 sense and antisense plasmid DNA was injected intramuscularly in mice. Lymphocyte proliferation was quantified by 3H-thymidine uptake assay; IL-2 mRNA expression was studied by RT-PCR and using Real Time PCR assay, we monitored the CD3, p21, TNF-alpha and IFN-gamma mRNA expression. RESULTS: Transfection of CD3p21 sense and antisense in mouse thyoma cell line (EL4-IL-2) resulted in modulation of mitogen-induced proliferation. The intramuscular injection of CD3p21 sense and antisense plasmid DNA into mice also modulated lymphocyte proliferation and mRNA expression of pro-inflammatory cytokines. CONCLUSION: These results demonstrate a novel strategy of in vitro and in vivo transfer of p21 gene to T cells using CD3-promoter to achieve targeted inhibition of lymphocyte proliferation and immune activation. [Abstract/Link to Full Text]

Azizi A, Anderson DE, Ghorbani M, Gee K, Diaz-Mitoma F
Immunogenicity of a polyvalent HIV-1 candidate vaccine based on fourteen wild type gp120 proteins in golden hamsters.
BMC Immunol. 2006;725.
BACKGROUND: One of the major obstacles in the design of an effective vaccine against HIV-1 is the hypervariability of the HIV-1 envelope glycoprotein. Most HIV-1 vaccine candidates have utilized envelope glycoprotein from a single virus isolate, but to date, none of them elicited broadly reactive humoral immunity. Herein, we hypothesised that a cocktail of HIV-1 gp120 proteins containing multiple epitopes may increase the breadth of immune responses against HIV-1. We compared and evaluated the immunogenicity of HIV-1 vaccines containing either gp120 protein alone or in combinations of four or fourteen gp120s from different primary HIV-1 isolates in immunized hamsters. RESULTS: We amplified and characterized 14 different gp120s from primary subtype B isolates with both syncytium and non-syncytium inducing properties, and expressed the proteins in Chinese Hamster Ovary (CHO) cell lines. Purified proteins were used either alone or in combinations of four or fourteen different gp120s to vaccinate golden hamsters. The polyvalent vaccine showed higher antibody titers to HIV-1 subtype B isolates MN and SF162 compared to the groups that received one or four gp120 proteins. However, the polyvalent vaccine was not able to show higher neutralizing antibody responses against HIV-1 primary isolates. Interestingly, the polyvalent vaccine group had the highest proliferative immune responses and showed a substantial proportion of cross-subtype CD4 reactivity to HIV-1 subtypes B, C, and A/E CONCLUSION: Although the polyvalent approach achieved only a modest increase in the breadth of humoral and cellular immunity, the qualitative change in the vaccine (14 vs. 1 gp120) resulted in a quantitative improvement in vaccine-induced immunity. [Abstract/Link to Full Text]

Nielsen UB, Kirpotin DB, Pickering EM, Drummond DC, Marks JD
A novel assay for monitoring internalization of nanocarrier coupled antibodies.
BMC Immunol. 2006;724.
BACKGROUND: Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA. RESULTS: The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay. CONCLUSION: The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers. [Abstract/Link to Full Text]

van Lieshout AW, van der Voort R, le Blanc LM, Roelofs MF, Schreurs BW, van Riel PL, Adema GJ, Radstake TR
Novel insights in the regulation of CCL18 secretion by monocytes and dendritic cells via cytokines, toll-like receptors and rheumatoid synovial fluid.
BMC Immunol. 2006;723.
BACKGROUND: The T cell attracting chemokine CCL18 is produced by antigen presenting cells and a role for CCL18 has been suggested in the pathogenesis of a variety of diseases. Rheumatoid arthritis (RA) is one of these conditions, in which abundant CCL18 production is present. Although Th2 cytokines and IL-10 are known to have an effect on CCL18 production, there are several gaps in our knowledge regarding the exact regulation of CCL18 secretion, both in general and in RA. In this study we provide new insights in the regulation of CCL18 secretion by monocytes and dendritic cells. RESULTS: In contrast to a large panel of pro-inflammatory stimuli (IL-1beta, TNF-alpha, IL-10, IL-13, IL-15, IL-17, IL-18, IFN-gamma), T cell mimicking molecules (RANKL, CD40L) or TLR driven maturation, the anti-inflammatory IL-10 strongly stimulated DC to secrete CCL18. On freshly isolated monocytes, CCL18 secretion was induced by IL-4 and IL-13, in strong synergy with IL-10. This synergistic effect could already be observed after only 24 hours, indicating that not only macrophages and dendritic cells, but also monocytes secrete CCL18 under these stimulatory conditions. A high CCL18 expression was detected in RA synovial tissue and incubation of monocytes with synovial fluid from RA patients clearly enhanced the effects of IL-4, IL-13 and IL-10. Surprisingly, the effect of synovial fluid was not driven by IL-10 of IL-13, suggesting the presence of another CCL18 inducing factor in synovial fluid. CONCLUSION: In summary, IL-10 synergistically induces CCL18 secretion in combination with IL-4 of IL-13 on monocytes and monocyte derived cells. The effects of IL-14, IL-13 and IL-10 are strongly enhanced by synovial fluid. This synergy may contribute to the high CCL18 expression in RA. [Abstract/Link to Full Text]

Lee DH, Park KS, Kong ID, Kim JW, Han BG
Expression of P2 receptors in human B cells and Epstein-Barr virus-transformed lymphoblastoid cell lines.
BMC Immunol. 2006;722.
BACKGROUND: Epstein-Barr virus (EBV) infection immortalizes primary B cells in vitro and generates lymphoblastoid cell lines (LCLs), which are used for several purposes in immunological and genetic studies. Purinergic receptors, consisting of P2X and P2Y, are activated by extracellular nucleotides in most tissues and exert various physiological effects. In B cells, especially EBV-induced LCLs, their expression and function have not been well studied. We investigated the expression of P2 receptors on primary human B cells and LCLs using the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for revealing the gene expression profile of the P2 receptor subtypes and their changes during transformation. RESULTS: The mRNA transcripts of most P2 receptors were detected in primary B cells; the expression of P2X3 and P2X7 receptors was the lowest of all the P2 receptors. By contrast, LCLs expressed several dominant P2 receptors--P2X4, P2X5, and P2Y11--in amounts similar to those seen in B cells infected with EBV for 2 weeks. The amount of most P2 subtypes in LCLs or EBV-infected B cells was lower than in normal B cells. However, the amount of P2X7 receptor expressed in LCLs was higher. Protein expression was studied using Western blotting to confirm the mRNA findings for P2X1, P2X4, P2X7, P2Y1, and P2Y11 receptors. ATP increased the intracellular free Ca2+ concentration ([Ca2+]i) by enhancing the Ca2+ influx in both B cells and LCLs in a dose-dependent manner. CONCLUSION: These findings describe P2 receptor expression profiles and the effects of purinergic stimuli on B cells and suggest some plasticity in the expression of the P2 receptor phenotype. This may help explain the nature and effect of P2 receptors on B cells and their role in altering the characteristics of LCLs. [Abstract/Link to Full Text]

Brichacek B, Vanpouille C, Trachtenberg AJ, Pushkarsky T, Dubrovsky L, Martin G, Simon G, Bukrinsky M
Long-term changes of serum chemokine levels in vaccinated military personnel.
BMC Immunol. 2006;721.
BACKGROUND: Members of the United States Armed Forces receive a series of vaccinations during their course of service. To investigate the influence of multiple vaccinations on innate immunity, we measured concentrations of a panel of immunomodulatory and pro-inflammatory cytokines in serum samples from a group of such individuals. RESULTS: Significantly increased levels of macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta and interleukin 8 (IL-8) were detected. Since these cytokines are known to have anti-human immunodeficiency virus (HIV) activity, we tested the effect of serum from these individuals on HIV-1 infectivity and susceptibility of their peripheral blood mononuclear cells (PBMCs) to HIV-1 infection in vitro. Sera from vaccinated military personnel inhibited, and their PBMCs were partially resistant to, infection by HIV-1 strains tropic to CCR5 (R5), but not to CXCR4 (X4), chemokine receptor. CONCLUSION: These findings demonstrate that increased anti-HIV chemokines can be detected in vaccine recipients up to 68 weeks following immunization. [Abstract/Link to Full Text]

Tsiakalou V, Tsangaridou E, Polioudaki H, Nifli AP, Koulentaki M, Akoumianaki T, Kouroumalis E, Castanas E, Theodoropoulos PA
Optimized detection of circulating anti-nuclear envelope autoantibodies by immunofluorescence.
BMC Immunol. 2006;720.
BACKGROUND: Antinuclear antibodies are useful diagnostic tools in several autoimmune diseases. However, the routine detection of nuclear envelope autoantibodies using immunofluorescence (IF) is not always easy to perform in patients' sera because of the presence of autoantibodies to other nuclear and cytoplasmic components which could mask the characteristic rim-like pattern of nuclear envelope autoantibodies. This is particularly common in sera from patients with primary biliary cirrhosis (PBC), which generaly have high titres of anti-mitochondrial antibodies. Therefore, we have assayed a number of commercial slides and alternative fixation conditions to optimize the detection of anti-nuclear envelope antibodies (ANEA) in PBC sera. METHODS: We have explored the presence of ANEA in 33 sera from patients with established PBC using three different Hep2 commercial slides and home-made slides with HeLa and Hep2 cells fixed with methanol, ethanol, 1% or 4% formaldehyde. RESULTS: We observed that the IF pattern was related to the cell type used (Hep2 or HeLa), the manufacturer and the cell fixation scheme. When both cell lines were fixed with 1% formaldehyde, the intensity of the cytoplasmic staining was considerably decreased regardless to the serum sample, whereas the prevalence of cytoplasmic autoantibodies was significantly lowered, as compared to any of the Hep2 commercial slide and fixation used. In addition, the prevalence of ANEA was importantly increased in formaldehyde-fixed cells. CONCLUSION: Immunofluorescence using appropriately fixed cells represent an easy, no time-consuming and low cost technique for the routine screening of sera for ANEA. Detection of ANEA is shown to be more efficient using formaldehyde-fixed cells instead of commercially available Hep2 cells. [Abstract/Link to Full Text]

Koo GC, Gan YH
The innate interferon gamma response of BALB/c and C57BL/6 mice to in vitro Burkholderia pseudomallei infection.
BMC Immunol. 2006;719.
BACKGROUND: Burkholderia pseudomallei is the causative agent for melioidosis. For many bacterial infections, cytokine dysregulation is one of the contributing factors to the severe clinical outcomes in the susceptible hosts. The C57BL/6 and BALB/c mice have been established as a differential model of susceptibility in murine melioidosis. In this study, we compared the innate IFN-gamma response to B. pseudomallei between the C57BL/6 and BALB/c splenocytes and characterized the hyperproduction of IFN-gamma in the relatively susceptible BALB/c mice in vitro. RESULTS: Naďve BALB/c splenocytes were found to produce more IFN-gamma in response to live bacterial infection compared to C57BL/6 splenocytes. Natural killer cells were found to be the major producers of IFN-gamma, while T cells and Gr-1intermediate cells also contributed to the IFN-gamma response. Although anti-Gr-1 depletion substantially reduced the IFN-gamma response, this was not due to the contribution of Gr-1high, Ly-6G expressing neutrophils. We found no differences in the cell types making IFN-gamma between BALB/c and C57BL/6 splenocytes. Although IL-12 is essential for the IFN-gamma response, BALB/c and C57BL/6 splenocytes made similar amounts of IL-12 after infection. However, BALB/c splenocytes produced higher proinflammatory cytokines such as IL-1beta, TNF-alpha, IL-6, IL-18 than C57BL/6 splenocytes after infection with B. pseudomallei. CONCLUSION: Higher percentages of Gr-1 expressing NK and T cells, poorer ability in controlling bacteria growth, and higher IL-18 could be the factors contributing to IFN-gamma hyperproduction in BALB/c mice. [Abstract/Link to Full Text]

Oko L, Aduddell-Swope B, Willis D, Hamor R, Coons TA, Hjelle B, Schountz T
Profiling helper T cell subset gene expression in deer mice.
BMC Immunol. 2006;718.
BACKGROUND: Deer mice (Peromyscus maniculatus) are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV), the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS) in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. RESULTS: We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNgamma, TNF, LT), Th2 cells (GATA-3, STAT6, IL-4, IL-5) and regulatory T cells (Fox-p3, IL-10, TGFbeta1). These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. CONCLUSION: We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice. [Abstract/Link to Full Text]

Singh V, Agrewala JN
Regulatory role of pro-Th1 and pro-Th2 cytokines in modulating the activity of Th1 and Th2 cells when B cell and macrophages are used as antigen presenting cells.
BMC Immunol. 2006;717.
BACKGROUND: Presence of antigen presenting cells, expression of costimulatory molecules, the strength of first signal and cytokine milieu are quite important in influencing the reactivation of differentiated Th1 and Th2 cells. RESULTS: In the present study, we have analyzed the concerted action of pro-Th1 and pro-Th2 cytokines in the presence of B cells, peritoneal and splenic macrophages as antigen presenting cells and varied concentration of first (anti-CD3 Ab) and second (B7-1 transfectant) signals on the proliferation and cytokine secretion by Th1 and Th2 cells. Interesting observations were made that IFN-gamma significantly augmented the secretion of IL-4 by Th2 cells when either B cells or splenic or peritoneal macrophages were used as APC. Further, IFN-gamma significantly inhibited the proliferation of Th1 cells only in the presence of peritoneal macrophages. We have also observed that B cells could significantly respond to cytokines to further enhance the proliferation and cytokine release by Th1 and Th2 cells. But not much effect on addition of exogenous cytokines IL-1, IL-4, IL-5, IL-12 was observed on the proliferation of Th1 and Th2 cells in the presence of macrophages. In contrast, both IFN-gamma and IL-2 significantly enhanced the production of IL-4 and IL-5 respectively, by Th2 cells in presence of B cells, splenic and peritoneal macrophages. Another important observation was that the addition of B7-1 transfectants in the cultures, which were stimulated with low dose of anti-CD3 Ab significantly, enhanced the proliferation and cytokine secretion. CONCLUSION: This study indicates involvement of different type of APCs, cytokine milieu, dose of first and second signals in a concerted manner in the outcome of the immune response. The significance of this study is that the immunization with antigen along with costimulatory molecules may significantly reduce the dose of antigen and can generate better immune response than antigen alone. [Abstract/Link to Full Text]

Biggerstaff J, Weidow B, Amirkhosravi A, Francis JL
Enumeration of leukocyte infiltration in solid tumors by confocal laser scanning microscopy.
BMC Immunol. 2006;716.
BACKGROUND: Leukocytes commonly infiltrate solid tumors, and have been implicated in the mechanism of spontaneous regression in some cancers. Conventional techniques for the quantitative estimation of leukocyte infiltrates in tumors rely on light microscopy of immunostained thin tissue sections, in which an arbitrary assessment (based on low, medium or high levels of infiltration) of antigen density is made by the pathologist. These estimates are relatively subjective and often require the opinion of a second pathologist. In addition, since thin tissue sections are cut, no data regarding the three-dimensional distribution of antigen can be obtained. RESULTS: To overcome these problems, we have designed a method to enumerate leukocyte infiltration into tumors, using confocal laser scanning microscopy of fluorescently immunostained leukocytes in thick tissue sections. Using image analysis software, a threshold was applied to eliminate unstained tissue and residual noise. The total antigen volume in the scanned tissue was calculated and divided by the mean cell volume (calculated by "seeding" ten individual cells) to obtain the cell count. Using this method, we compared the calculated leukocyte counts with those obtained manually by ten laboratory personnel. There was no significant difference (P > 0.05) between the cell counts obtained by either method.We then compared leukocyte infiltration into seven tumors and matched non-malignant tissue obtained from the periphery of the resected tissue. There was a significant increase in the infiltration of all leukocyte subsets into the tumors compared to minimal numbers in the non-malignant tissue. CONCLUSION: From these results we conclude that this method may be of considerable use for the enumeration of cells in tissues. Furthermore, since it can be performed by laboratory technical staff, less time input is required by the pathologist in assessing the degree of leukocyte infiltration into tumors. [Abstract/Link to Full Text]

Granum S, Sundvold-Gjerstad V, Dai KZ, Kolltveit KM, Hildebrand K, Huitfeldt HS, Lea T, Spurkland A
Structure function analysis of SH2D2A isoforms expressed in T cells reveals a crucial role for the proline rich region encoded by SH2D2A exon 7.
BMC Immunol. 2006;715.
BACKGROUND: The activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd. RESULTS: TSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239-256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239-334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239-334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells. CONCLUSION: These data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239-334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined. [Abstract/Link to Full Text]

Kivisäkk P, Tucky B, Wei T, Campbell JJ, Ransohoff RM
Human cerebrospinal fluid contains CD4+ memory T cells expressing gut- or skin-specific trafficking determinants: relevance for immunotherapy.
BMC Immunol. 2006;714.
BACKGROUND: Circulating memory T cells can be divided into tissue-specific subsets, which traffic through distinct tissue compartments during physiologic immune surveillance, based on their expression of adhesion molecules and chemokine receptors. We reasoned that a bias (either enrichment or depletion) of CSF T cell expression of known organ-specific trafficking determinants might suggest that homing of T cells to the subarachnoid space could be governed by a CNS-specific adhesion molecule or chemokine receptor. RESULTS: The expression of cutaneous leukocyte antigen (CLA) and CC-chemokine receptor 4 (CCR4; associated with skin-homing) as well as the expression of integrin alpha4beta7 and CCR9 (associated with gut-homing) was analyzed on CD4+ memory T cells in CSF from individuals with non-inflammatory neurological diseases using flow cytometry. CSF contained similar proportions of CD4+ memory T cells expressing CLA, CCR4, integrin alpha4beta7 and CCR9 as paired blood samples. CONCLUSION: The results extend our previous findings that antigen-experienced CD4+ memory T cells traffic through the CSF in proportion to their abundance in the peripheral circulation. Furthermore, the ready access of skin- and gut-homing CD4+ memory T cells to the CNS compartment via CSF has implications for the mechanisms of action of immunotherapeutic strategies, such as oral tolerance or therapeutic immunization, where immunogens are administered using an oral or subcutaneous route. [Abstract/Link to Full Text]

Dřsen G, Tenstad E, Nygren MK, Stubberud H, Funderud S, Rian E
Wnt expression and canonical Wnt signaling in human bone marrow B lymphopoiesis.
BMC Immunol. 2006;713.
BACKGROUND: The early B lymphopoiesis in mammals is regulated through close interactions with stromal cells and components of the intracellular matrix in the bone marrow (BM) microenvironment. Although B lymphopoiesis has been studied for decades, the factors that are implicated in this process, both autocrine and paracrine, are inadequately explored. Wnt signaling is known to be involved in embryonic development and growth regulation of tissues and cancer. Wnt molecules are produced in the BM, and we here ask whether canonical Wnt signaling has a role in regulating human BM B lymphopoiesis. RESULTS: Examination of the mRNA expression pattern of Wnt ligands, Fzd receptors and Wnt antagonists revealed that BM B progenitor cells and stromal cells express a set of ligands and receptors available for induction of Wnt signaling as well as antagonists for fine tuning of this signaling. Furthermore, different B progenitor maturation stages showed differential expression of Wnt receptors and co-receptors, beta-catenin, plakoglobin, LEF-1 and TCF-4 mRNAs, suggesting canonical Wnt signaling as a regulator of early B lymphopoiesis. Exogenous Wnt3A induced stabilization and nuclear accumulation of beta-catenin in primary lineage restricted B progenitor cells. Also, Wnt3A inhibited B lymphopoiesis of CD133+CD10- hematopoietic progenitor cells and CD10+ B progenitor cells in coculture assays using a supportive layer of stromal cells. This effect was blocked by the Wnt antagonists sFRP1 or Dkk1. Examination of early events in the coculture showed that Wnt3A inhibits cell division of B progenitor cells. CONCLUSION: These results indicate that canonical Wnt signaling is involved in human BM B lymphopoiesis where it acts as a negative regulator of cell proliferation in a direct or stroma dependent manner. [Abstract/Link to Full Text]

Yang J, Rosen SD, Bendele P, Hemmerich S
Induction of PNAd and N-acetylglucosamine 6-O-sulfotransferases 1 and 2 in mouse collagen-induced arthritis.
BMC Immunol. 2006;712.
BACKGROUND: Leukocyte recruitment across blood vessels is fundamental to immune surveillance and inflammation. Lymphocyte homing to peripheral lymph nodes is mediated by the adhesion molecule, L-selectin, which binds to sulfated carbohydrate ligands on high endothelial venules (HEV). These glycoprotein ligands are collectively known as peripheral node addressin (PNAd), as defined by the function-blocking monoclonal antibody known as MECA-79. The sulfation of these ligands depends on the action of two HEV-expressed N-acetylglucosamine 6-O-sulfotransferases: GlcNAc6ST-2 and to a lesser degree GlcNAc6ST-1. Induction of PNAd has also been shown to occur in a number of human inflammatory diseases including rheumatoid arthritis (RA). RESULTS: In order to identify an animal model suitable for investigating the role of PNAd in chronic inflammation, we examined the expression of PNAd as well as GlcNAc6ST-1 and -2 in collagen-induced arthritis in mice. Here we show that PNAd is expressed in the vasculature of arthritic synovium in mice immunized with collagen but not in the normal synovium of control animals. This de novo expression of PNAd correlates strongly with induction of transcripts for both GlcNAc6ST-1 and GlcNAc6ST-2, as well as the expression of GlcNAc6ST-2 protein. CONCLUSION: Our results demonstrate that PNAd and the sulfotransferases GlcNAc6ST-1 and 2 are induced in mouse collagen-induced arthritis and suggest that PNAd antagonists or inhibitors of the enzymes may have therapeutic benefit in this widely-used mouse model of RA. [Abstract/Link to Full Text]

Ruitenberg JJ, Mulder CB, Maino VC, Landay AL, Ghanekar SA
VACUTAINER CPT and Ficoll density gradient separation perform equivalently in maintaining the quality and function of PBMC from HIV seropositive blood samples.
BMC Immunol. 2006;711.
BACKGROUND: For immune monitoring studies during HIV vaccine clinical trials, whole blood specimens from HIV seropositive (HIV+) patients may be collected at multiple sites and sent to a central location for peripheral blood mononuclear cell (PBMC) isolation, cryopreservation and functional evaluation. In this study we show a comparison of two PBMC preparation options, Ficoll density gradient separation (Ficoll) and Cell Preparation Tubes (CPT) using shipped whole blood specimens from 19 HIV+ patients (CD4 > 350, viral load < 50). The pre- and post- cryopreservation performance of samples collected by these two methods were compared by assessment of antigen-specific IFNgamma expression in CD8+ and CD8- T cells, cellular viability, and cellular recovery. RESULTS: The results indicate that cryopreserved PBMC samples tested for CMV- and HIV-specific interferon-gamma (IFNgamma) expression performed equivalent to the respective fresh PBMC processed under both collection conditions. Compared to fresh PBMC, the viability was significantly lower for cryopreserved PBMC derived using Ficoll, although it was never less than 90%. There were no significant differences in the IFNgamma response, viability, or recovery between cryopreserved PBMC derived by Ficoll and by CPT. CONCLUSION: These data suggest that CPT is an efficient system for the collection and cryopreservation of functionally active HIV+ PBMC, as well as a viable alternative to Ficoll gradient separation. [Abstract/Link to Full Text]

Boysen P, Olsen I, Berg I, Kulberg S, Johansen GM, Storset AK
Bovine CD2-/NKp46+ cells are fully functional natural killer cells with a high activation status.
BMC Immunol. 2006;710.
BACKGROUND: Natural killer (NK) cells in the cow have been elusive due to the lack of specific NK cell markers, and various criteria including a CD3-/CD2+ phenotype have been used to identify such cells. The recent characterization of the NK-specific NKp46 receptor has allowed a more precise definition of bovine NK cells. NK cells are known as a heterogeneous cell group, and we here report the first functional study of bovine NK cell subsets, based on the expression of CD2. RESULTS: Bovine CD2- NK cells, a minor subset in blood, proliferated more rapidly in the presence of IL-2, dominating the cultures after a few days. Grown separately with IL-2, CD2- and CD2+ NK cell subsets did not change CD2 expression for at least two weeks. In blood, CD2- NK cells showed a higher expression of CD44 and CD25, consistent with a high activation status. A higher proportion of CD2- NK cells had intracellular interferon-gamma in the cytoplasm in response to IL-2 and IL-12 stimulation, and the CD2- subset secreted more interferon-gamma when cultured separately. Cytotoxic capacity was similar in both subsets, and both carried transcripts for the NK cell receptors KIR, CD16, CD94 and KLRJ. Ligation by one out of two tested anti-CD2 monoclonal antibodies could trigger interferon-gamma production from NK cells, but neither of them could alter cytotoxicity. CONCLUSION: These results provide evidence that bovine CD2- as well as CD2+ cells of the NKp46+ phenotype are fully functional NK cells, the CD2- subset showing signs of being more activated in the circulation. [Abstract/Link to Full Text]

Graca L, Daley S, Fairchild PJ, Cobbold SP, Waldmann H
Co-receptor and co-stimulation blockade for mixed chimerism and tolerance without myelosuppressive conditioning.
BMC Immunol. 2006;79.
BACKGROUND: A major challenge in the application of marrow transplantation as a route to immunological tolerance of a transplanted organ is to achieve hematopoietic stem cell (HSC) engraftment with minimal myelosuppressive treatments. RESULTS: We here describe a combined antibody protocol which can achieve long-term engraftment with clinically relevant doses of MHC-mismatched bone marrow, without the need for myelosuppressive drugs. Although not universally applicable in all strains, we achieved reliable engraftment in permissive strains with a two-stage strategy: involving first, treatment with anti-CD8 and anti-CD4 in advance of transplantation; and second, treatment with antibodies targeting CD4, CD8 and CD40L (CD154) at the time of marrow transplantation. Long-term mixed chimerism through co-receptor and co-stimulation blockade facilitated tolerance to donor-type skin grafts, without any evidence of donor-antigen driven regulatory T cells. CONCLUSION: We conclude that antibodies targeting co-receptor and co-stimulatory molecules synergise to enable mixed hematopoietic chimerism and central tolerance, showing that neither cytoreductive conditioning nor 'megadoses' of donor bone marrow are required for donor HSC to engraft in permissive strains. [Abstract/Link to Full Text]

Currier JR, Visawapoka U, Tovanabutra S, Mason CJ, Birx DL, McCutchan FE, Cox JH
CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: use of multiple peptide sets increases the detectable breadth of the CTL response.
BMC Immunol. 2006;78.
BACKGROUND: Subtype A is a major strain in the HIV-1 pandemic in eastern Europe, central Asia and in certain regions of east Africa, notably in rural Kenya. While considerable effort has been focused upon mapping and defining immunodominant CTL epitopes in HIV-1 subtype B and subtype C infections, few epitope mapping studies have focused upon subtype A. RESULTS: We have used the IFN-gamma ELIspot assay and overlapping peptide pools to show that the pattern of CTL recognition of the Gag and Nef proteins in subtype A infection is similar to that seen in subtypes B and C. The p17 and p24 proteins of Gag and the central conserved region of Nef were targeted by CTL from HIV-1-infected Kenyans. Several epitope/HLA associations commonly seen in subtype B and C infection were also observed in subtype A infections. Notably, an immunodominant HLA-C restricted epitope (Gag 296-304; YL9) was observed, with 8/9 HLA-CW0304 subjects responding to this epitope. Screening the cohort with peptide sets representing subtypes A, C and D (the three most prevalent HIV-1 subtypes in east Africa), revealed that peptide sets based upon an homologous subtype (either isolate or consensus) only marginally improved the capacity to detect CTL responses. While the different peptide sets detected a similar number of responses (particularly in the Gag protein), each set was capable of detecting unique responses not identified with the other peptide sets. CONCLUSION: Hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the HIV-1-specific CTL response. Interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given CTL response. Therefore, reagent choice and knowledge of the exact sequences that prime CTL responses will be important factors in experimentally defining cross-reactive CTL responses and their role in HIV-1 disease pathogenesis and validating vaccines aimed at generating broadly cross-reactive CTL responses. [Abstract/Link to Full Text]

Huang J, Honda W
CED: a conformational epitope database.
BMC Immunol. 2006;77.
BACKGROUND: Antigen epitopes provide valuable information useful for disease prevention, diagnosis, and treatment. Recently, more and more databases focusing on different types of epitopes have become available. Conformational epitopes are an important form of epitope formed by residues that are sequentially discontinuous but close together in three-dimensional space. These epitopes have implicit structural information, making them attractive for both theoretical and applied biomedical research. However, most existing databases focus on linear rather than conformational epitopes. DESCRIPTION: We describe CED, a special database of well defined conformational epitopes. CED provides a collection of conformational epitopes and related information including the residue make up and location of the epitope, the immunological property of the epitope, the source antigen and corresponding antibody of the epitope. All entries in this database are manually curated from articles published in peer review journals. The database can be browsed or searched through a user-friendly web interface. Most epitopes in CED can also be viewed interactively in the context of their 3D structures. In addition, the entries are also hyperlinked to various databases such as Swiss-Prot, PDB, KEGG and PubMed, providing wide background information. CONCLUSION: A conformational epitope database called CED has been developed as an information resource for investigators involved in both theoretical and applied immunology research. It complements other existing specialised epitope databases. The database is freely available at http://web.kuicr.kyoto-u.ac.jp/~ced. [Abstract/Link to Full Text]

Jiang Q, Su H, Knudsen G, Helms W, Su L
Delayed functional maturation of natural regulatory T cells in the medulla of postnatal thymus: role of TSLP.
BMC Immunol. 2006;76.
BACKGROUND: Generation of functional (CD4+)(CD8-)CD25+ regulatory T cells (Treg) in the murine thymus depends on FoxP3. Removal of the thymus from neonatal mice has been shown to result in a multiple organ autoimmune disease phenotype that can be prevented by introducing the FoxP3+ Treg population to the animal. It has therefore, been proposed that functional FoxP3+ Treg cells are not made in the neonatal thymus; however, it remains unclear when and where functional (FoxP3+)(CD4+)(CD8-)CD25+ thymocytes are generated in postnatal thymus. RESULTS: We report that neither FoxP3 mRNA nor protein is expressed in (CD4+)(CD8-)CD25+, or (CD4+)(CD8-)CD25- thymocytes until 3-4 days post birth, despite the presence of mature (CD4+)(CD8-)CD25+/- thymocytes in the thymus by 1-2 days after birth. (FoxP3-)(CD4+)(CD8-)CD25+ thymocytes from day 2 newborn mice show no Treg activity. Interestingly, we are able to detect low numbers of FoxP3+ thymocytes dispersed throughout the medullary region of the thymus as early as 3-4 days post birth. Expression of FoxP3 is induced in embryonic day 17 fetal thymus organ culture (FTOC) after 4-6 days of in vitro culture. Treatment of FTOCs with thymic stromal derived lymphopoietin (TSLP) enhanced expression of FoxP3, and blocking the TSLP receptor reduces FoxP3 expression in FTOC. Furthermore, TSLP stimulates FoxP3 expression in purified (CD4+)CD8- thymocytes, but not in (CD4+)CD8+, (CD4-)CD8+ and (CD4-)CD8- thymocytes. CONCLUSION: Expression of FoxP3 or Treg maturation is ontogenically distinct and kinetically delayed from the generation of (CD4+)(CD8-)CD25+ or (CD4+)(CD8-)CD25- thymocytes in the postnatal thymus. TSLP produced from medullary thymic epithelia cells (mTEC) contributes to the expression of FoxP3 and the maturation of natural regulatory T cells. Overall, these results suggest that the development of Treg cells requires paracrine signaling during late stages of thymocyte maturation that is distinct from signaling during positive or negative selection. [Abstract/Link to Full Text]

Luhm J, Langenkamp U, Hensel J, Frohn C, Brand JM, Hennig H, Rink L, Koritke P, Wittkopf N, Williams DL, Mueller A
Beta-(1-->3)-D-glucan modulates DNA binding of nuclear factors kappaB, AT and IL-6 leading to an anti-inflammatory shift of the IL-1beta/IL-1 receptor antagonist ratio.
BMC Immunol. 2006;75.
BACKGROUND: Beta-1-->3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses. Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal beta-1-->3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1). RESULTS: Despite an activation of nuclear factor (NF) kappaB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1beta, IL-6, tumor necrosis factor alpha or interferon gamma induced by glucan phosphate. Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h: 5000 pg/ml), likely due to binding of NFkappaB to a consensus site in the IL-8 promoter. An increase in IL-1receptor antagonist (RA) production (peak at 24 h: 12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels. Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments. When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFkappaB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA. Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-1beta and IL-6. As a consequence, glucan phosphate shifted the TSST-1-induced IL-1beta/IL-1RA ratio towards an anti-inflammatory phenotype. Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2. CONCLUSION: Thus, beta-1-->3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction. Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a beta-1-->3-D-glucan. [Abstract/Link to Full Text]

Dutsch-Wicherek M, Tomaszewska R, Strek P, Wicherek L, Skladzien J
The analysis of RCAS1 and DFF-45 expression in nasal polyps with respect to immune cells infiltration.
BMC Immunol. 2006;74.
BACKGROUND: Nasal polyp constitutes a benign growth process in the nasal and sinus mucosa. RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) is a protein expressed mainly by various human cancer cells. It is not only the marker of cancer process and its expression can also be observed in physiological processes. It is responsible for the regulation of immune cells activity. DFF45 (DNA fragmenting factor) has been described as a substrate for caspase-3. DFF45 seems to play an important role in the onset of apoptotic process by acting probably through the regulation of DNA fragmentation. The aim of the study was to evaluate the ability of nasal polyps to regulate the cytotoxic immune response and to determine their resistance to apoptosis. RESULTS: The higher RCAS1 level was identified in lymphocytic nasal polyps, the medium one in eosinophilic while the lowest was identified in neutrophilic. DFF-45 expression was higher in eosinophilic than in neutrophilic and lymphocytic nasal polyps. CONCLUSION: The changes in DFF-45 level in nasal polyps might indicate a different resistance to apoptosis mediated by immune cells. The alterations in RCAS1 expression indicate that nasal polyps have the ability to regulate the cytotoxic immune response. The breaking of resistance to immune mediated apoptosis in nasal polyps might have a new therapeutic impact. [Abstract/Link to Full Text]

Zahorsky-Reeves JL, Gregory CR, Cramer DV, Patanwala IY, Kyles AE, Borie DC, Kearns-Jonker MK
Similarities in the immunoglobulin response and VH gene usage in rhesus monkeys and humans exposed to porcine hepatocytes.
BMC Immunol. 2006;73.
BACKGROUND: The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose alpha (1,3) galactose (alphaGal) present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s) with bioartficial liver devices (BALs), composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine alphaGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH) immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta) were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies. RESULTS: Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH) cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has not been previously identified, and encodes xenoantibodies at later time points post-transplant. Sequencing of IgG clones revealed increased usage of the monkey germline progenitor most similar to human IGHV3-11 and the onset of mutations. CONCLUSION: The small number of IGVH genes encoding xenoantibodies to porcine hepatocytes in non-human primates and humans is highly conserved. Rhesus monkeys are an appropriate preclinical model for testing novel reagents such as those developed using structure-based drug design to target and deplete antibodies to porcine xenografts. [Abstract/Link to Full Text]

Lamason R, Zhao P, Rawat R, Davis A, Hall JC, Chae JJ, Agarwal R, Cohen P, Rosen A, Hoffman EP, Nagaraju K
Sexual dimorphism in immune response genes as a function of puberty.
BMC Immunol. 2006;72.
BACKGROUND: Autoimmune diseases are more prevalent in females than in males, whereas males have higher mortality associated with infectious diseases. To increase our understanding of this sexual dimorphism in the immune system, we sought to identify and characterize inherent differences in immune response programs in the spleens of male and female mice before, during and after puberty. RESULTS: After the onset of puberty, female mice showed a higher expression of adaptive immune response genes, while males had a higher expression of innate immune genes. This result suggested a requirement for sex hormones. Using in vivo and in vitro assays in normal and mutant mouse strains, we found that reverse signaling through FasL was directly influenced by estrogen, with downstream consequences of increased CD8+ T cell-derived B cell help (via cytokines) and enhanced immunoglobulin production. CONCLUSION: These results demonstrate that sexual dimorphism in innate and adaptive immune genes is dependent on puberty. This study also revealed that estrogen influences immunoglobulin levels in post-pubertal female mice via the Fas-FasL pathway. [Abstract/Link to Full Text]

Mosenson JA, McNulty JA
Characterization of lymphocyte subsets over a 24-hour period in Pineal-Associated Lymphoid Tissue (PALT) in the chicken.
BMC Immunol. 2006;71.
BACKGROUND: Homeostatic trafficking of lymphocytes in the brain has important relevance to the understanding of CNS disease processes. The pineal gland of the chicken contains large accumulations of lymphocytes that suggest an important role related to homeostatic circadian neuro-immune interactions. The purpose of this initial study was to characterize the lymphocyte subsets in the pineal gland and quantitate the distribution and frequency of lymphocyte phenotypes at two time points over the 24-hour light:dark cycle. RESULTS: PALT comprised approximately 10% of the total pineal area. Image analysis of immunocytochemically stained sections showed that the majority of lymphocytes were CD3+ (80%) with the remaining 20% comprising B-cells and monocytes (Bu-1+), which tended to distribute along the periphery of the PALT. T-cell subsets in PALT included CD4+ (75-80%), CD8+ (20-25%), TCRalphabeta/Vbeta1+ (60%), and TCRgammadelta+ (15%). All of the T-cell phenotypes were commonly found within the interfollicular septa and follicles of the pineal gland. However, the ratios of CD8+/CD4+ and TCRgammadelta+/TCRalphabeta/Vbeta1+ within the pineal tissue were each 1:1, in contrast to the PALT where the ratios of CD8+/CD4+ and TCRgammadelta+/TCRalphabeta/Vbeta1+ each approximated 1:4. Bu-1+ cells were only rarely seen in the pineal interstitial spaces, but ramified Bu-1+ microglia/macrophages were common in the pineal follicles. Effects of the 24-h light:dark cycle on these lymphocyte-pineal interactions were suggested by an increase in the area of PALT, a decline in the density of TCRalphabeta/Vbeta1+ cells, and a decline in the area density of Bu-1+ microglia at the light:dark interphase (1900 h) compared to the dark:light interphase (0700 h). CONCLUSION: The degree of lymphocyte infiltration in the pineal suggests novel mechanisms of neuro-immune interactions in this part of the brain. Our results further suggest that these interactions have a temporal component related to the 24-hour light:dark cycle and that CD8+ and TCRgammadelta+ T-cells are preferentially recruited to the pineal follicles. Pineal microglia/macrophages were common and represent an important candidate for mediating these lymphocyte-pineal interactions via secretion of cytokines and chemokines. [Abstract/Link to Full Text]


Recent Articles in BMC Infectious Diseases

Alam MM, Zaidi SZ, Malik SA, Shaukat S, Naeem A, Sharif S, Angez M, Butt JA
Molecular epidemiology of Hepatitis B virus genotypes in Pakistan.
BMC Infect Dis. 2007;7115.
BACKGROUND: Eight genotypes of Hepatitis B virus designated A-H, have been known but in Pakistan, no such data is available on the prevalent HBV genotypes. Therefore, the subject study was conducted to determine HBV genotypes in the indigenous Pakistani population. METHODS: A total of 690 individuals were enrolled for HBV screening with EIA and nested PCR. Positive samples were further analyzed to determine HBV genotypes (A-F) by multiplex-PCR using type specific primers. RESULTS: 110 (15.94%) individuals were positive for HBV, including 64% males and 36% females. Out of these, 66 samples (65.34%) were classified into genotype D, 27 (26.73%) were of genotype B while 5(4.95%) had genotype A. In 3 (2.98%) samples, multiple genotypes were detected (genotype A+B; 2(1.99%) and genotypes B+D; 1(0.99%). Nine (8.18%) samples remained untyable. CONCLUSION: In Asia, genotypes B and C are the most prevalent but our study reveals that genotype D is predominant and HBV infection constitutes a significant health problem in Pakistan. [Abstract/Link to Full Text]

Kankova S, Flegr J
Longer pregnancy and slower fetal development in women with latent "asymptomatic" toxoplasmosis.
BMC Infect Dis. 2007 Oct 4;7(1):114.
ABSTRACT: BACKGROUND: The purpose of this study was to confirm that women with latent toxoplasmosis have developmentally younger fetuses at estimated pregnancy week 16 and to test four exclusive hypotheses that could explain the observed data. METHODS: In the present retrospective cohort study we analysed by the GLM (general linear model) method data from 730 Toxoplasma-free and 185 Toxoplasma-infected pregnant women. RESULTS: At pregnancy week 16 estimated from the date of the last menstruation, the mothers with latent toxoplasmosis had developmentally younger fetuses based on ultrasound scan (P = 0.014). Pregnancy of Toxoplasma-positive compared to Toxoplasma-negative women was by about 1.3 days longer, as estimated both from the date of the last menstruation (P = 0.015) and by ultrasonography (P = 0.025). CONCLUSION: The most parsimonious explanation for the observed data is retarded fetal growth during the first weeks of pregnancy in Toxoplasma-positive women. The phenomenon was only detectable in multiparous women, suggesting that the immune system may play some role in it. [Abstract/Link to Full Text]

Moss NJ, Harper CC, Ahrens K, Scott K, Kao S, Padian N, Raine T, Klausner JD
Predictors of incident herpes simplex virus type 2 infections in young women at risk for unintended pregnancy in San Francisco.
BMC Infect Dis. 2007;7113.
BACKGROUND: Young women receiving family planning services are at risk for both unintended pregnancy and herpes simplex virus type 2 (HSV-2) infection. METHODS: We performed a secondary analysis using data from a previously published randomized controlled trial evaluating access to emergency contraception on reproductive health outcomes. Women aged 15 to 24 years were recruited from two Planned Parenthood clinics and two community health clinics in San Francisco. Demographic information and sexual history were obtained by interview. HSV-2 seropositivity was determined by fingerstick blood test. New pregnancies were measured by self-report, urine testing and medical chart review. Subjects were evaluated for incident HSV-2 infection and pregnancy at a 6-month follow-up appointment. Women who were pregnant or intending to become pregnant at enrolment were excluded. RESULTS: At enrolment 2,104 women were screened for HSV-2 and 170 (8.1%) were seropositive. Eighty-seven percent of initially seronegative women completed the study (n = 1,672) and 73 (4.4%) became HSV-2 seropositive. HSV-2 seroincidence was 7.8 cases per 100 person-years. One hundred and seventeen women (7%) became pregnant and 7 (6%) of these had a seroincident HSV-2 infection during the study. After adjustment for confounders, predictors of incident HSV-2 infection were African American race and having multiple partners in the last six months. Condom use at last sexual encounter was protective. CONCLUSION: HSV-2 seroincidence and the unintended pregnancy rate in young women were high. Providers who counsel women on contraceptive services and sexually transmitted infection prevention could play an expanded role in counselling women about HSV-2 prevention given the potential sequelae in pregnancy. The potential benefit of targeted screening and future vaccination against HSV-2 needs to be assessed in this population. [Abstract/Link to Full Text]

Salehi Omran A, Karimi A, Ahmadi SH, Davoodi S, Marzban M, Movahedi N, Abbasi K, Boroumand MA, Davoodi S, Moshtaghi N
Superficial and deep sternal wound infection after more than 9000 coronary artery bypass graft (CABG): incidence, risk factors and mortality.
BMC Infect Dis. 2007;7112.
BACKGROUND: Sternal wound infection (SWI) is an uncommon but potentially life-threatening complication of cardiac surgery. Predisposing factors for SWI are multiple with varied frequencies in different studies. The purpose of this study was to assess the incidence, risk factors, and mortality of SWI after coronary artery bypass grafting (CABG) at Tehran Heart Center. METHODS: This study prospectively evaluated multiple risk factors for SWI in 9201 patients who underwent CABG at Tehran Heart Center between January 2002 and February 2006. Cases of SWI were confirmed based on the criteria of the Centers for Disease Control and Prevention. Deep SWI (bone and mediastinitis) was categorized according to the Oakley classification. RESULTS: In the study period, 9201 CABGs were performed with a total SWI rate of 0.47 percent (44 cases) and deep SWI of 0.22 percent (21 cases). Perioperative (in-hospital) mortality was 9.1% for total SWI and about 14% for deep SWI versus 1.1% for non-SWI CABG patients. Female gender, preoperative hypertension, high functional class, diabetes mellitus, obesity, prolonged intubation time (more than 48 h), and re-exploration for bleeding were significant risk factors for developing SWI (p = 0.05) in univariate analysis. In multivariate analysis, hypertension (OR = 10.7), re-exploration (OR = 13.4), and female gender (OR = 2.7) were identified as significant predictors of SWI (p < 0.05 for all). The rate of SWI was relatively similar in 3 groups of prophylactic antibiotic regimen (Cefazolin, Cefazolin + Gentamycin and Cefazolin + Amikacin: 0.5%, 0.5%, and 0.34% respectively). CONCLUSION: Rarely reported previously, the two risk factors of hypertension and the female gender were significant risk factors in our study. Conversely, some other risk factors such as cigarette smoking and age mentioned as significant in other reports were not significant in our study. Further studies are needed for better documentation. [Abstract/Link to Full Text]

Drago L, De Vecchi E, Nicola L, Gismondo MR
In vitro evaluation of antibiotics' combinations for empirical therapy of suspected methicillin resistant Staphylococcus aureus severe respiratory infections.
BMC Infect Dis. 2007;7111.
BACKGROUND: Methicillin resistant Staphylococcus aureus (MRSA) is an increasingly common cause of nosocomial infections, causing severe morbidity and mortality worldwide, and accounting in some hospitals for more than 50% of all S. aureus diseases. Treatment of infections caused by resistant bacterial pathogens mainly relies on two therapeutic modalities: development of new antimicrobials and use of combinations of available antibiotics. Combinations of antibiotics used in the empiric treatment of infections with suspected methicillin resistant Staphylococcus aureus etiology were investigated. METHODS: Double (vancomycin or teicoplanin with either levofloxacin or cefotaxime) and triple (vancomycin or teicoplanin + levofloxacin + one among amikacin, ceftazidime, cefepime, imipenem, piperacillin/tazobactam) combinations were evaluated by means of checkerboard assay and time kill curves. Mutational rates of single and combined drugs at antimicrobial concentrations equal to the resistance breakpoints were also calculated. RESULTS: Vancomycin or teicoplanin + levofloxacin showed synergy in 16/50 and in 9/50 strains respectively, while vancomycin or teicoplanin + cefotaxime resulted synergic for 43/50 and 23/50 strains, respectively. Triple combinations, involving teicoplanin, levofloxacin and ceftazidime or piperacillin/tazobactam gave synergy in 20/25 strains. Teicoplanin + levofloxacin gave synergy in triple combinations more frequently than vancomycin + levofloxacin. For single antibiotics, mutational frequencies ranged between 10(-5) and <10(-9) for levofloxacin, cefotaxime, amikacin and imipenem, and <10(-9) for vancomycin and teicoplanin. When tested in combinations, mutational frequencies fell below 10(-9) for all the combinations. CONCLUSION: In vitro evidence of synergy between glycopeptides, fluoroquinolones (levofloxacin) and beta-lactams and of reduction of mutational frequencies by combinations are suggestive for a potential role in empirical therapy of severe pneumonia with suspected MRSA etiology. [Abstract/Link to Full Text]

de la Fuente J, Manzano-Roman R, Blouin EF, Naranjo V, Kocan KM
Sp110 transcription is induced and required by Anaplasma phagocytophilum for infection of human promyelocytic cells.
BMC Infect Dis. 2007;7110.
BACKGROUND: The tick-borne intracellular pathogen, Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) causes human granulocytic anaplasmosis after infection of polymorphonuclear leucocytes. The human Sp110 gene is a member of the nuclear body (NB) components that functions as a nuclear hormone receptor transcriptional coactivator and plays an important role in immunoprotective mechanisms against pathogens in humans. In this research, we hypothesized that Sp110 may be involved in the infection of human promyelocytic HL-60 cells with A. phagocytophilum. METHODS: The human Sp110 and A. phagocytophilum msp4 mRNA levels were evaluated by real-time RT-PCR in infected human HL-60 cells sampled at 0, 12, 24, 48, 72 and 96 hours post-infection. The effect of Sp110 expression on A. phagocytophilum infection was determined by RNA interference (RNAi). The expression of Sp110 was silenced in HL-60 cells by RNAi using pre-designed siRNAs using the Nucleofector 96-well shuttle system (Amaxa Biosystems, Gaithersburg, MD, USA). The A. phagocytophilum infection levels were evaluated in HL-60 cells after RNAi by real-time PCR of msp4 and normalizing against human Alu sequences. RESULTS: While Sp110 mRNA levels increased concurrently with A. phagocytophilum infections in HL-60 cells, the silencing of Sp110 expression by RNA interference resulted in decreased infection levels. CONCLUSION: These results demonstrated that Sp110 expression is required for A. phagocytophilum infection and multiplication in HL-60 cells, and suggest a previously undescribed mechanism by which A. phagocytophilum modulates Sp110 mRNA levels to facilitate establishment of infection of human HL-60 cells. [Abstract/Link to Full Text]

Vree M, Hoa NB, Sy DN, Co NV, Cobelens FG, Borgdorff MW
Low tuberculosis notification in mountainous Vietnam is not due to low case detection: a cross-sectional survey.
BMC Infect Dis. 2007;7109.
BACKGROUND: Studies show that tuberculosis notification declines with increasing altitude. This can be due to declining incidence or declining case detection. In Vietnam notification rates of new smear-positive tuberculosis in the central mountainous provinces (26/100,000 population) are considerably lower than in Vietnam in general (69/100,000 population). In order to clarify whether this is explained by low incidence or low case detection, we aimed to assess the prevalence of new smear-positive tuberculosis among adults with prolonged cough in three mountainous provinces in central Vietnam. METHODS: A house-to-house survey of persons (> or = 15 years) was carried out in twelve randomly selected districts in 2003. Three sputum specimens were microscopically examined of persons reporting a prolonged cough (> or = 3 weeks). Case detection was assessed by the ratio between notification and prevalence. RESULTS: Of 68,946 included persons (95% response), 1,298 (1.9% 95%CI 1.8-2.2) reported a prolonged cough. Of these, eighteen were sputum smear-positive of whom two had had anti-tuberculosis treatment. The prevalence of new smear-positive tuberculosis was 27/100,000 (95%CI 11-44/100,000) and the notification rate was 44/100,000 among persons > or = 15 years. The estimated case detection rate was 76%. CONCLUSION: Low tuberculosis notification in this mountainous setting is probably a true reflection of low tuberculosis incidence. Possible causes for low incidence in mountainous areas include low transmission rates or altitude-related differences in pathology. [Abstract/Link to Full Text]

Jessen KM, Lindboe SB, Petersen AL, Eugen-Olsen J, Benfield T
Common TNF-alpha, IL-1 beta, PAI-1, uPA, CD14 and TLR4 polymorphisms are not associated with disease severity or outcome from Gram negative sepsis.
BMC Infect Dis. 2007;7108.
BACKGROUND: Several studies have investigated single nucleotide polymorphisms (SNPs) in candidate genes associated with sepsis and septic shock with conflicting results. Only few studies have combined the analysis of multiple SNPs in the same population. METHODS: Clinical data and DNA from consecutive adult patients with culture proven Gram negative bacteremia admitted to a Danish hospital between 2000 and 2002. Analysis for commonly described SNPs of tumor necrosis-alpha, (TNF-alpha), interleukin-1 beta (IL-1 beta), plasminogen activator-1 (PAI-1), urokinase plasminogen activator (uPA), CD14 and toll-like receptor 4 (TLR4) was done. RESULTS: Of 319 adults, 74% had sepsis, 19% had severe sepsis and 7% were in septic shock. No correlation between severity or outcome of sepsis was observed for the analyzed SNPs of TNF-alpha, IL-1 beta, PAI-1, uPA, CD14 or TLR-4. In multivariate Cox proportional hazard regression analysis, increasing age, polymicrobial infection and haemoglobin levels were associated with in-hospital mortality. CONCLUSION: We did not find any association between TNF-alpha, IL-1 beta, PAI-1, uPA, CD14 and TLR4 polymorphisms and outcome of Gram negative sepsis. Other host factors appear to be more important than the genotypes studied here in determining the severity and outcome of Gram negative sepsis. [Abstract/Link to Full Text]

Hufnagel M, Liese C, Loescher C, Kunze M, Proempeler H, Berner R, Krueger M
Enterococcal colonization of infants in a neonatal intensive care unit: associated predictors, risk factors and seasonal patterns.
BMC Infect Dis. 2007;7107.
BACKGROUND: During and shortly after birth, newborn infants are colonized with enterococci. This study analyzes predictors for early enterococcal colonization of infants in a neonatal intensive care unit and describes risk factors associated with multidrugresistant enterococci colonization and its seasonal patterns. METHODS: Over a 12-month period, we performed a prospective epidemiological study in 274 infants admitted to a neonatal intensive care unit. On the first day of life, we compared infants with enterococcal isolates detected in meconium or body cultures to those without. We then tested the association of enterococcal colonization with peripartal predictors/risk factors by using bivariate and multivariate statistical methods. RESULTS: Twenty-three percent of the infants were colonized with enterococci. The three most common enterococcal species were E. faecium (48% of isolates), E. casseliflavus (25%) and E. faecalis (13%). Fifty-seven percent of the enterococci found were resistant to three of five antibiotic classes, but no vancomycin-resistant isolates were observed. During winter/spring months, the number of enterococci and multidrug-resistant enterococci were higher than in summer/fall months (p = 0.002 and p < 0.0001, respectively). With respect to enterococcal colonization on the first day of life, predictors were prematurity (p = 0.043) and low birth weight (p = 0.011). With respect to colonization with multidrug-resistant enterococci, risk factors were prematurity (p = 0.0006), low birth weight (p < 0.0001) and prepartal antibiotic treatment (p = 0.019). Using logistic regression, we determined that gestational age was the only parameter significantly correlated with multidrug-resistant enterococci colonization. No infection with enterococci or multidrugresistant enterococci in the infants was detected. The outcome of infants with and without enterococcal colonization was the same with respect to death, necrotizing enterocolitis, intracerebral hemorrhage and bronchopulmonary dysplasia. CONCLUSION: In neonatal intensive care units, an infant's susceptibility to early colonization with enterococci in general, and his or her risk for colonization with multidrug-resistant enterococci in particular, is increased in preterm newborns, especially during the winter/spring months. The prepartal use of antibiotics with no known activity against enterococci appears to increase the risk for colonization with multidrug-resistant enterococci. [Abstract/Link to Full Text]

Vandijck DM, Hoste EA, Blot SI, Depuydt PO, Peleman RA, Decruyenaere JM
Dynamics of C-reactive protein and white blood cell count in critically ill patients with nosocomial Gram positive vs. Gram negative bacteremia: a historical cohort study.
BMC Infect Dis. 2007;7106.
BACKGROUND: Nosocomial bacteremia is associated with a poor prognosis. Early adequate therapy has been shown to improve outcome. Consequently, rapid detection of a beginning sepsis is therefore of the utmost importance. This historical cohort study was designed to evaluate if different patterns can be observed in either C-reactive protein (CRP) and white blood cell count (WCC) between Gram positive bacteremia (GPB) vs. Gram negative bacteremia (GNB), and to assess the potential benefit of serial measurements of both biomarkers in terms of early antimicrobial therapy initiation. METHODS: A historical study (2003-2004) was conducted, including all adult intensive care unit patients with a nosocomial bacteremia. CRP and WCC count measurements were recorded daily from two days prior (d(-2)) until one day after onset of bacteremia (d(+1)). Delta (Delta) CRP and Delta WCC levels from the level at d-2 onward were calculated. RESULTS: CRP levels and WCC counts were substantially higher in patients with GNB. Logistic regression analysis demonstrated that GNB and Acute Physiology and Chronic Health Evaluation (APACHE) II score were independently associated with a CRP increase of 5 mg/dL from d-2 to d+1, and both were also independently associated with an increase of WCC levels from d(-2) to d(+1) of 5,000 x 10(3) cells/mm3. CONCLUSION: Increased levels of CRP and WCC are suggestive for GNB, while almost unchanged CRP and WCC levels are observed in patients with GPB. However, despite the different patterns observed, antimicrobial treatment as such cannot be guided based on both biomarkers. [Abstract/Link to Full Text]

Bhalla A, Aron DC, Donskey CJ
Staphylococcus aureus intestinal colonization is associated with increased frequency of S. aureus on skin of hospitalized patients.
BMC Infect Dis. 2007;7105.
BACKGROUND: Intestinal colonization by Staphylococcus aureus among hospitalized patients has been associated with increased risk of staphylococcal infection and could potentially contribute to transmission. We hypothesized that S. aureus intestinal colonization is associated with increased frequency of S. aureus on patients' skin and nearby environmental surfaces. METHODS: Selected inpatients were cultured weekly for S. aureus from stool, nares, skin (groin and axilla), and environmental surfaces (bed rail and bedside table). Investigator's hands were cultured after contacting the patients' skin and the environmental surfaces. RESULTS: Of 71 subjects, 32 (45.1%) had negative nares and stool cultures, 23 (32.4%) had positive nares and stool cultures, 13 (18.3%) were nares carriers only, and 3 (4.2%) were stool carriers only. Of the 39 patients with S. aureus carriage, 30 (76.9%) had methicillin-resistant isolates. In comparison to nares colonization only, nares and intestinal colonization was associated with increased frequency of positive skin cultures (41% versus 77%; p = 0.001) and trends toward increased environmental contamination (45% versus 62%; p = 0.188) and acquisition on investigator's hands (36% versus 60%; p = 0.057). Patients with negative nares and stool cultures had low frequency of S. aureus on skin and the environment (4.8% and 11.3%, respectively). CONCLUSION: We found that hospitalized patients with S. aureus nares and/or stool carriage frequently had S. aureus on their skin and on nearby environmental surfaces. S. aureus intestinal colonization was associated with increased frequency of positive skin cultures, which could potentially facilitate staphylococcal infections and nosocomial transmission. [Abstract/Link to Full Text]

Reither K, Ignatius R, Weitzel T, Seidu-Korkor A, Anyidoho L, Saad E, Djie-Maletz A, Ziniel P, Amoo-Sakyi F, Danikuu F, Danour S, Otchwemah RN, Schreier E, Bienzle U, Stark K, Mockenhaupt FP
Acute childhood diarrhoea in northern Ghana: epidemiological, clinical and microbiological characteristics.
BMC Infect Dis. 2007;7104.
BACKGROUND: Acute diarrhoea is a major cause of childhood morbidity and mortality in sub-Saharan Africa. Its microbiological causes and clinico-epidemiological aspects were examined during the dry season 2005/6 in Tamale, urban northern Ghana. METHODS: Stool specimens of 243 children with acute diarrhoea and of 124 control children were collected. Patients were clinically examined, and malaria and anaemia were assessed. Rota-, astro-, noro- and adenoviruses were identified by (RT-) PCR assays. Intestinal parasites were diagnosed by microscopy, stool antigen assays and PCR, and bacteria by culturing methods. RESULTS: Watery stools, fever, weakness, and sunken eyes were the most common symptoms in patients (mean age, 10 months). Malaria occurred in 15% and anaemia in 91%; underweight (22%) and wasting (19%) were frequent. Intestinal micro-organisms were isolated from 77% of patients and 53% of controls (P < 0.0001). The most common pathogens in patients were rotavirus (55%), adenovirus (28%) and norovirus (10%); intestinal parasites (5%) and bacteria (5%) were rare. Rotavirus was the only pathogen found significantly more frequently in patients than in controls (odds ratio 7.7; 95%CI, 4.2-14.2), and was associated with young age, fever and watery stools. Patients without an identified cause of diarrhoea more frequently had symptomatic malaria (25%) than those with diagnosed intestinal pathogens (12%, P = 0.02). CONCLUSION: Rotavirus-infection is the predominant cause of acute childhood diarrhoea in urban northern Ghana. The abundance of putative enteropathogens among controls may indicate prolonged excretion or limited pathogenicity. In this population with a high burden of diarrhoeal and other diseases, sanitation, health education, and rotavirus-vaccination can be expected to have substantial impact on childhood morbidity. [Abstract/Link to Full Text]

Alam FF, Mustafa AS, Khan ZU
Comparative evaluation of (1, 3)-beta-D-glucan, mannan and anti-mannan antibodies, and Candida species-specific snPCR in patients with candidemia.
BMC Infect Dis. 2007;7103.
BACKGROUND: Candidemia is a major infectious complication of seriously immunocompromised patients. In the absence of specific signs and symptoms, there is a need to evolve an appropriate diagnostic approach. A number of methods based on the detection of Candida mannan, nucleic acid and (1,3)-beta- D- glucan (BDG) have been used with varying specificities and sensitivities. In this retrospective study, attention has been focused to evaluate the usefulness of two or more disease markers in the diagnosis of candidemia. METHODS: Diagnostic usefulness of Platelia Candida Ag for the detection of mannan, Platelia Candida Ab for the detection of anti-mannan antibodies, Fungitell for the detection of BDG, and of a semi-nested PCR (snPCR) for the detection Candida species-specific DNA have been retrospectively evaluated using 32 sera from 27 patients with culture-proven candidemia, 51 sera from 39 patients with clinically suspected candidemia, sera of 10 women with C. albicans vaginitis, and sera of 16 healthy controls. RESULTS: Using cut-off values recommended by the manufacturers, the sensitivity of the assays for candidemia patients were as follows: Candida snPCR 88%, BDG 47%, mannan 41%, anti-mannan antibodies 47%, respectively. snPCR detected 5 patients who had candidemia due to more than one Candida species. The sensitivities of the combined tests were as follows: Candida mannan and anti-mannan antibodies 75%, and Candida mannan and BDG 56%. Addition of snPCR data improved the sensitivity further to 88%, thus adding 10 sera that were negative by BDG and/or mannan. In clinically suspected, blood culture negative patients; the positivities of the tests were as follows: Candida DNA was positive in 53%, BDG in 29%, mannan in 16%, and anti-mannan antibodies in 29%. The combined detection of mannan and BDG, and mannan, BDG and Candida DNA enhanced the positivity to 36% and 54%, respectively. None of the sera from Candida vaginitis patients and healthy subjects were positive for Candida DNA and mannan. CONCLUSION: The observations made in this study reinforce the diagnostic value of snPCR in the sensitive and specific diagnosis of candidemia and detection of more than one Candida species in a given patient. Additionally, in the absence of a positive blood culture, snPCR detected Candida DNA in sera of more than half of the clinically suspected patients. While detection of BDG, mannan and anti-mannan antibodies singly or in combination could help enhancing sensitivity and eliminating false positive tests, a more extensive evaluation of these assays in sequentially collected serum samples is required to assess their value in the early diagnosis of candidemia. [Abstract/Link to Full Text]

Kuvandik G, Cetin M, Genctoy G, Horoz M, Duru M, Akcali C, Satar S, Kiykim AA, Kaya H
The prevalance, epidemiology and risk factors for onychomycosis in hemodialysis patients.
BMC Infect Dis. 2007 Aug 30;7(1):102.
ABSTRACT: BACKGROUND: Onychomycosis has a high prevalance among immunocompromised patients such as diabetics and hemodialysis patients. In the present study, we aimed to investigate the prevalence of onychomycosis among hemodialysis patients with and without diabetes mellitus, and to find out the factors likely to be associated with the development of onychomycosis among hemodialysis patients. METHODS: One hundred and nine hemodialysis patients were enrolled. Fifty-seven of hemodialysis patients had the diagnosis of diabetes mellitus. Nail scrapings were obtained from 76 patients who had dystrophic nail changes. Samples were examined with 20% potassium hydroxide solution and all of the samples were inoculated on Saboraud's dextrose agar, potateus dextrose agar and mycobiotic agar. Diagnosis of onychomycosis was based on the presence of both positive clinical signs and positive potassium hydroxide test. RESULTS: Onychomycosis was diagnosed in 26.6% of hemodialysis patients. Diabetes mellitus was present in 68.9% of patients with onychomycosis. Toenail scraping cultures were reported to be positive in 19.7% of patients with dystrophic nail changes. Logistic regression analysis revealed that the presence of diabetes mellitus and the mean duration of hemodialysis were the significant predictors associated with the development of onychomycosis. CONCLUSION: The prevalence of dystrophic nail changes and onychomycosis is increased among hemodialysis patients. The dialysis duration and the presence of diabetes mellitus are the independent risk factors associated with the development of onychomycosis in uraemic patients. [Abstract/Link to Full Text]

Theodoridou MN, Vasilopoulou VA, Atsali EE, Pangalis AM, Mostrou GJ, Syriopoulou VP, Hadjichristodoulou CS
Meningitis registry of hospitalized cases in children: epidemiological patterns of acute bacterial meningitis throughout a 32-year period.
BMC Infect Dis. 2007;7101.
BACKGROUND: Bacterial meningitis remains a source of substantial morbidity and mortality in childhood. During the last decades gradual changes have been observed in the epidemiology of bacterial meningitis, related to the introduction of new polysaccharide and conjugate vaccines. The study presents an overview of the epidemiological patterns of acute bacterial meningitis in a tertiary children 's hospital during a 32-year period, using information from a disease registry. Moreover, it discusses the contribution of communicable disease registries in the study of acute infectious diseases. METHODS: In the early 1970s a Meningitis Registry (MR) was created for patients admitted with meningitis in Aghia Sofia Children's Hospital in Athens. The MR includes demographic, clinical and laboratory data as well as treatment, complications and outcome of the patients. In 2000 a database was created and the collected data were entered, analyzed and presented in three chronological periods: A (1974-1984), B (1985-1994) and C (1995-2005). RESULTS: Of the 2,477 cases of bacterial meningitis registered in total, 1,146 cases (46.3%) were classified as "probable" and 1,331 (53.7%) as "confirmed" bacterial meningitis. The estimated mean annual Incidence Rate (IR) was 16.9/100,000 for bacterial meningitis, 8.9/100,000 for Neisseria meningitidis, 1.3/100,000 for Streptococcus pneumoniae, 2.5/100,000 for Haemophilus influenzae type b (Hib) before vaccination and 0.4/100,000 for Hib after vaccination. Neisseria meningitis constituted the leading cause of childhood bacterial meningitis for all periods and in all age groups. Hib was the second most common cause of bacterial meningitis before the introduction of Hib conjugate vaccine, in periods A and B. The incidence of bacterial meningitis due to Streptococcus pneumoniae was stable. The long-term epidemiological pattern of Neisseria meningitidis appears in cycles of approximately 10 years, confirmed by a significant rise of IR in period C. The Case Fatality Rate (CFR) from all causes was 3.8%, while higher CFR were estimated for Streptococcus pneumoniae (7.5%, RR=2.1, 95% CI 1.2-3.7) and Neisseria meningitidis (4.8%, RR=1.7, 95% CI 1.1-2.5) compared to other pathogens. Moreover, overall CFR varied significantly among the three time periods (p = 0.0015), and was estimated to be higher in period C. CONCLUSION: By using the MR we were able to delineate long-term changes in the epidemiology of bacterial meningitis. Thus the MR proved to be a useful tool in the study and the prevention of communicable diseases in correlation with prevention strategies, such as vaccinations. [Abstract/Link to Full Text]

La Torre G, Miele L, Chiaradia G, Mannocci A, Reali M, Gasbarrini G, De Vito E, Grieco A, Ricciardi W
Socio-demographic determinants of coinfections by HIV, hepatitis B and hepatitis C viruses in central Italian prisoners.
BMC Infect Dis. 2007;7100.
BACKGROUND: The coinfections HIV/HCV/HBV are an important health issue in penitentiary communities. The aim of the study was to examine HIV, HBV and HCV coinfections determinants amongst prisoners in the jails of Southern Lazio (Central Italy), in the period 1995-2000. METHODS: Diagnosis of seropositivities for HIV, HBV and HCV was made using ELISA method. A multiple logistic regression analysis was conducted to verify the influence of socio-demographic factors on the HIV/HBV/HCV coinfections. RESULTS: HIV/HCV, HBV/HCV and HIV/HBV coinfections were detected in 42 (4%), 203 (17.9%) and 31 (2.9%) inmates, respectively. These coinfections are significantly associated with the status of drug addiction (OR = 16.02; p = 0.012; OR = 4.15; p < 0.001; OR = 23.57; p = 0.002), smoking habits (OR = 3.73; p = 0.033; OR = 1.42; p = 0.088; OR = 4.25; p = 0.053) and Italian nationality (OR = 7.05; p = 0.009; OR = 2.31; p < 0.001; OR = 4.61; p = 0.04). CONCLUSION: The prevalence of HIV, HBV and HCV seropositivity in jails suggests that information and education programs for inmates could be useful to reduce the spread of such infections. [Abstract/Link to Full Text]

Lawn SD, Bangani N, Vogt M, Bekker LG, Badri M, Ntobongwana M, Dockrell HM, Wilkinson RJ, Wood R
Utility of interferon-gamma ELISPOT assay responses in highly tuberculosis-exposed patients with advanced HIV infection in South Africa.
BMC Infect Dis. 2007;799.
BACKGROUND: Interferon-gamma (IFN-gamma) ELISPOT assays incorporating Mycobacterium tuberculosis-specific antigens are useful in the diagnosis of tuberculosis (TB) or latent infection. However, their utility in patients with advanced HIV is unknown. We studied determinants of ELISPOT responses among patients with advanced HIV infection (but without active TB) living in a South African community with very high TB notification rates. METHODS: IFN-gamma responses to ESAT-6 and CFP-10 in overnight ELISPOT assays and in 7-day whole blood assays (WBA) were compared in HIV-infected patients (HIV+, n = 40) and healthy HIV-negative controls (HIV-, n = 30) without active TB. Tuberculin skin tests (TSTs) were also done. RESULTS: ELISPOTs, WBAs and TSTs were each positive in >70% of HIV- controls, reflecting very high community exposure to M. tuberculosis. Among HIV+ patients, quantitative WBA responses and TSTs (but not the proportion of positive ELISPOT responses) were significantly impaired in those with CD4 cell counts <100 cells/mul compared to those with higher counts. In contrast, ELISPOT responses (but not WBA or TST) were strongly related to history of TB treatment; a much lower proportion of HIV+ patients who had recently completed treatment for TB (n = 19) had positive responses compared to those who had not been treated (11% versus 62%, respectively; P < 0.001). Multivariate analysis confirmed that ELISPOT responses had a strong inverse association with a history of recent TB treatment (adjusted OR = 0.06, 95%CI = 0.10-0.40, P < 0.01) and that they were independent of CD4 cell count and viral load. Among HIV+ individuals who had not received TB treatment both the magnitude and proportion of positive ELISPOT responses (but not TST or WBA) were similar to those of HIV-negative controls. CONCLUSION: The proportion of positive ELISPOT responses in patients with advanced HIV infection was independent of CD4 cell count but had a strong inverse association with history of TB treatment. This concurs with the previously documented low TB risk among patients in this cohort with a history of recent treatment for TB. These data suggest ELISPOT assays may be useful for patient assessment and as an immuno-epidemiological research tool among patients with advanced HIV and warrant larger scale prospective evaluation. [Abstract/Link to Full Text]

Bono JL, Keen JE, Clawson ML, Durso LM, Heaton MP, Laegreid WW
Association of Escherichia coli O157:H7 tir polymorphisms with human infection.
BMC Infect Dis. 2007;798.
BACKGROUND: Emerging molecular, animal model and epidemiologic evidence suggests that Shiga-toxigenic Escherichia coli O157:H7 (STEC O157) isolates vary in their capacity to cause human infection and disease. The translocated intimin receptor (tir) and intimin (eae) are virulence factors and bacterial receptor-ligand proteins responsible for tight STEC O157 adherence to intestinal epithelial cells. They represent logical genomic targets to investigate the role of sequence variation in STEC O157 pathogenesis and molecular epidemiology. The purposes of this study were (1) to identify tir and eae polymorphisms in diverse STEC O157 isolates derived from clinically ill humans and healthy cattle (the dominant zoonotic reservoir) and (2) to test any observed tir and eae polymorphisms for association with human (vs bovine) isolate source. RESULTS: Five polymorphisms were identified in a 1,627-bp segment of tir. Alleles of two tir polymorphisms, tir 255 T>A and repeat region 1-repeat unit 3 (RR1-RU3, presence or absence) had dissimilar distributions among human and bovine isolates. More than 99% of 108 human isolates possessed the tir 255 T>A T allele and lacked RR1-RU3. In contrast, the tir 255 T>A T allele and RR1-RU3 absence were found in 55% and 57%, respectively, of 77 bovine isolates. Both polymorphisms associated strongly with isolate source (p < 0.0001), but not by pulsed field gel electrophoresis type or by stx1 and stx2 status (as determined by PCR). Two eae polymorphisms were identified in a 2,755-bp segment of 44 human and bovine isolates; 42 isolates had identical eae sequences. The eae polymorphisms did not associate with isolate source. CONCLUSION: Polymorphisms in tir but not eae predict the propensity of STEC O157 isolates to cause human clinical disease. The over-representation of the tir 255 T>A T allele in human-derived isolates vs the tir 255 T>A A allele suggests that these isolates have a higher propensity to cause disease. The high frequency of bovine isolates with the A allele suggests a possible bovine ecological niche for this STEC O157 subset. [Abstract/Link to Full Text]

Hautala T, Ikäheimo I, Husu H, Säily M, Siitonen T, Koistinen P, Vuopio-Varkila J, Koskela M, Kujala P
A cluster of Candida krusei infections in a haematological unit.
BMC Infect Dis. 2007;797.
BACKGROUND: Candida krusei infections are associated with high mortality. In order to explore ways to prevent these infections, we investigated potential routes for nosocomial spread and possible clonality of C. krusei in a haematological unit which had experienced an unusually high incidence of cases. METHODS: We searched for C. krusei contamination of the hospital environment and determined the level of colonization in patients and health care workers. We also analyzed the possible association between exposure to prophylactic antifungals or chemotherapeutic agents and occurrence of C. krusei. The C. krusei isolates found were genotyped by pulsed-field electrophoresis method in order to determine possible relatedness of the cases. RESULTS: Twelve patients with invasive C. krusei infection and ten patients with potentially significant infection or mucosal colonization were documented within nine months. We were unable to identify any exogenic source of infection or colonization. Genetic analysis of the isolates showed little evidence of clonal transmission of C. krusei strains between the patients. Instead, each patient was colonized or infected by several different closely related genotypes. No association between medications and occurrence of C. krusei was found. CONCLUSION: Little evidence of nosocomial spread of a single C. krusei clone was found. The outbreak may have been controlled by cessation of prophylactic antifungals and by intensifying infection control measures, e.g. hand hygiene and cohorting of the patients, although no clear association with these factors was demonstrated. [Abstract/Link to Full Text]

Lala S, Dheda K, Chang JS, Huggett JF, Kim LU, Johnson MA, Rook GA, Keshav S, Zumla A
The pathogen recognition sensor, NOD2, is variably expressed in patients with pulmonary tuberculosis.
BMC Infect Dis. 2007;796.
BACKGROUND: NOD2, an intracellular pathogen recognition sensor, modulates innate defences to muropeptides derived from various bacterial species, including Mycobacterium tuberculosis (MTB). Experimentally, NOD2 attenuates two key putative mycobactericidal mechanisms. TNF-alpha synthesis is markedly reduced in MTB-antigen stimulated-mononuclear cells expressing mutant NOD2 proteins. NOD2 agonists also induce resistance to apoptosis, and may thus facilitate the survival of MTB in infected macrophages. To further define a role for NOD2 in disease pathogenesis, we analysed NOD2 transcriptional responses in pulmonary leucocytes and mononuclear cells harvested from patients with pulmonary tuberculosis (PTB). METHODS: We analysed NOD2 mRNA expression by real-time polymerase chain-reaction in alveolar lavage cells obtained from 15 patients with pulmonary tuberculosis and their matched controls. We compared NOD2 transcriptional responses, in peripheral leucocytes, before and after anti-tuberculous treatment in 10 patients. In vitro, we measured NOD2 mRNA levels in MTB-antigen stimulated-mononuclear cells. RESULTS: No significant differences in NOD2 transcriptional responses were detected in patients and controls. In some patients, however, NOD2 expression was markedly increased and correlated with toll-like-receptor 2 and 4 expression. In whole blood, NOD2 mRNA levels increased significantly after completion of anti-tuberculosis treatment. NOD2 expression levels did not change significantly in mononuclear cells stimulated with mycobacterial antigens in vitro. CONCLUSION: There are no characteristic NOD2 transcriptional responses in PTB. Nonetheless, the increased levels of NOD2 expression in some patients with severe tuberculosis, and the increases in expression levels within peripheral leucocytes following treatment merit further studies in selected patient and control populations. [Abstract/Link to Full Text]

Tien HC, Battad A, Bryce EA, Fuller J, Mulvey M, Bernard K, Brisebois R, Doucet JJ, Rizoli SB, Fowler R, Simor A
Multi-drug resistant Acinetobacter infections in critically injured Canadian forces soldiers.
BMC Infect Dis. 2007;795.
BACKGROUND: Military members, injured in Afghanistan or Iraq, have returned home with multi-drug resistant Acinetobacter baumannii infections. The source of these infections is unknown. METHODS: Retrospective study of all Canadian soldiers who were injured in Afghanistan and who required mechanical ventilation from January 1 2006 to September 1 2006. Patients who developed A. baumannii ventilator associated pneumonia (VAP) were identified. All A. baumannii isolates were retrieved for study patients and compared with A. baumannii isolates from environmental sources from the Kandahar military hospital using pulsed-field gel electrophoresis (PFGE). RESULTS: During the study period, six Canadian Forces (CF) soldiers were injured in Afghanistan, required mechanical ventilation and were repatriated to Canadian hospitals. Four of these patients developed A. baumannii VAP. A. baumannii was also isolated from one environmental source in Kandahar - a ventilator air intake filter. Patient isolates were genetically indistinguishable from each other and from the isolates cultured from the ventilator filter. These isolates were resistant to numerous classes of antimicrobials including the carbapenems. CONCLUSION: These results suggest that the source of A. baumannii infection for these four patients was an environmental source in the military field hospital in Kandahar. A causal linkage, however, was not established with the ventilator. This study suggests that infection control efforts and further research should be focused on the military field hospital environment to prevent further multi-drug resistant A. baumannii infections in injured soldiers. [Abstract/Link to Full Text]

Kerttula AM, Lyytikäinen O, Kardén-Lilja M, Ibrahem S, Salmenlinna S, Virolainen A, Vuopio-Varkila J
Nationwide trends in molecular epidemiology of methicillin-resistant Staphylococcus aureus, Finland, 1997-2004.
BMC Infect Dis. 2007;794.
BACKGROUND: In Finland, the annual number of MRSA notifications to the National Infectious Disease Register (NIDR) has constantly increased since 1995, and molecular typing has revealed numerous outbreak isolates of MRSA. We analyzed the data on MRSA notifications of the NIDR, and MRSA isolates were identified mainly by pulsed-field gel electrophoresis (PFGE) at the National Reference Laboratory (NRL) in Finland during 1997-2004. One isolate representative of each major PFGE type was further characterized by multilocus sequence (MLST)-, staphylococcal cassette chromosome mec (SCCmec)-, and Panton-Valentine leukocidin (PVL)-typing. RESULTS: The annual number of MRSA notifications to the NIDR rose over ten-fold, from 120 in 1997 to 1458 in 2004, and the proportion of MRSA among S. aureus blood isolates tripled, from <1% during 1997-2003 to 2.8% in 2004. During the same period of time, 253 different strains among 4091 MRSA isolates were identified by PFGE: 215 were sporadic and 38 outbreak/epidemic strains, including 24 new strains. Two epidemic strains resembling internationally recognized MRSA clones accounted for most of the increase: FIN-16 (ST125:IA) from <1% in 1997 to 25% in 2004, and FIN-21 (ST228:I) from 6% in 2002 to 28% in 2004. Half of the ten most common strains carried SCCmec IV or V. CONCLUSION: The predominant MRSA strains seem to change over time, which encourages us to continue implementing active control measures with each new MRSA case. [Abstract/Link to Full Text]

O'Loughlin RE, Kightlinger L, Werpy MC, Brown E, Stevens V, Hepper C, Keane T, Benson RF, Fields BS, Moore MR
Restaurant outbreak of Legionnaires' disease associated with a decorative fountain: an environmental and case-control study.
BMC Infect Dis. 2007;793.
BACKGROUND: From June to November 2005, 18 cases of community-acquired Legionnaires' disease (LD) were reported in Rapid City South Dakota. We conducted epidemiologic and environmental investigations to identify the source of the outbreak. METHODS: We conducted a case-control study that included the first 13 cases and 52 controls randomly selected from emergency department records and matched on underlying illness. We collected information about activities of case-patients and controls during the 14 days before symptom onset. Environmental samples (n = 291) were cultured for Legionella. Clinical and environmental isolates were compared using monoclonal antibody subtyping and sequence based typing (SBT). RESULTS: Case-patients were significantly more likely than controls to have passed through several city areas that contained or were adjacent to areas with cooling towers positive for Legionella. Six of 11 case-patients (matched odds ratio (mOR) 32.7, 95% CI 4.7-infinity) reported eating in Restaurant A versus 0 controls. Legionella pneumophila serogroup 1 was isolated from four clinical specimens: 3 were Benidorm type strains and 1 was a Denver type strain. Legionella were identified from several environmental sites including 24 (56%) of 43 cooling towers tested, but only one site, a small decorative fountain in Restaurant A, contained Benidorm, the outbreak strain. Clinical and environmental Benidorm isolates had identical SBT patterns. CONCLUSION: This is the first time that small fountain without obvious aerosol-generating capability has been implicated as the source of a LD outbreak. Removal of the fountain halted transmission. [Abstract/Link to Full Text]

Moyo SJ, Maselle SY, Matee MI, Langeland N, Mylvaganam H
Identification of diarrheagenic Escherichia coli isolated from infants and children in Dar es Salaam, Tanzania.
BMC Infect Dis. 2007;792.
BACKGROUND: Relatively few studies have been done in Tanzania to detect and classify diarrheagenic Escherichia coli (DEC) strains among children with diarrhea. This study aimed at investigating DEC among children in Dar es Salaam aged less than five years hospitalized due to acute/persistent diarrhea. METHODS: DEC were isolated from stool samples collected from two hundred and eighty children with acute/persistent diarrhea at Muhimbili National Hospital and Ilala and Mwananyamala Municipal Hospitals in Dar es Salaam. A multiplex PCR system method was used to detect a species specific gene for E.coli and ten different virulence genes for detection of five pathogroups of DEC namely enteroaggregative- (EAEC), enteropathogenic- (EPEC), enterotoxigenic- (ETEC), enteroinvasive- (EIEC) and enterohemorghagic- Escherichia coli (EHEC). RESULTS: Sixty-four patients (22.9%) harbored DEC. Forty-one of them (14.6%) were categorized as EAEC. Most of the EAEC (82.9%) were classified as typical EAEC possessing the aggR gene, and 92.6% carried the aat gene. Isolates from thirteen patients were EPEC (4.6%) and most of these (92.3%) were typical EPEC with both eae and bfpA genes. Ten isolates were identified as ETEC (3.6%) with only the heat stable toxin; either st1a or st1b but not both. Age wise, EAEC and EPEC were significantly more prevalent among the age group 0-6 months (p < 0.05). Genes for EHEC (stx1 and stx2) and EIEC (ial) were not detected in this study group. CONCLUSION: The results show a high proportion of DEC among Tanzanian children with diarrhea, with typical EAEC and typical EPEC predominating. The use of primers for both variants of ST1 (st1a and st1b) increased the sensitivity for detection of ETEC strains. [Abstract/Link to Full Text]

Ray KJ, Porco TC, Hong KC, Lee DC, Alemayehu W, Melese M, Lakew T, Yi E, House J, Chidambaram JD, Whitcher JP, Gaynor BD, Lietman TM
A rationale for continuing mass antibiotic distributions for trachoma.
BMC Infect Dis. 2007;791.
BACKGROUND: The World Health Organization recommends periodic mass antibiotic distributions to reduce the ocular strains of chlamydia that cause trachoma, the world's leading cause of infectious blindness. Their stated goal is to control infection, not to completely eliminate it. A single mass distribution can dramatically reduce the prevalence of infection. However, if infection is not eliminated in every individual in the community, it may gradually return back into the community, so often repeated treatments are necessary. Since public health groups are reluctant to distribute antibiotics indefinitely, we are still in need of a proven long-term rationale. Here we use mathematical models to demonstrate that repeated antibiotic distributions can eliminate infection in a reasonable time period. METHODS: We fit parameters of a stochastic epidemiological transmission model to data collected before and 6 months after a mass antibiotic distribution in a region of Ethiopia that is one of the most severely affected areas in the world. We validate the model by comparing our predicted results to Ethiopian data which was collected biannually for two years past the initial mass antibiotic distribution. We use the model to simulate the effect of different treatment programs in terms of local elimination of infection. RESULTS: Simulations show that the average prevalence of infection across all villages progressively decreases after each treatment, as long as the frequency and coverage of antibiotics are high enough. Infection can be eliminated in more villages with each round of treatment. However, in the communities where infection is not eliminated, it returns to the same average level, forming the same stationary distribution. This phenomenon is also seen in subsequent epidemiological data from Ethiopia. Simulations suggest that a biannual treatment plan implemented for 5 years will lead to elimination in 95% of all villages. CONCLUSION: Local elimination from a community is theoretically possible, even in the most severely infected communities. However, elimination from larger areas may require repeated biannual treatments and prevention of re-introduction from outside to treated areas. [Abstract/Link to Full Text]

French DJ, Jones D, McDowell DG, Thomson JA, Debenham PG
Analysis of multiple single nucleotide polymorphisms closely positioned in the ovine PRNP gene using linear fluorescent probes and melting curve analysis.
BMC Infect Dis. 2007;790.
BACKGROUND: Resistance and susceptibility to scrapie has been associated with single nucleotide polymorphisms located within codons 136, 154 and 171 of the ovine prion protein gene (PRNP). Dual-labelled HyBeacon probes were developed to analyse single and clustered polymorphisms within these and neighbouring codons. METHODS: Extracted DNAs and unpurified blood samples were genotyped with respect to polymorphisms in PRNP codons 136, 141, 154 and 171. PCR amplicons were investigated using a LightTyper instrument, measuring the stability of probe/target hybridisation through peak melting temperatures and determining the sequence of nucleotides at polymorphic sites. RESULTS: The performance of HyBeacon assays was evaluated in a validation study comparing genotypes with those obtained using a primer extension assay (Sequenom MassEXTEND) analysed on a MALDI-ToF mass spectrometer. Over 12,000 sheep samples were successfully genotyped, reliably detecting A136, V136, T136, T137, L141, F141 R154, H154, L168, R171, Q171, H171 and K171 sequence variants using only 4 HyBeacon probes. CONCLUSION: HyBeacon assays provide an extremely robust and accurate method for the analysis of single and clustered PRNP polymorphisms in a high-throughput format. The flexibility of the diagnostic tests ensures that samples are correctly genotyped even in the presence of additional sequence variations that flank the polymorphisms of interest. Such sequence variations may also be neutralised using universal bases such as 5-nitroindole if required. [Abstract/Link to Full Text]

Karakousis PC, Sifakis FG, de Oca RM, Amorosa VC, Page KR, Manabe YC, Campbell JD
U.S. medical resident familiarity with national tuberculosis guidelines.
BMC Infect Dis. 2007;789.
BACKGROUND: The ability of medical residents training at U.S. urban medical centers to diagnose and manage tuberculosis cases has important public health implications. We assessed medical resident knowledge about tuberculosis diagnosis and early management based on American Thoracic Society guidelines. METHODS: A 20-question tuberculosis knowledge survey was administered to 131 medical residents during a single routinely scheduled teaching conference at four different urban medical centers in Baltimore and Philadelphia. Survey questions were divided into 5 different subject categories. Data was collected pertaining to institution, year of residency training, and self-reported number of patients managed for tuberculosis within the previous year. The Kruskal-Wallis test was used to detect differences in median percent of questions answered correctly based on these variables. RESULTS: The median percent of survey questions answered correctly for all participating residents was 55%. Medical resident knowledge about tuberculosis did not improve with increasing post-graduate year of training or greater number of patients managed for tuberculosis within the previous year. Common areas of knowledge deficiency included the diagnosis and management of latent tuberculosis infection (median percent correct, 40.7%), as well as the interpretation of negative acid-fast sputum smear samples. CONCLUSION: Many medical residents lack adequate knowledge of recommended guidelines for the management of tuberculosis. Since experience during training influences future practice patterns, education of medical residents on guidelines for detection and early management of tuberculosis may be important for future improvements in national tuberculosis control strategies. [Abstract/Link to Full Text]

Junior MS, Correa L, Marra AR, Camargo LF, Pereira CA
Analysis of vancomycin use and associated risk factors in a university teaching hospital: a prospective cohort study.
BMC Infect Dis. 2007;788.
BACKGROUND: Vancomycin use is considered inappropriate in most hospitals. A particular concern is the recent emergence of S. aureus with decreased susceptibility to vancomycin, making it important to reduce overall exposure to vancomycin to minimize the incidence of VRE (vancomycin-resistant enterococci). The aim of this work was to analyze the use of vancomycin and the risk factors associated with inappropriate treatment. METHODS: A prospective survey was conducted on all patients receiving vancomycin between 1st March 2002 and 30th September 2002 in a university-school hospital. Appropriateness of vancomycin use was assessed, according to the criteria established by the Centers for Disease Control and Prevention (CDC), at two time points: first, at the beginning of therapy, and second, continuing after 72 hours. RESULTS: A total of 557 patients received vancomycin. Three hundred seventy-four (67.1%) were under 60 years old, 374 (67.1%) had prolonged stays (>two weeks) in hospital, and 455 (81.7%) were in the intensive care unit (ICU). Two hundred sixty-three patients (47.2%) had some invasive device. In 324 (58.2%) patients the duration of vancomycin treatment was up to two weeks. Vancomycin was inappropriately used in 65.7% during the first 24 hours and in 67% at the 72 hours point according to CDC criteria 4. The inappropriateness of vancomycin use during the first 24 hours was related to: patients aged less than 60 (OR 1.7; CI 95% 1.1-2.5), non-ICU patients (OR 1.5; CI 95% 1.0-2.4) and patients without neutropenia (OR 7.5; CI 95% 2.4-22.7). At 72 hours, the inappropriateness of vancomycin use was related to: patients aged less than 60 (OR 1.5; CI 95% 1.0-2.3), non-ICU patients (OR 1.7; CI 95% 1.1-2.7) and patients without neutropenia (OR 8.0; CI 95% 2.6-24.3). CONCLUSION: Vancomycin was abused. Patients aged less than 60, non-ICU patients and those who did not present neutropenia were the principal groups at risk of inappropriate use. [Abstract/Link to Full Text]

Aydin S, Ertugrul B, Gultekin B, Uyar G, Kir E
Treatment of two postoperative endophthalmitis cases due to Aspergillus flavus and Scopulariopsis spp. with local and systemic antifungal therapy.
BMC Infect Dis. 2007;787.
BACKGROUND: Endophthalmitis is the inflammatory response to invasion of the eye with bacteria or fungi. The incidence of endophthalmitis after cataract surgery varies between 0.072-0.13 percent. Treatment of endophthalmitis with fungal etiology is difficult. CASE PRESENTATION: Case 1: A 71-year old male diabetic patient developed postoperative endophthalmitis due to Aspergillus flavus. The patient was treated with topical amphotericin B ophthalmic solution, intravenous (IV) liposomal amphotericin-B and caspofungin following vitrectomy.Case 2: A 72-year old male cachectic patient developed postoperative endophthalmitis due to Scopulariopsis spp. The patient was treated with topical and IV voriconazole and caspofungin. CONCLUSION: Aspergillus spp. are responsible of postoperative fungal endophthalmitis. Endophthalmitis caused by Scopulariopsis spp. is a very rare condition. The two cases were successfully treated with local and systemic antifungal therapy. [Abstract/Link to Full Text]

Krishnan MY, Radhakrishnan I, Joseph BV, Madhavi Latha GK, Ajay Kumar R, Mundayoor S
Combined use of Amplified Fragment Length Polymorphism and IS6110-RFLP in fingerprinting clinical isolates of Mycobacterium tuberculosis from Kerala, South India.
BMC Infect Dis. 2007;786.
BACKGROUND: DNA fingerprinting by IS6110-RFLP has shown a high incidence of Mycobacterium tuberculosis isolates having no and low copies of the insertion sequence in Kerala, South India. Amplified Fragment Length Polymorphism (AFLP) would scan the entire genome rather than a few repetitive elements, we thought that this technique would help us in differentiating the large reservoir of isolates from an endemic region. Here we evaluate the ability of Amplified Fragment Length Polymorphism (AFLP) to type clinical isolates. METHODS: Fifty clinical isolates of M. tuberculosis were analysed by conventional radioactive AFLP and IS6110- RFLP. M. bovis, M. bovis BCG and two non tuberculous mycobacteria were also analysed to see species specific differences generated by AFLP. Cluster analysis was performed using the AFLP profile that showed the maximum polymorphism within M. tuberculosis and this was compared to the number of copies of IS6110 insertions. RESULTS: For AFLP, out of ten primer pairs tested, the EO/MC pair generated maximum polymorphism among the clinical isolates of M. tuberculosis. The similarity between the isolates ranged between 88 and 99.5%. Majority (nearly 85%) of the 'low copy' IS6110 isolates clustered together, while the rest clustered irrespective of the copy numbers. AFLP could show rare differences between isolates of M. tuberculosis, M. bovis and M. bovis BCG. The AFLP profiles for non-tuberculous mycobacteria were highly different from those of M. tuberculosis. CONCLUSION: Polymorphism generated by AFLP within the M. tuberculosis species is limited and hence AFLP alone seems to have limited use in fingerprinting the isolates in Kerala. The combined use of AFLP and IS6110-RFLP showed relatively better differentiation of 'high copy' IS6110 isolates, but failed to differentiate the 'low copy' isolates. However, the technique may be efficient in inter-species differentiation, and hence potentially useful in identifying and developing species-specific markers. [Abstract/Link to Full Text]


Recent Articles in BMC Microbiology

Osterhout RE, Figueroa IA, Keasling JD, Arkin AP
Global analysis of host response to induction of a latent bacteriophage.
BMC Microbiol. 2007 Aug 31;7(1):82.
ABSTRACT: BACKGROUND: The transition from viral latency to lytic growth involves complex interactions among host and viral factors, and the extent to which host physiology is buffered from the virus during induction of lysis is not known. A reasonable hypothesis is that the virus should be evolutionarily selected to ensure host health throughout induction to minimize its chance of reproductive failure. To address this question, we collected transcriptional profiles of Escherichia coli and bacteriophage lambda throughout lysogenic induction by UV light. RESULTS: We observed a temporally coordinated program of phage gene expression, with distinct early, middle and late transcriptional classes. Our study confirmed known host-phage interactions of induction of the heat shock regulon, escape replication, and suppression of genes involved in cell division and initiation of replication. We identified 728 E. coli genes responsive to prophage induction, which included pleiotropic stress response pathways, the Arc and Cpx regulons, and global regulators crp and lrp. Several hundred genes involved in central metabolism, energy metabolism, translation and transport were down-regulated late in induction. Though statistically significant, most of the changes in these genes were mild, with only 140 genes showing greater than two-fold change. CONCLUSIONS: Overall, we observe that prophage induction has a surprisingly low impact on host physiology. This study provides the first global dynamic picture of how host processes respond to lambda phage induction. [Abstract/Link to Full Text]

Kariyawasam S, Scaccianoce JA, Nolan LK
Common and specific genomic sequences of avian and human extraintestinal pathogenic Escherichia coli as determined by genomic subtractive hybridization.
BMC Microbiol. 2007;781.
BACKGROUND: Suppression subtractive hybridization (SSH) strategy was used with extraintestinal pathogenic Escherichia coli (EXPEC) that cause avian colibacillosis (avian pathogenic E. coli or APEC) and human urinary tract infections (uropathogenic E. coli or UPEC) to determine if they possessed genes that were host and/or niche specific. Both APEC and UPEC isolates were used as tester and driver strains in 4 different SSHs in order to obtain APEC- and UPEC-specific subtraction fragments (SFs). RESULTS: These procedures yielded a total of 136 tester-specific SFs of which 85 were APEC-derived and 51 were UPEC-derived. Most of the APEC-derived SFs were associated with plasmids; whereas, the majority of UPEC-derived sequences matched to the bacterial chromosome. We further determined the distribution of these tester-derived sequences in a collection of UPEC and APEC isolates using polymerase chain reaction techniques. Plasmid-borne, APEC-derived sequences (tsh, cvaB, traR, traC and sopB) were predominantly present in APEC, as compared to UPEC. Of the UPEC-derived SFs, those encoding hemolysin D and F1C major and minor fimbrial subunits were present only in UPEC. However, two UPEC-derived SFs that showed strong similarity to the uropathgenic-specific protein gene (usp) occurred in APEC, demonstrating that usp is not specific to UPEC. CONCLUSION: This study provides evidence of the genetic variability of ExPEC as well as genomic similarities between UPEC and APEC; it did not identify any single marker that would dictate host and/or niche specificity in APEC or UPEC. However, further studies on the genes that encode putative or hypothetical proteins might offer important insight into the pathogenesis of disease, as caused by these two ExPEC. [Abstract/Link to Full Text]

Embry A, Hinojosa E, Orihuela CJ
Regions of Diversity 8, 9 and 13 contribute to Streptococcus pneumoniae virulence.
BMC Microbiol. 2007;780.
BACKGROUND: Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. Previously, using comparative genomic analyses, 13 regions of genomic plasticity have been identified in the S. pneumoniae genome. These "Regions of Diversity" (RDs) accounted for half the genomic variation observed amongst all pneumococci tested, moreover, were determined to encode a variety of putative virulence factors. To date, genes within 5 RDs have been unequivocally demonstrated to contribute to S. pneumoniae virulence. It is unknown if the remaining RDs also contribute to virulence. RESULTS: Using allelic exchange, we created S. pneumoniae mutants that were deficient in RD2, 5, 7, 8, 9, 12 and 13. Mutants deficient in RD8, 9 and 13 were attenuated in a mouse model of disease. RD8 is 40,358 nucleotides in length and encodes 37 genes. Using a panel of isogenic mutants, we determined that RD8b3 is the operon within RD8 that is responsible for virulence. Mice infected with mutants deficient in RD8, RD8b3, RD9 and RD13 had significantly less bacteria in the blood two days after intranasal challenge and improved survival over time versus mice infected with wild type. In all instances mutants colonized the nasopharynx at levels equivalent to wild type. CONCLUSION: Genes within RD1, 3, 4, 6, and 10 have previously been shown to contribute to virulence. This study demonstrates that genes within RD8, 9 and 13 also contribute to virulence. The ability of mutants deficient in RD2, 5, 7, 8, 9, 12, and 13 to colonize the nasopharynx indicates that genes within these RDs are not required for asymptomatic carriage. Nonetheless, the observation that mutants deficient in RD8b3, 9 and 13 are attenuated indicates that genes within these loci are necessary for spread of the bacteria beyond the nasopharynx to normally sterile sites. [Abstract/Link to Full Text]

von Ah U, Mozzetti V, Lacroix C, Kheadr EE, Fliss I, Meile L
Classification of a moderately oxygen-tolerant isolate from baby faeces as Bifidobacterium thermophilum.
BMC Microbiol. 2007;779.
BACKGROUND: Bifidobacteria are found at varying prevalence in human microbiota and seem to play an important role in the human gastrointestinal tract (GIT). Bifidobacteria are highly adapted to the human GIT which is reflected in the genome sequence of a Bifidobacterim longum isolate. The competitiveness against other bacteria is not fully understood yet but may be related to the production of antimicrobial compounds such as bacteriocins. In a previous study, 34 Bifidobacterium isolates have been isolated from baby faeces among which six showed proteinaceous antilisterial activity against Listeria monocytogenes. In this study, one of these isolates, RBL67, was further identified and characterized. RESULTS: Bifidobacterium isolate RBL67 was classified and characterized using a polyphasic approach. RBL67 was classified as Bifidobacterium thermophilum based on phenotypic and DNA-DNA hybridization characteristics, although 16S rDNA analyses and partial groEL sequences showed higher homology with B. thermacidophilum subsp. porcinum and B. thermacidophilum subsp. thermacidophilum, respectively. RBL67 was moderately oxygen-tolerant and was able to grow at pH 4 and at a temperature of 47 degrees C. CONCLUSION: In order to assign RBL67 to a species, a polyphasic approach was used. This resulted in the classification of RBL67 as a Bifidobacterium thermophilum strain. To our knowledge, this is the first report about B. thermophilum isolated from baby faeces since the B. thermophilum strains were related to ruminants and swine faeces before. B. thermophilum was previously only isolated from animal sources and was therefore suggested to be used as differential species between animal and human contamination. Our findings may disapprove this suggestion and further studies are now conducted to determine whether B. thermophilum is distributed broader in human faeces. Furthermore, the postulated differentiation between human and animal strains by growth above 45 degrees C is no longer valid since B. thermophilum is able to grow at 47 degrees C. In our study, 16S rDNA and partial groEL sequence analysis were not able to clearly assign RBL67 to a species and were contradictory. Our study suggests that partial groEL sequences may not be reliable as a single tool for species differentiation. [Abstract/Link to Full Text]

Wiesinger-Mayr H, Vierlinger K, Pichler R, Kriegner A, Hirschl AM, Presterl E, Bodrossy L, Noehammer C
Identification of human pathogens isolated from blood using microarray hybridisation and signal pattern recognition.
BMC Microbiol. 2007;778.
BACKGROUND: Pathogen identification in clinical routine is based on the cultivation of microbes with subsequent morphological and physiological characterisation lasting at least 24 hours. However, early and accurate identification is a crucial requisite for fast and optimally targeted antimicrobial treatment. Molecular biology based techniques allow fast identification, however discrimination of very closely related species remains still difficult. RESULTS: A molecular approach is presented for the rapid identification of pathogens combining PCR amplification with microarray detection. The DNA chip comprises oligonucleotide capture probes for 25 different pathogens including Gram positive cocci, the most frequently encountered genera of Enterobacteriaceae, non-fermenter and clinical relevant Candida species. The observed detection limits varied from 10 cells (e.g. E. coli) to 10(5) cells (S. aureus) per mL artificially spiked blood. Thus the current low sensitivity for some species still represents a barrier for clinical application. Successful discrimination of closely related species was achieved by a signal pattern recognition approach based on the k-nearest-neighbour method. A prototype software providing this statistical evaluation was developed, allowing correct identification in 100 % of the cases at the genus and in 96.7 % at the species level (n = 241). CONCLUSION: The newly developed molecular assay can be carried out within 6 hours in a research laboratory from pathogen isolation to species identification. From our results we conclude that DNA microarrays can be a useful tool for rapid identification of closely related pathogens particularly when the protocols are adapted to the special clinical scenarios. [Abstract/Link to Full Text]

Abd Rahman RN, Leow TC, Salleh AB, Basri M
Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia.
BMC Microbiol. 2007;777.
BACKGROUND: Thermophilic Bacillus strains of phylogenetic Bacillus rRNA group 5 were described as a new genus Geobacillus. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus Geobacillus have a growth temperatures ranging from 35 to 78 degrees C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids. The members of Geobacillus have similarity in their 16S rRNA gene sequences (96.5-99.2%). Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification. RESULTS: Twenty-nine putative lipase producers were screened and isolated from palm oil mill effluent in Malaysia. Of these, isolate T1T was chosen for further study as relatively higher lipase activity was detected quantitatively. The crude T1 lipase showed high optimum temperature of 70 degrees C and was also stable up to 60 degrees C without significant loss of crude enzyme activity. Strain T1T was a Gram-positive, rod-shaped, endospore forming bacterium. On the basic of 16S rDNA analysis, strain T1T was shown to belong to the Bacillus rRNA group 5 related to Geobacillus thermoleovorans (DSM 5366T) and Geobacillus kaustophilus (DSM 7263T). Chemotaxonomic data of cellular fatty acids supported the affiliation of strain T1T to the genus Geobacillus. The results of physiological and biochemical tests, DNA/DNA hybridization, RiboPrint analysis, the length of lipase gene and protein pattern allowed genotypic and phenotypic differentiation of strain T1T from its validly published closest phylogenetic neighbors. Strain T1T therefore represents a novel species, for which the name Geobacillus zalihae sp. nov. is proposed, with the type strain T1T (=DSM 18318T; NBRC 101842T). CONCLUSION: Strain T1T was able to secrete extracellular thermostable lipase into culture medium. The strain T1T was identified as Geobacillus zalihae T1T as it differs from its type strains Geobacillus kaustophilus (DSM 7263T) and Geobacillus thermoleovorans (DSM 5366T) on some physiological studies, cellular fatty acids composition, RiboPrint analysis, length of lipase gene and protein profile. [Abstract/Link to Full Text]

Ali A, Hasan Z, Tanveer M, Siddiqui AR, Ghebremichael S, Kallenius G, Hasan R
Characterization of Mycobacterium tuberculosis Central Asian Strain 1 using mycobacterial interspersed repetitive unit genotyping.
BMC Microbiol. 2007;776.
BACKGROUND: The Central Asian Strain 1 (CAS1) genogroup of Mycobacterium tuberculosis (MTB) is the most prevalent in Pakistan, India and Bangladesh. Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing is a reliable and reproducible method for differentiation of MTB isolates. However, information of its utility in determining the diversity of CAS1 strain is limited. We performed standard 12 loci based MIRU-VNTR typing on previously spoligotyped CAS1 strains and 'unique' strains in order to evaluate its discriminatory power for these isolates. METHODS: Twelve loci based MIRU- VNTR typing was used to type 178 CAS1 and 189 'unique' MTB strains. The discriminatory index for each of the loci was calculated using the Hunter Gaston Discriminatory Index (HGDI). A subset of these strains (n = 78) were typed using IS6110 restriction fragment length polymorphism (RFLP). MIRU-VNTR profiles were studied together with their drug susceptibility patterns. RESULTS: A total of 349 MIRU patterns were obtained for the 367 strains tested. The CAS1 strains were subdivided into 160 distinct patterns; 15 clusters of 2 strains each, 1 cluster of four strains and 144 unique patterns. Using HGDI, seven MIRU loci, (numbers 26, 31, 27, 16, 10, 39, and 40) were found to be "highly discriminatory" (DI: >or=0.6), four MIRU loci (numbers 20, 24, 23, and 4) were "moderately discriminatory" (DI: 0.3-0.59), and one locus (number 2) was "poorly discriminatory" (DI< 0.3). Loci 26 and 31 were the most discriminatory for the CAS1 isolates. Amongst 'unique' strains in addition to loci 26, 31, 27, 16, 10, 39, and 40, locus 23 was highly discriminatory, while no locus was poorly discriminating. DI values for loci 4, 10 and 26 were significantly lower (P-value < .01) in CAS1 strains than in 'unique' strains. The association between CAS1 strains and MDR was not found to be significant (p value = 0.21). CONCLUSION: We propose that MIRU typing could be used to estimate the phylogenetic relatedness amongst prevalent CAS1 strains, for which MIRU loci 26, 31, 16, 10, 27, 39 and 40 were found to be the most discriminatory. [Abstract/Link to Full Text]

Candia N, Lopez B, Zozio T, Carrivale M, Diaz C, Russomando G, de Romero NJ, Jara JC, Barrera L, Rastogi N, Ritacco V
First insight into Mycobacterium tuberculosis genetic diversity in Paraguay.
BMC Microbiol. 2007;775.
BACKGROUND: We present a picture of the biodiversity of Mycobacterium tuberculosis in Paraguay, an inland South American country harboring 5 million inhabitants with a tuberculosis notification rate of 38/100,000. RESULTS: A total of 220 strains collected throughout the country in 2003 were classified by spoligotyping into 79 different patterns. Spoligopatterns of 173 strains matched 51 shared international types (SITs) already present in an updated version of SpolDB4, the global spoligotype database at Pasteur Institute, Guadeloupe. Our study contributed to the database 13 new SITs and 15 orphan spoligopatterns. Frequencies of major M. tuberculosis spoligotype lineages in our sample were as follows: Latin-American & Mediterranean (LAM) 52.3%, Haarlem 18.2%, S clade 9.5%, T superfamily 8.6%, X clade 0.9% and Beijing clade 0.5%. Concordant clustering by IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping identified transmission in specific settings such as the Tacumbu jail in Asuncion and aboriginal communities in the Chaco. LAM genotypes were ubiquitous and predominated among both RFLP clusters and new patterns, suggesting ongoing transmission and adaptative evolution in Paraguay. We describe a new and successfully evolving clone of the Haarlem 3 sub-lineage, SIT2643, which is thus far restricted to Paraguay. We confirmed its clonality by RFLP and mycobacterial interspersed repetitive unit (MIRU) typing; we named it "Tacumbu" after the jail where it was found to be spreading. One-fifth of the spoligopatterns in our study are rarely or never seen outside Paraguay and one-tenth do not fit within any of the major phylogenetic clades in SpolDB4. CONCLUSION: Lineages currently thriving in Paraguay may reflect local host-pathogen adaptation of strains introduced during past migrations from Europe. [Abstract/Link to Full Text]

Matsumoto K, Parola P, Rolain JM, Jeffery K, Raoult D
Detection of "Rickettsia sp. strain Uilenbergi" and "Rickettsia sp. strain Davousti" in Amblyomma tholloni ticks from elephants in Africa.
BMC Microbiol. 2007;774.
BACKGROUND: To date, 6 tick-borne rickettsiae pathogenic for humans are known to occur in Africa and 4 of them were first identified in ticks before being recognized as human pathogens. RESULTS: We examined 33 and 5 Amblyomma tholloni ticks from African elephants in the Central African Republic and Gabon, respectively, by PCR amplification and sequencing of a part of gltA and ompA genes of the genus Rickettsia. The partial sequences of gltA and ompA genes detected in tick in Gabon had 99.1% similarity with those of R. heilongjiangensis and 97.1% with those of Rickettsia sp. HL-93 strain, respectively. The partial gltA and ompA gene sequences detected in tick in the Central African Republic were 98.9% and 95.1% similar to those of Rickettsia sp. DnS14 strain and R. massiliae, respectively. Phylogenetic analysis showed Rickettsia sp. detected in Gabon clusters with R. japonica and R. heilongjiangensis in a phylogenetic tree based on the partial gltA and ompA genes. The genotype of the Rickettsia sp. detected in the Central African Republic is close to those of R. massiliae group in the phylogenetic tree based on partial gltA gene sequences, and distantly related to other rickettsiae in the tree based on partial ompA gene. CONCLUSION: The degrees of similarity of partial gltA and ompA genes with recognized species indicate the rickettsiae detected in this study may be new species although we could only study the partial sequences of 2 genes regarding the amount of DNA that was available. We propose the Rickettsia sp. detected in Gabon be provisionally named "Rickettsia sp. stain Davousti" and Rickettsia sp. detected in the Central African Republic be named "Rickettsia sp. strain Uilenbergi". [Abstract/Link to Full Text]

Martín A, Herránz M, Serrano MJ, Bouza E, de Viedma DG
Rapid clonal analysis of recurrent tuberculosis by direct MIRU-VNTR typing on stored isolates.
BMC Microbiol. 2007;773.
BACKGROUND: The application of molecular tools to the analysis of tuberculosis has revealed examples of clonal complexity, such as exogenous reinfection, coinfection, microevolution or compartmentalization. The detection of clonal heterogeneity by standard genotyping approaches is laborious and often requires expertise. This restricts the rapid availability of Mycobacterium tuberculosis (MTB) genotypes for clinical or therapeutic decision-making. A new PCR-based technique, MIRU-VNTR, has made it possible to genotype MTB in a time frame close to real-time fingerprinting. Our purpose was to evaluate the capacity of this technique to provide clinicians with a rapid discrimination between reactivation and exogenous reinfection and whether MIRU-VNTR makes it possible to obtain data directly from stored MTB isolates from recurrent episodes. RESULTS: We detected differences, between the MIRUtypes of recurrent isolates in 38.5% (5/13) of the cases studied. These included cases of i) exogenous reinfection, often with more resistant strains, ii) likely examples of microevolution, leading to the appearance of new clonal variants and iii) a combination of microevolution, coinfection and competition. CONCLUSION: MIRU-VNTR rapidly obtained clinically useful genotyping data in a challenging situation, directly from stored MTB isolates without subculturing them or purifying their DNA. Our results also mean that MIRU-VNTR could be applied for easy, rapid and affordable massive screening of collections of stored MTB isolates, which could establish the real dimension of clonal heterogeneity in MTB infection. [Abstract/Link to Full Text]

Fournier PE, Raoult D
Identification of rickettsial isolates at the species level using multi-spacer typing.
BMC Microbiol. 2007;772.
BACKGROUND: In order to estimate whether multi-spacer typing (MST), based on the sequencing of variable intergenic spacers, could serve for the identification of Rickettsia at the species level, we applied it to 108 rickettsial isolates or arthropod amplicons that include representatives of 23 valid Rickettsia species. RESULTS: MST combining the dksA-xerC, mppA-purC, and rpmE-tRNAfMet spacer sequences identified 61 genotypes, allowing the differentiation of each species by at least one distinct genotype. In addition, MST was discriminatory at the strain level in six species for which several isolates or arthropod amplicons were available. CONCLUSION: MST proved to be a reproducible and high-resolution genotyping method allowing clear identification of rickettsial isolates at the species level and further additional differentiation of strains within some species. [Abstract/Link to Full Text]

Voisard R, Krügers T, Reinhardt B, Vaida B, Baur R, Herter T, Lüske A, Weckermann D, Weingärtner K, Rössler W, Hombach V, Mertens T
HCMV-infection in a human arterial organ culture model: effects on cell proliferation and neointimal hyperplasia.
BMC Microbiol. 2007;768.
BACKGROUND: The impact of infections with the human cytomegalovirus (HCMV) for the development of atherosclerosis and restenosis is still unclear. Both a clear correlation and no correlation at all have been reported in clinical, mostly serological studies. In our study we employed a human non-injury ex vivo organ culture model to investigate the effect of an in vitro permissive HCMV-infection on cell proliferation and neointimal hyperplasia for a period of 56 days. RESULTS: During routine-nephrectomies parts of renal arteries from 71 patients were obtained and prepared as human organ cultures. Cell free HCMV infection was performed with the fibroblast adapted HCMV strain AD169, the endotheliotropic strain TB40E, and a clinical isolate (AN 365). After 3, 7, 14, 21, 28, 35, and 56 days in culture staining of HCMV-antigens was carried out and reactive cell proliferation and neointimal thickening were analysed. Successful HCMV-infection was accomplished with all three virus strains studied. During the first 21 days in organ culture no cell proliferation or neointimal hyperplasia was detected. At day 35 and day 56 moderate cell proliferation and neointimal hyperplasia was found both in HCMV-infected segments and mock infected controls. Neointimal hyperplasia in productively HCMV-infected segments was lower than in non infected at day 35 and day 56, but relatively higher after infection with the endotheliotropic TB40E in comparison with the two other strains. CONCLUSION: The data do not support the hypothesis that HCMV-infection triggers restenosis via a stimulatory effect on cell proliferation and neointimal hyperplasia in comparison to non infected controls. Interestingly however, even after lytic infection, a virus strain specific difference was observed. [Abstract/Link to Full Text]

Bertani I, Rampioni G, Leoni L, Venturi V
The Pseudomonas putida Lon protease is involved in N-acyl homoserine lactone quorum sensing regulation.
BMC Microbiol. 2007;771.
BACKGROUND: In Pseudomonas putida and Pseduomonas aeruginosa, the similar PpuR/RsaL/PpuI and LasR/RsaL/LasI acyl homoserine lactones (AHLs) quorum sensing (QS) systems have been shown to be under considerable regulation by other global regulators. A major regulator is the RsaL protein which strongly directly represses the transcription of the P. putida ppuI and P. aeruginosa lasI AHL synthases. In this study we screened a transposon mutant bank of P. putida in order to identify if any other regulators were involved in negative regulation of AHL QS. RESULTS: In our screen we identified three Tn5 mutants which displayed overproduction of AHLs in P. putida strain WCS358. Two of the mutants had a Tn5 located in the rsaL gene, whereas in one mutant the transposon was located in the lon protease gene. Lon proteases play important roles in protein quality control via degradation of misfolded proteins. It was determined that in the P. putida lon mutant, AHL levels, PpuR levels and ppuI promoter activity all increased significantly; we therefore postulated that PpuR is a target for Lon. The Lon protease had no effect on AHL production in P. aeruginosa. CONCLUSION: The Lon protease is a negative regulator of AHL production in P. putida WCS358. The Lon protease has also been shown by others to influence AHL QS in Vibrio fischeri and Agrobacterium tumefaciens and can thus become an important regulator of AHL QS timing and regulation in bacteria. [Abstract/Link to Full Text]

Jonas K, Tomenius H, Kader A, Normark S, Römling U, Belova LM, Melefors O
Roles of curli, cellulose and BapA in Salmonella biofilm morphology studied by atomic force microscopy.
BMC Microbiol. 2007;770.
BACKGROUND: Curli, cellulose and the cell surface protein BapA are matrix components in Salmonella biofilms. In this study we have investigated the roles of these components for the morphology of bacteria grown as colonies on agar plates and within a biofilm on submerged mica surfaces by applying atomic force microscopy (AFM) and light microscopy. RESULTS: AFM imaging was performed on colonies of Salmonella Typhimurium grown on agar plates for 24 h and on biofilms grown for 4, 8, 16 or 24 h on mica slides submerged in standing cultures. Our data show that in the wild type curli were visible as extracellular material on and between the cells and as fimbrial structures at the edges of biofilms grown for 16 h and 24 h. In contrast to the wild type, which formed a three-dimensional biofilm within 24 h, a curli mutant and a strain mutated in the global regulator CsgD were severely impaired in biofilm formation. A mutant in cellulose production retained some capability to form cell aggregates, but not a confluent biofilm. Extracellular matrix was observed in this mutant to almost the same extent as in the wild type. Overexpression of CsgD led to a much thicker and a more rapidly growing biofilm. Disruption of BapA altered neither colony and biofilm morphology nor the ability to form a biofilm within 24 h on the submerged surfaces. Besides curli, the expression of flagella and pili as well as changes in cell shape and cell size could be monitored in the growing biofilms. CONCLUSION: Our work demonstrates that atomic force microscopy can efficiently be used as a tool to monitor the morphology of bacteria grown as colonies on agar plates or within biofilms formed in a liquid at high resolution. [Abstract/Link to Full Text]

Yoshimura M, Oshima T, Ogasawara N
Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both Bacillus subtilis and Escherichia coli.
BMC Microbiol. 2007;769.
BACKGROUND: Phospholipid biosynthesis commences with the acylation of glycerol-3-phosphate (G3P) to form 1-acyl-G3P. This step is catalyzed by the PlsB protein in Escherichia coli. The gene encoding this protein has not been identified, however, in the majority of bacterial genome sequences, including that of Bacillus subtilis. Recently, a new two-step pathway catalyzed by PlsX and PlsY proteins for the initiation of phospholipid formation in Streptococcus pneumoniae has been reported. RESULTS: In B. subtilis, 271 genes have been reported to be indispensable, when inactivated singly, for growth in LB medium. Among these, 11 genes encode proteins with unknown functions. As part of a genetic study to identify the functions of these genes, we show here that the B. subtilis ortholog of S. pneumoniae PlsY, YneS, is required for G3P acyltransferase activity, together with PlsX. The B. subtilis genome lacks plsB, and we show in vivo that the PlsX/Y pathway is indeed essential for the growth of bacteria lacking plsB. Interestingly, in addition to plsB, E. coli possesses plsX and the plsY ortholog, ygiH. We therefore explored the functional relationship between PlsB, PlsX and YgiH in E. coli, and found that plsB is essential for E. coli growth, indicating that PlsB plays an important role in 1-acyl-G3P synthesis in E. coli. We also found, however, that the simultaneous inactivation of plsX and ygiH was impossible, revealing important roles for PlsX and YgiH in E. coli growth. CONCLUSION: Both plsX and yneS are essential for 1-acyl-G3P synthesis in B. subtilis, in agreement with recent reports on their biochemical functions. In E. coli, PlsB plays a principal role in 1-acyl-G3P synthesis and is also essential for bacterial growth. PlsX and YgiH also, however, play important roles in E. coli growth, possibly by regulating the intracellular concentration of acyl-ACP. These proteins are therefore important targets for development of new antibacterial agents. [Abstract/Link to Full Text]

Piekarowicz A, K?yz A, Majchrzak M, Adamczyk-Pop?awska M, Maugel TK, Stein DC
Characterization of the dsDNA prophage sequences in the genome of Neisseria gonorrhoeae and visualization of productive bacteriophage.
BMC Microbiol. 2007;766.
BACKGROUND: Bioinformatic analysis of the genome sequence of Neisseria gonorrhoeae revealed the presence of nine probable prophage islands. The distribution, conservation and function of many of these sequences, and their ability to produce bacteriophage particles are unknown. RESULTS: Our analysis of the genomic sequence of FA1090 identified five genomic regions (NgoPhi1 - 5) that are related to dsDNA lysogenic phage. The genetic content of the dsDNA prophage sequences were examined in detail and found to contain blocks of genes encoding for proteins homologous to proteins responsible for phage DNA replication, structural proteins and proteins responsible for phage assembly. The DNA sequences from NgoPhi1, NgoPhi2 and NgoPhi3 contain some significant regions of identity. A unique region of NgoPhi2 showed very high similarity with the Pseudomonas aeruginosa generalized transducing phage F116. Comparative analysis at the nucleotide and protein levels suggests that the sequences of NgoPhi1 and NgoPhi2 encode functionally active phages, while NgoPhi3, NgoPhi4 and NgoPhi5 encode incomplete genomes. Expression of the NgoPhi1 and NgoPhi2 repressors in Escherichia coli inhibit the growth of E. coli and the propagation of phage lambda. The NgoPhi2 repressor was able to inhibit transcription of N. gonorrhoeae genes and Haemophilus influenzae HP1 phage promoters. The holin gene of NgoPhi1 (identical to that encoded by NgoPhi2), when expressed in E. coli, could serve as substitute for the phage lambda s gene. We were able to detect the presence of the DNA derived from NgoPhi1 in the cultures of N. gonorrhoeae. Electron microscopy analysis of culture supernatants revealed the presence of multiple forms of bacteriophage particles. CONCLUSION: These data suggest that the genes similar to dsDNA lysogenic phage present in the gonococcus are generally conserved in this pathogen and that they are able to regulate the expression of other neisserial genes. Since phage particles were only present in culture supernatants after induction with mitomycin C, it indicates that the gonococcus also regulates the expression of bacteriophage genes. [Abstract/Link to Full Text]

Dittmann S, Schmid A, Richter S, Trülzsch K, Heesemann J, Wilharm G
The Yersinia enterocolitica type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1.
BMC Microbiol. 2007;767.
BACKGROUND: Pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) share a virulence plasmid encoding a type three secretion system (T3SS). This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins), the specific Yop chaperones (Sycs), and the Ysc (Yop secretion) proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study. RESULTS: We have established the large-scale production of recombinant SycO in its outright form. We confirm that Y. enterocolitica SycO forms homodimers which is typical for Syc chaperones. SycO overproduction in Y. enterocolitica decreases secretion of Yops into the culture supernatant suggesting a regulatory role of SycO in type III secretion. We demonstrate that in vitro SycO interacts with YscM1, a negative regulator of Yop expression in Y. enterocolitica. However, the SycO overproduction phenotype was not mediated by YscM1, YscM2, YopO or YopP as revealed by analysis of isogenic deletion mutants. CONCLUSION: We present evidence that SycO is integrated into the regulatory network of the Yersinia T3SS. Our picture of the Yersinia T3SS interactome is supplemented by identification of the SycO/YscM1 interaction. Further, our results suggest that at least one additional interaction partner of SycO has to be identified. [Abstract/Link to Full Text]

Bullard B, Lipski S, Lafontaine ER
Regions important for the adhesin activity of Moraxella catarrhalis Hag.
BMC Microbiol. 2007;765.
BACKGROUND: The Moraxella catarrhalis Hag protein, an Oca autotransporter adhesin, has previously been shown to be important for adherence of this respiratory tract pathogen to human middle ear and A549 lung cells. RESULTS: The present study demonstrates that adherence of M. catarrhalis isogenic hag mutant strains to the human epithelial cell lines Chang (conjunctival) and NCIH292 (lung) is reduced by 50-93%. Furthermore, expressing Hag in a heterologous Escherichia coli background substantially increased the adherence of recombinant bacteria to NCIH292 cells and murine type IV collagen. Hag did not, however, increase the attachment of E. coli to Chang cells. These results indicate that Hag directly mediates adherence to NCIH292 lung cells and collagen, but is not sufficient to confer binding to conjunctival monolayers. Several in-frame deletions were engineered within the hag gene of M. catarrhalis strain O35E and the resulting proteins were tested for their ability to mediate binding to NCIH292 monolayers, middle ear cells, and type IV collagen. These experiments revealed that epithelial cell and collagen binding properties are separable, and that residues 385-705 of this ~2,000 amino acid protein are important for adherence to middle ear and NCIH292 cells. The region of O35E-Hag encompassing aa 706 to 1194 was also found to be required for adherence to collagen. In contrast, beta-roll repeats present in Hag, which are structural features conserved in several Oca adhesins and responsible for the adhesive properties of Yersinia enterocolitica YadA, are not important for Hag-mediated adherence. CONCLUSION: Hag is a major adherence factor for human cells derived from various anatomical sites relevant to pathogenesis by M. catarrhalis and its structure-function relationships differ from those of other, closely-related autotransporter proteins. [Abstract/Link to Full Text]

Mundodi V, Kucknoor AS, Alderete JF
Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis.
BMC Microbiol. 2007;764.
BACKGROUND: Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach. RESULTS: We successfully select stable trichomonads with sense (S) and antisense (AS) plasmids. RT-PCR confirmed decreased amounts of ap33 mRNA in AS-transfected parasites, and decreased amounts of AP33 had no effect on growth and viability when compared to wild-type (wt) trichomonads. Immunoblots of proteins from AS-transfectants gave significant decreased amounts of functional AP33 capable of binding to host cells compared to wt- and S-transfected trichomonads. As expected, AS-transfectants had lower levels of adherence to VECs, which was related to reduction in surface expression of AP33. Stable expression of T. vaginalis AP33::HA fusion in T. foetus was confirmed by immunoblots and fluorescence. The episomally-expressed surface AP33::HA fusion increased adherence of trichomonads to human VECs, which was abrogated with anti-AP33 serum. CONCLUSION: These results using both antisense inhibition of gene expression and AP33 synthesis and the heterologous expression of AP33 in T. foetus confirms a role for this protein as an adhesin in T. vaginalis. [Abstract/Link to Full Text]

Su HC, Hutchison CA, Giddings MC
Mapping phosphoproteins in Mycoplasma genitalium and Mycoplasma pneumoniae.
BMC Microbiol. 2007;763.
BACKGROUND: Little is known regarding the extent or targets of phosphorylation in mycoplasmas, yet in many other bacterial species phosphorylation is known to play an important role in signaling and regulation of cellular processes. To determine the prevalence of phosphorylation in mycoplasmas, we examined the CHAPS-soluble protein fractions of Mycoplasma genitalium and Mycoplasma pneumoniae by two-dimensional gel electrophoresis (2-DE), using a combination of Pro-Q Diamond phosphoprotein stain and 33P labeling. Protein spots that were positive for phosphorylation were identified by peptide mass fingerprinting using MALDI-TOF-TOF mass spectrometry. RESULTS: We identified a total of 24 distinct phosphoproteins, about 3% and 5% of the total protein complement in M. pneumoniae and M. genitalium, respectively, indicating that phosphorylation occurs with prevalence similar to many other bacterial species. Identified phosphoproteins include pyruvate dehydrogenase E1 alpha and beta subunits, enolase, heat shock proteins DnaK and GroEL, elongation factor Tu, cytadherence accessory protein HMW3, P65, and several hypothetical proteins. These proteins are involved in energy metabolism, carbohydrate metabolism, translation/transcription and cytadherence. Interestingly, fourteen of the 24 phosphoproteins we identified (58%) were previously reported as putatively associated with a cytoskeleton-like structure that is present in the mycoplasmas, indicating a potential regulatory role for phosphorylation in this structure. CONCLUSION: This study has shown that phosphorylation in mycoplasmas is comparable to that of other bacterial species. Our evidence supports a link between phosphorylation and cytadherence and/or a cytoskeleton-like structure, since over half of the proteins identified as phosphorylated have been previously associated with these functions. This opens the door to further research into the purposes and mechanisms of phosphorylation for mycoplasmas. [Abstract/Link to Full Text]

Ender M, Berger-Bächi B, McCallum N
Variability in SCCmecN1 spreading among injection drug users in Zurich, Switzerland.
BMC Microbiol. 2007;762.
BACKGROUND: An extremely low level methicillin resistant Staphylococcus aureus (MRSA) belonging to ST45, circulates among intravenous drug users in the Zurich area. This clone can be misinterpreted as an MSSA by phenotypic oxacillin resistance tests, although it carries a staphylococcal cassette chromosome mec (SCCmec) element encoding a functional mecA gene and it produces PBP2a. RESULTS: This clone carried a new 45.7-kb element, termed SCCmecN1, containing a class B mec complex (mecA-DeltamecR1::IS1272), a truncated Tn4003 harbouring the dfrA gene, and a fusB1 gene, conferring methicillin, trimethoprim and low level fusidic acid resistance, respectively. In addition to the two insertion site sequences (ISS) framing the SCCmec, a third ISS (ISS*) was identified within the element. SCCmecN1 also harboured two distinct ccrAB complexes belonging to the class 4 subtype, both of which were shown to be active and to be able to excise the SCCmecN1 or parts thereof. Slight variations in the SmaI-PFGE pattern of the clinical MRSA isolates belonging to this clone were traced back to differences in the sizes of the SCCmec J2 regions and/or to a 6.4-kb deletion extending from ISS* to the right end ISS. This latter deletion led to a variant right SCCmec-chromosomal junction site. MRSA clones carrying the shorter SCCmec with the 6.4-kb deletion were usually ciprofloxacin resistant, while strains with the complete SCCmecN1 were co-trimoxazole resistant or had no additional resistances. This suggested that the genetic backbone of the host S. aureus, although identical by PFGE pattern, had at some stage diverged with one branch acquiring a sulfonomide resistance mutation and the other ciprofloxacin resistance. CONCLUSION: This description of the structure and variations of SCCmecN1 will allow for quicker and easier molecular detection of this clone and monitoring of its spread. [Abstract/Link to Full Text]

Desjardins CA, Gundersen-Rindal DE, Hostetler JB, Tallon LJ, Fuester RW, Schatz MC, Pedroni MJ, Fadrosh DW, Haas BJ, Toms BS, Chen D, Nene V
Structure and evolution of a proviral locus of Glyptapanteles indiensis bracovirus.
BMC Microbiol. 2007;761.
BACKGROUND: Bracoviruses (BVs), a group of double-stranded DNA viruses with segmented genomes, are mutualistic endosymbionts of parasitoid wasps. Virus particles are replication deficient and are produced only by female wasps from proviral sequences integrated into the wasp genome. Virus particles are injected along with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and therefore perpetuation of proviral DNA. Here we describe a 223 kbp region of Glyptapanteles indiensis genomic DNA which contains a part of the G. indiensis bracovirus (GiBV) proviral genome. RESULTS: Eighteen of ~24 GiBV viral segment sequences are encoded by 7 non-overlapping sets of BAC clones, revealing that some proviral segment sequences are separated by long stretches of intervening DNA. Two overlapping BACs, which contain a locus of 8 tandemly arrayed proviral segments flanked on either side by ~35 kbp of non-packaged DNA, were sequenced and annotated. Structural and compositional analyses of this cluster revealed it exhibits a G+C and nucleotide composition distinct from the flanking DNA. By analyzing sequence polymorphisms in the 8 GiBV viral segment sequences, we found evidence for widespread selection acting on both protein-coding and non-coding DNA. Comparative analysis of viral and proviral segment sequences revealed a sequence motif involved in the excision of proviral genome segments which is highly conserved in two other bracoviruses. CONCLUSION: Contrary to current concepts of bracovirus proviral genome organization our results demonstrate that some but not all GiBV proviral segment sequences exist in a tandem array. Unexpectedly, non-coding DNA in the 8 proviral genome segments which typically occupies ~70% of BV viral genomes is under selection pressure suggesting it serves some function(s). We hypothesize that selection acting on GiBV proviral sequences maintains the genetic island-like nature of the cluster of proviral genome segments described herein. In contrast to large differences in the predicted gene composition of BV genomes, sequences that appear to mediate processes of viral segment formation, such as proviral segment excision and circularization, appear to be highly conserved, supporting the hypothesis of a single origin for BVs. [Abstract/Link to Full Text]

Chaudhuri B, Rojek J, Vickerman MM, Tanzer JM, Scannapieco FA
Interaction of salivary alpha-amylase and amylase-binding-protein A (AbpA) of Streptococcus gordonii with glucosyltransferase of S. gordonii and Streptococcus mutans.
BMC Microbiol. 2007;760.
BACKGROUND: Glucosyltransferases (Gtfs), enzymes that produce extracellular glucans from dietary sucrose, contribute to dental plaque formation by Streptococcus gordonii and Streptococcus mutans. The alpha-amylase-binding protein A (AbpA) of S. gordonii, an early colonizing bacterium in dental plaque, interacts with salivary amylase and may influence dental plaque formation by this organism. We examined the interaction of amylase and recombinant AbpA (rAbpA), together with Gtfs of S. gordonii and S. mutans. RESULTS: The addition of salivary alpha-amylase to culture supernatants of S. gordonii precipitated a protein complex containing amylase, AbpA, amylase-binding protein B (AbpB), and the glucosyltransferase produced by S. gordonii (Gtf-G). rAbpA was expressed from an inducible plasmid, purified from Escherichia coli and characterized. Purified rAbpA, along with purified amylase, interacted with and precipitated Gtfs from culture supernatants of both S. gordonii and S. mutans. The presence of amylase and/or rAbpA increased both the sucrase and transferase component activities of S. mutans Gtf-B. Enzyme-linked immunosorbent assay (ELISA) using anti-Gtf-B antibody verified the interaction of rAbpA and amylase with Gtf-B. A S. gordonii abpA-deficient mutant showed greater biofilm growth under static conditions than wild-type in the presence of sucrose. Interestingly, biofilm formation by every strain was inhibited in the presence of saliva. CONCLUSION: The results suggest that an extracellular protein network of AbpA-amylase-Gtf may influence the ecology of oral biofilms, likely during initial phases of colonization. [Abstract/Link to Full Text]

Bonhomme CJ, Nappez C, Raoult D
Microarray for serotyping of Bartonella species.
BMC Microbiol. 2007;759.
BACKGROUND: Bacteria of the genus Bartonella are responsible for a large variety of human and animal diseases. Serological typing of Bartonella is a method that can be used for differentiation and identification of Bartonella subspecies. RESULTS: We have developed a novel multiple antigenic microarray to serotype Bartonella strains and to select poly and monoclonal antibodies. It was validated using mouse polyclonal antibodies against 29 Bartonella strains. We then tested the microarray for serotyping of Bartonella strains and defining the profile of monoclonal antibodies.Bartonella strains gave a strong positive signal and all were correctly identified. Screening of monoclonal antibodies towards the Gro EL protein of B. clarridgeiae identified 3 groups of antibodies, which were observed with variable affinities against Bartonella strains. CONCLUSION: We demonstrated that microarray of spotted bacteria can be a practical tool for serotyping of unidentified strains or species (and also for affinity determination) by polyclonal and monoclonal antibodies. This could be used in research and for identification of bacterial strains. [Abstract/Link to Full Text]

Lima-Bittencourt CI, Astolfi-Filho S, Chartone-Souza E, Santos FR, Nascimento AM
Analysis of Chromobacterium sp. natural isolates from different Brazilian ecosystems.
BMC Microbiol. 2007;758.
BACKGROUND: Chromobacterium violaceum is a free-living bacterium able to survive under diverse environmental conditions. In this study we evaluate the genetic and physiological diversity of Chromobacterium sp. isolates from three Brazilian ecosystems: Brazilian Savannah (Cerrado), Atlantic Rain Forest and Amazon Rain Forest. We have analyzed the diversity with molecular approaches (16S rRNA gene sequences and amplified ribosomal DNA restriction analysis) and phenotypic surveys of antibiotic resistance and biochemistry profiles. RESULTS: In general, the clusters based on physiological profiles included isolates from two or more geographical locations indicating that they are not restricted to a single ecosystem. The isolates from Brazilian Savannah presented greater physiologic diversity and their biochemical profile was the most variable of all groupings. The isolates recovered from Amazon and Atlantic Rain Forests presented the most similar biochemical characteristics to the Chromobacterium violaceum ATCC 12472 strain. Clusters based on biochemical profiles were congruent with clusters obtained by the 16S rRNA gene tree. According to the phylogenetic analyses, isolates from the Amazon Rain Forest and Savannah displayed a closer relationship to the Chromobacterium violaceum ATCC 12472. Furthermore, 16S rRNA gene tree revealed a good correlation between phylogenetic clustering and geographic origin. CONCLUSION: The physiological analyses clearly demonstrate the high biochemical versatility found in the C. violaceum genome and molecular methods allowed to detect the intra and inter-population diversity of isolates from three Brazilian ecosystems. [Abstract/Link to Full Text]

Bandara AB, Contreras A, Contreras-Rodriguez A, Martins AM, Dobrean V, Poff-Reichow S, Rajasekaran P, Sriranganathan N, Schurig GG, Boyle SM
Brucella suis urease encoded by ure1 but not ure2 is necessary for intestinal infection of BALB/c mice.
BMC Microbiol. 2007;757.
BACKGROUND: In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. RESULTS: The deduced amino acid sequence of urease-alpha subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Deltaure1K (generated by deleting ureD and ureA in ure1 operon), strain 1330Deltaure2K (ureB and ureC in ure2 operon), strain 1330Deltaure2C (ureA, ureB, and ureC in ure2 operon), and strain 1330Deltaure1KDeltaure2C (ureD and ureA in ure1 operon and ureA, ureB, and ureC in ure2 operon). When grown in urease test broth, strains 1330, 1330Deltaure2K and 1330Deltaure2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Deltaure1K and 1330Deltaure1KDeltaure2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Deltaure1K and 1330Deltaure1KDeltaure2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Deltaure1KDeltaure2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Deltaure1K was completely killed, strain 1330Deltaure2C was partially killed, but strains 1330 and 1330Deltaure2K were not killed. CONCLUSION: These findings suggest that the ure1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure2 operon apparently enhances the resistance to low-pH killing in-vitro. [Abstract/Link to Full Text]

Buchinsky FJ, Forbes ML, Hayes JD, Shen K, Ezzo S, Compliment J, Hogg J, Hiller NL, Hu FZ, Post JC, Ehrlich GD
Virulence phenotypes of low-passage clinical isolates of nontypeable Haemophilus influenzae assessed using the chinchilla laniger model of otitis media.
BMC Microbiol. 2007;756.
BACKGROUND: The nontypeable Haemophilus influenzae (NTHi) are associated with a spectrum of respiratory mucosal infections including: acute otitis media (AOM); chronic otitis media with effusion (COME); otorrhea; locally invasive diseases such as mastoiditis; as well as a range of systemic disease states, suggesting a wide range of virulence phenotypes. Genomic studies have demonstrated that each clinical strain contains a unique genic distribution from a population-based supragenome, the distributed genome hypothesis. These diverse clinical and genotypic findings suggest that each NTHi strain possesses a unique set of virulence factors that contributes to the course of the disease. RESULTS: The local and systemic virulence patterns of ten genomically characterized low-passage clinical NTHi strains (PittAA - PittJJ) obtained from children with COME or otorrhea were stratified using the chinchilla model of otitis media (OM). Each isolate was used to bilaterally inoculate six animals and thereafter clinical assessments were carried out daily for 8 days by blinded observers. There was no statistical difference in the time it took for any of the 10 NTHi strains to induce otologic (local) disease with respect to any or all of the other strains, however the differences in time to maximal local disease and the severity of local disease were both significant between the strains. Parameters of systemic disease indicated that the strains were not all equivalent: time to development of the systemic disease, maximal systemic scores and mortality were all statistically different among the strains. PittGG induced 100% mortality while PittBB, PittCC, and PittEE produced no mortality. Overall Pitt GG, PittII, and Pitt FF produced the most rapid and most severe local and systemic disease. A post hoc determination of the clinical origins of the 10 NTHi strains revealed that these three strains were of otorrheic origin, whereas the other 7 were from patients with COME. CONCLUSION: Collectively these data suggest that the chinchilla OM model is useful for discriminating between otorrheic and COME NTHi strains as to their disease-producing potential in humans, and combined with whole genome analyses, point the way towards identifying classes of virulence genes. [Abstract/Link to Full Text]

Bo Andersen J, Roldgaard BB, Christensen BB, Licht TR
Oxygen restriction increases the infective potential of Listeria monocytogenes in vitro in Caco-2 cells and in vivo in guinea pigs.
BMC Microbiol. 2007;755.
BACKGROUND: Listeria monocytogenes has been implicated in several food borne outbreaks as well as sporadic cases of disease. Increased understanding of the biology of this organism is important in the prevention of food borne listeriosis.The infectivity of Listeria monocytogenes ScottA, cultivated with and without oxygen restriction, was compared in vitro and in vivo. Fluorescent protein labels were applied to allow certain identification of Listeria cells from untagged bacteria in in vivo samples, and to distinguish between cells grown under different conditions in mixed infection experiments. RESULTS: Infection of Caco-2 cells revealed that Listeria cultivated under oxygen-restricted conditions were approximately 100 fold more invasive than similar cultures grown without oxygen restriction. This was observed for exponentially growing bacteria, as well as for stationary-phase cultures.Oral dosage of guinea pigs with Listeria resulted in a significantly higher prevalence (p < 0.05) of these bacteria in jejunum, liver and spleen four and seven days after challenge, when the bacterial cultures had been grown under oxygen-restricted conditions prior to dosage. Additionally, a 10-100 fold higher concentration of Listeria in fecal samples was observed after dosage with oxygen-restricted bacteria. These differences were seen after challenge with single Listeria cultures, as well as with a mixture of two cultures grown with and without oxygen restriction. CONCLUSION: Our results show for the first time that the environmental conditions to which L. monocytogenes is exposed prior to ingestion are decisive for its in vivo infective potential in the gastrointestinal tract after passage of the gastric barrier. This is highly relevant for safety assessment of this organism in food. [Abstract/Link to Full Text]

Kivi M, Rodin S, Kupershmidt I, Lundin A, Tindberg Y, Granström M, Engstrand L
Helicobacter pylori genome variability in a framework of familial transmission.
BMC Microbiol. 2007;754.
BACKGROUND: Helicobacter pylori infection is exceptionally prevalent and is considered to be acquired primarily early in life through person-to-person transmission within the family. H. pylori is a genetically diverse bacterial species, which may facilitate adaptation to new hosts and persistence for decades. The present study aimed to explore the genetic diversity of clonal isolates from a mother and her three children in order to shed light on H. pylori transmission and host adaptation. RESULTS: Two different H. pylori strains and strain variants were identified in the family members by PCR-based molecular typing and sequencing of five loci. Genome diversity was further assessed for 15 isolates by comparative microarray hybridizations. The microarray consisted of 1,745 oligonucleotides representing the genes of two previously sequenced H. pylori strains. The microarray analysis detected a limited mean number (+/- standard error) of divergent genes between clonal isolates from the same and different individuals (1 +/- 0.4, 0.1%, and 3 +/- 0.3, 0.2%, respectively). There was considerable variability between the two different strains in the family members (147 +/- 4, 8%) and for all isolates relative to the two sequenced reference strains (314 +/- 16, 18%). The diversity between different strains was associated with gene functional classes related to DNA metabolism and the cell envelope. CONCLUSION: The present data from clonal H. pylori isolates of family members do not support that transmission and host adaptation are associated with substantial sequence diversity in the bacterial genome. However, important phenotypic modifications may be determined by additional genetic mechanisms, such as phase-variation. Our findings can aid further exploration of H. pylori genetic diversity and adaptation. [Abstract/Link to Full Text]

Gutierrez-Ríos RM, Freyre-Gonzalez JA, Resendis O, Collado-Vides J, Saier M, Gosset G
Identification of regulatory network topological units coordinating the genome-wide transcriptional response to glucose in Escherichia coli.
BMC Microbiol. 2007;753.
BACKGROUND: Glucose is the preferred carbon and energy source for Escherichia coli. A complex regulatory network coordinates gene expression, transport and enzyme activities in response to the presence of this sugar. To determine the extent of the cellular response to glucose, we applied an approach combining global transcriptome and regulatory network analyses. RESULTS: Transcriptome data from isogenic wild type and crp- strains grown in Luria-Bertani medium (LB) or LB + 4 g/L glucose (LB+G) were analyzed to identify differentially transcribed genes. We detected 180 and 200 genes displaying increased and reduced relative transcript levels in the presence of glucose, respectively. The observed expression pattern in LB was consistent with a gluconeogenic metabolic state including active transport and interconversion of small molecules and macromolecules, induction of protease-encoding genes and a partial heat shock response. In LB+G, catabolic repression was detected for transport and metabolic interconversion activities. We also detected an increased capacity for de novo synthesis of nucleotides, amino acids and proteins. Cluster analysis of a subset of genes revealed that CRP mediates catabolite repression for most of the genes displaying reduced transcript levels in LB+G, whereas Fis participates in the upregulation of genes under this condition. An analysis of the regulatory network, in terms of topological functional units, revealed 8 interconnected modules which again exposed the importance of Fis and CRP as directly responsible for the coordinated response of the cell. This effect was also seen with other not extensively connected transcription factors such as FruR and PdhR, which showed a consistent response considering media composition. CONCLUSION: This work allowed the identification of eight interconnected regulatory network modules that includes CRP, Fis and other transcriptional factors that respond directly or indirectly to the presence of glucose. In most cases, each of these modules includes genes encoding physiologically related functions, thus indicating a connection between regulatory network topology and related cellular functions involved in nutrient sensing and metabolism. [Abstract/Link to Full Text]


Recent Articles in Clinical and Diagnostic Laboratory Immunology

Han SJ, Jeong SY, Nam YJ, Yang KH, Lim HS, Chung J
Xylitol inhibits inflammatory cytokine expression induced by lipopolysaccharide from Porphyromonas gingivalis.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1285-91.
Porphyromonas gingivalis is one of the suspected periodontopathic bacteria. The lipopolysaccharide (LPS) of P. gingivalis is a key factor in the development of periodontitis. Inflammatory cytokines play important roles in the gingival tissue destruction that is a characteristic of periodontitis. Macrophages are prominent at chronic inflammatory sites and are considered to contribute to the pathogenesis of periodontitis. Xylitol stands out and is widely believed to possess anticaries properties. However, to date, little is known about the effect of xylitol on periodontitis. The aim of the present study was to determine tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) expression when RAW 264.7 cells were stimulated with P. gingivalis LPS (hereafter, LPS refers to P. gingivalis LPS unless stated otherwise) and the effect of xylitol on the LPS-induced TNF-alpha and IL-1beta expression. The kinetics of TNF-alpha and IL-1beta levels in culture supernatant after LPS treatment showed peak values at 1 h (TNF-alpha) and 2 to 4 h (IL-1beta), respectively. NF-kappaB, a transcription factor, was also activated by LPS treatment. These cytokine expressions and NF-kappaB activation were suppressed by pretreatment with pyrrolidine dithiocarbamate (an inhibitor of NF-kappaB). Pretreatment with xylitol inhibited LPS-induced TNF-alpha and IL-1beta gene expression and protein synthesis. LPS-induced mobilization of NF-kappaB was also inhibited by pretreatment with xylitol in a dose-dependent manner. Xylitol also showed inhibitory effect on the growth of P. gingivalis. Taken together, these findings suggest that xylitol may have good clinical effect not only for caries but also for periodontitis by its inhibitory effect on the LPS-induced inflammatory cytokine expression. [Abstract/Link to Full Text]

Okuda M, Sugiyama T, Fukunaga K, Kondou M, Miyashiro E, Nakazawa T
A strain-specific antigen in Japanese Helicobacter pylori recognized in sera of Japanese children.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1280-4.
An enzyme immuno assay (EIA) test based on Japanese strain-derived high-molecular-weight cell-associated proteins (JHM-CAP) was evaluated by comparing with a previously developed EIA test based on a U.S. strain-derived high-molecular-weight cell-associated proteins (HM-CAP). Serum samples of 131 Japanese asymptomatic children (mean age, 5.5 years; range, 0 to 21 years) were tested that include 43 positive and 88 negative children as judged by Helicobacter pylori stool antigen test (HpSA test). Both tests showed comparable and reliable specificities, but the sensitivity of JHM-CAP EIA, at 93.0%, was much higher than that of HM-CAP EIA, at 67.4%. More false-negative results of HM-CAP were obtained in children under 10 years of age. Immunoblot analysis revealed that the JHM-CAP but not the HM-CAP preparation had a 100-kDa antigen recognized by JHM-CAP positive sera. It was concluded that JHM-CAP EIA is highly accurate for the serodiagnosis of H. pylori infection in Japanese young children and that the high sensitivity of JHM-CAP EIA in contrast to HM-CAP EIA is due to the presence of a 100-kDa antigen in Japanese strains that may be recognized by the host immune system at an early stage of infection. [Abstract/Link to Full Text]

Jiang Y, Shang H, Zhang Z, Diao Y, Dai D, Geng W, Zhang M, Han X, Wang Y, Liu J
Alterations of natural killer cell and T-lymphocyte counts in adults infected with human immunodeficiency virus through blood and plasma sold in the past in China and in whom infection has progressed slowly over a long period.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1275-9.
Natural killer (NK) cells, natural killer T (NKT) cells, and T lymphocytes were analyzed by using a flow cytometer in 225 human immunodeficiency virus (HIV)-positive individuals infected through the past sale of blood and plasma without receiving antiretroviral therapy in the People's Republic of China. According to CD4 T-cell counts these HIV-infected adults were stratified into three groups: long-term slow progressors, HIV-infected subjects, and AIDS patients. NK cell counts in long-term slow progressors were higher compared to HIV infection and AIDS patients (P < 0.05) and lower compared to normal controls (P < 0.05), whereas NKT cell counts in slow progressors and the HIV infection group were not different from those of normal controls. NK cell counts in HIV-seropositive subjects were positively correlated with CD4 T-cell counts (P < 0.05), and NKT cell counts were positively correlated with CD4 T-cell and CD8 T-cell counts (P < 0.05). The CD8 T-cell counts were higher in slow progressors compared to those with HIV infection, AIDS patients, and normal controls. These results indicated that HIV infection causes alterations of NK cells and T cells in slow progressors, HIV-infected subjects, and AIDS patient groups, but no difference was found in NKT cell counts and percentages in slow progressors and the HIV-infected group compared to normal controls. [Abstract/Link to Full Text]

Rawlins ML, Gerstner C, Hill HR, Litwin CM
Evaluation of a western blot method for the detection of Yersinia antibodies: evidence of serological cross-reactivity between Yersinia outer membrane proteins and Borrelia burgdorferi.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1269-74.
Yersinia enterocolitica and Yersinia pseudotuberculosis have been identified as causative organisms of reactive arthritis in humans. We evaluated a Western blot assay which uses Yersinia outer membrane proteins as antigens for the detection of Yersinia antibodies as a replacement for the complement fixation (CF) assay. Clinical agreement, sensitivity, and specificity were determined by testing 19 positive and 21 negative serum samples by the CF assay, Western blot assay, and enzyme-linked immunosorbent assay (ELISA). The CF assay and ELISA were compared to the Western blot assay, which was the reference method used in this study. Sera with antibodies that could potentially cross-react with Yersinia were also tested by the Western blot assay. The agreement, sensitivity, and specificity of the CF method were 61%, 26%, and 95%, respectively; and those for the ELISA were 89%, 95%, and 82%, respectively. The prevalences of Yersinia antibodies in 50 healthy donors were 6% for immunoglobulin G (IgG), 2% for IgA, and 2% for IgM. Sera positive for Bartonella henselae, Brucella, Chlamydia pneumoniae, and Rickettsia rickettsii antibodies showed cross-reactivity by the Western blot assay. The highest cross-reactivity was observed with Borrelia burgdorferi; 5 of 11 (45%) specimens were cross-reactive by the IgM-specific assay. Overall, the Western blot assay performs acceptably and is more sensitive than the CF assay, warranting replacement of the CF assay in the laboratory. Due to the evidence of cross-reactivity, particularly with B. burgdorferi, which can cause an oligoarthritis similar to reactive arthritis, the diagnosis of reactive arthritis should be based on clinical findings and complete serologic analysis of the potential causative infectious pathogens. [Abstract/Link to Full Text]

Devenish J, Brooks B, Perry K, Milnes D, Burke T, McCabe D, Duff S, Lutze-Wallace CL
Validation of a monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of Campylobacter fetus.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1261-8.
A monoclonal antibody (MAb)-based antigen capture enzyme-linked immunosorbent assay (ELISA) was compared with the routine culture methodology for the detection of Campylobacter fetus subspecies from bovine and ovine field samples inoculated into Clark's transport enrichment medium (TEM). The work was a collaboration between two different diagnostic laboratories, one in Canada and the other in England. In both labs, TEM samples were incubated for 4 days at 35 degrees C and then tested by culture and ELISA. The ELISA consisted of initial screening with MAb M1825 against C. fetus subspecies core lipopolysaccharide (LPS). All samples positive on ELISA screening were then retested by ELISA with MAb M1825 and MAbs M1177, M1183, and M1194, which recognize serotype A- and/or serotype B-specific C. fetus subspecies LPS epitopes. The Canadian samples consisted of 1,060 preputial washings from 529 bulls, of which 18 were positive by both culture and ELISA and 1,042 were negative by both methods. The English samples consisted of 321 tissue specimens, mostly stomach contents and placentas, from 190 aborted ovine and bovine fetuses. A total of 262 samples were negative by culture and ELISA, 52 samples were positive by culture and ELISA, and 7 samples were culture negative but ELISA positive. The results for all 70 culture-positive isolates were confirmed by conventional biochemical methods as C. fetus subsp. fetus, with 39 presumptively identified by the ELISA as serotype A and 30 presumptively identified as serotype B and with one sample containing isolates presumptively identified as serotype A and serotype B. A receiver operating characteristic analysis of the combined ELISA data from both countries resulted in an area under the curve of 0.997, with a sensitivity of 100% and a specificity of 99.5% relative to the results of culture. The data confirm that this ELISA method can be used as an excellent test for the screening of field samples in TEM for the presence of C. fetus subspecies. [Abstract/Link to Full Text]

Frémont M, Vaeyens F, Herst CV, De Meirleir K, Englebienne P
37-Kilodalton/83-kilodalton RNase L isoform ratio in peripheral blood mononuclear cells: analytical performance and relevance for chronic fatigue syndrome.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1259-60; author reply 1260. [Abstract/Link to Full Text]

Tiev KP, Demettre E, Ercolano P, Bastide L, Lebleu B, Cabane J
RNase L levels in peripheral blood mononuclear cells: 37-kilodalton/83-kilodalton isoform ratio is a potential test for chronic fatigue syndrome.
Clin Diagn Lab Immunol. 2003 Mar;10(2):315-6.
Chronic fatigue syndrome (CFS) is a disorder characterized by debilitating fatigue associated with immunological abnormalities. The etiology remains unclear. A low-molecular-mass (37 kDa) isoform of RNase L has been described in peripheral blood mononuclear cell (PBMC) extracts, and the ratio of two isoforms of RNase L (37 kDa/83 kDa) has been proposed as a potential biochemical marker of CFS. In a prospective case-control study, we tested whether the RNase L 37-kDa/83-kDa ratio could discriminate a SFC population. We compared the ratio of RNase L isoforms in PBMCs from 11 patients with CFS (6 women and 5 men; mean age +/- standard deviation, 43.2 +/- 13.8 years) and PBMCs from 14 healthy well-matched volunteers (10 women and 4 men; age, 39.1 +/- 11.6 years). A ratio of RNase L of 0.4 used as a threshold allowed diagnosis of CFS with high sensitivity (91%; 95% confidence interval [CI], 57 to 99%) and specificity (71%; 95% CI, 41 to 90%). The positive and negative prognostic values were 71% (95% CI, 41 to 90%) and 91% (95% CI, 57 to 99%), respectively. In the absence of acute infection or chronic inflammation, a high RNase L ratio could distinguish CFS patients from healthy volunteers. Additional large studies and follow-up studies are required to confirm the stability of this high ratio of RNase L isoforms in a CFS group. [Abstract/Link to Full Text]

Ohkawara T, Takeda H, Asaka M, Mizue Y, Nishihira J
Increased levels of macrophage migration inhibitory factor in sera of patients with Escherichia coli O157:H7-induced enterocolitis.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1257-8. [Abstract/Link to Full Text]

von Aulock S, Rupp J, Gueinzius K, Maass M, Hermann C
Critical investigation of the CD14 promoter polymorphism: lack of a role for in vitro cytokine response and membrane CD14 expression.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1254-6.
Blood of volunteers, genotyped for the CD14 C(-159)-->T polymorphism, showed no difference in cytokine release when stimulated with nine CD14-dependent immune stimuli. An analysis of the published data on the proposed association of CD14 genotype with membrane CD14 density revealed no significant correlation, questioning a functional impact of the CD14 polymorphism. [Abstract/Link to Full Text]

Khalil M, Al-Mazrou Y, Balmer P, Bramwell J, Andrews N, Borrow R
Immunogenicity of meningococcal ACYW135 polysaccharide vaccine in Saudi children 5 to 9 years of age.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1251-3.
Meningococcal tetravalent polysaccharide vaccines were observed to be immunogenic in Saudi children 5 to 9 years of age, with >90% having serum bactericidal antibody titers of > or = 8 for serogroups A, Y, and W135; for serogroup C, 77% were putatively protected after vaccination. [Abstract/Link to Full Text]

Stelmach I, Podsiad?owicz-Borzecka M, Grzelewski T, Majak P, Stelmach W, Jerzy?ska J, Pop?awska M, Dziadek J
Humoral and cellular immunity in children with Mycoplasma pneumoniae infection: a 1-year prospective study.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1246-50.
To determine whether children have persistent abnormalities in cellular and humoral immunity development after acute Mycoplasma pneumoniae infection, serum immunoglobulin G (IgG), IgA, IgM, and IgE levels and lymphocyte phenotypes were determined. There were no changes in the levels of IgG, IgM, IgA, or CD4+ or CD19+ lymphocytes that were measured in M. pneumoniae-positive patients after 3 months or after 12 months, but there were increases in these in M. pneumoniae-negative patients. Serum IgE increased in M. pneumoniae-positive patients. We have shown alterations in immunity development after M. pneumoniae infection. [Abstract/Link to Full Text]

Martella V, Cavalli A, Decaro N, Elia G, Desario C, Campolo M, Bozzo G, Tarsitano E, Buonavoglia C
Immunogenicity of an intranasally administered modified live canine parvovirus type 2b vaccine in pups with maternally derived antibodies.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1243-5.
The ability of a modified live canine parvovirus type 2b vaccine to elicit active immunization in pups with maternally derived antibodies (MDA) by intranasal administration was evaluated. The vaccine induced seroconversion in 100% of pups with MDA titers of < or = 80 and in 51.6% of pups with titers between 160 and 320. [Abstract/Link to Full Text]

Bieging KT, Rajam G, Holder P, Udoff R, Carlone GM, Romero-Steiner S
Fluorescent multivalent opsonophagocytic assay for measurement of functional antibodies to Streptococcus pneumoniae.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1238-42.
We developed fluorescent mono- and multivalent opsonophagocytic assays (fOPA and fmOPA, respectively) specific for seven Streptococcus pneumoniae serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F). Bacterial survival was quantitated with alamar blue, a fluorescent metabolic indicator. Both fOPA and fmOPA allow for determination of viability endpoints for up to seven serotypes with high levels of agreement to the reference method. The fmOPA eliminates colony counting, reduces serum volume, and produces results in 1 day. [Abstract/Link to Full Text]

Nawa M, Takasaki T, Ito M, Inoue S, Morita K, Kurane I
Immunoglobulin A antibody responses in dengue patients: a useful marker for serodiagnosis of dengue virus infection.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1235-7.
We determined the usefulness of an immunoglobulin A (IgA) antibody-capture enzyme-linked immunosorbent assay for serodiagnosis of dengue virus infections. The results indicate that the presence of IgA and IgM in serum samples assures recent primary dengue virus infection even with a single serum sample. [Abstract/Link to Full Text]

Marangoni A, Sambri V, Accardo S, Cavrini F, D'Antuono A, Moroni A, Storni E, Cevenini R
Evaluation of LIAISON Treponema Screen, a novel recombinant antigen-based chemiluminescence immunoassay for laboratory diagnosis of syphilis.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1231-4.
The purpose of this study was to evaluate the diagnostic performance of LIAISON Treponema Screen (DiaSorin, Saluggia, Italy), a new automated chemiluminescence immunoassay (CLIA), in comparison with that of rapid plasma reagin (RPR) and the following currently used treponemal tests: hemagglutination test (TPHA), immunoenzymatic assay (EIA), and Western blot (WB). First, a retrospective study was performed with a panel of 2,494 blood donor sera, a panel of 131 clinical and serologically characterized syphilitic sera, and 96 samples obtained from subjects with potentially interfering diseases or conditions. A prospective study was also performed by testing 1,800 unselected samples submitted to the Microbiology Laboratory of the St. Orsola Hospital in Bologna, Italy, for routine screening for syphilis. As expected, RPR was the least specific method, especially when potentially cross-reacting sera were tested. On the contrary, all of the treponemal tests proved to be very specific (99.9%) and they performed with the following sensitivities: 100% (WB), 99.2% (CLIA), 95.4% (EIA), and 94.7% (TPHA). [Abstract/Link to Full Text]

Boesen A, Sundar K, Coico R
Lassa fever virus peptides predicted by computational analysis induce epitope-specific cytotoxic-T-lymphocyte responses in HLA-A2.1 transgenic mice.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1223-30.
Lassa fever is a hemorrhagic disease caused by Lassa fever virus (LV). Although the precise host defense mechanism(s) that affords protection against LV is not completely understood, cellular immunity mediated by cytotoxic T lymphocytes (CTLs) plays a pivotal role in controlling viral replication and LV infection. To date, there have been no reports mapping major histocompatibility complex (MHC) class I-binding CTL epitopes for LV. Using computer-assisted algorithms, we identified five HLA-A2.1-binding peptides of LV glycoprotein (GP) and two peptides from LV nucleoprotein (NP). Synthesized peptides were examined for their ability to bind to MHC class I molecules using a flow cytometric assay that measures peptide stabilization of class I. Three of the LV-GP peptides tested (LLGTFTWTL, SLYKGVYEL, and YLISIFLHL) stabilized HLA-A2. The LV-NP peptides tested failed to stabilize this HLA-A2. We then investigated the ability of the HLA-A2-binding LV-GP peptides to generate peptide-specific CTLs in HLA-A2.1 transgenic mice. Functional assays used to confirm CTL activation included gamma interferon enzyme-linked immunospot (ELISPOT) assays and intracellular cytokine staining of CD8+ T cells from peptide-primed mice. CTL assays were also performed to verify the cytolytic activity of peptide-pulsed target cells. Each of the LV-GP peptides induced CTL responses in HLA-A2-transgenic mice. MHC class I tetramers prepared using one LV-GP peptide that showed the highest cytolytic index (LLGTFTWTL) confirmed that peptide-binding CD8+ T cells were present in pooled lymphocytes harvested from peptide-primed mice. These findings provide direct evidence for the existence of LV-derived GP epitopes that may be useful in the development of protective immunogens for this hemorrhagic virus. [Abstract/Link to Full Text]

Rose MA, Schubert R, Strnad N, Zielen S
Priming of immunological memory by pneumococcal conjugate vaccine in children unresponsive to 23-valent polysaccharide pneumococcal vaccine.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1216-22.
Pneumococcal polysaccharide vaccine (PPV) is of limited immunogenicity in infants and immunocompromised patients. Our prospective randomized controlled trial investigated whether priming with pneumococcal conjugate vaccine (PCV) induced specific immunological memory in previously nonresponders to PPV. Of a total of 33 children (2 to 18 years) with polysaccharide-specific immunodeficiency (PSI), group A (n = 16) received two doses of 7-valent PCV in a 4- to 6-week interval, and a booster dose of 23-valent PPV after one year. Group B (n = 17) received two doses of PPV in a 1-year interval exclusively. Specific antibody concentrations for serotypes 4, 5, 6B, 9V, 14, 18C, 19F, and 23F were determined (enzyme-linked immunosorbent assay) before and at 7 and 28 days after administration of the PPV booster and compared to an opsonophagocytosis assay. Of group A, 64 to 100% had antibody concentrations of > or = 1 microg/ml on day 28 after the booster versus 25 to 94% of group B. Group A had significantly higher antibody concentrations for all PCV-containing serotypes already on day 7, indicating early memory response. Antibody concentrations were in accordance with functional opsonic activity, although opsonic titers varied among individuals. Pneumococcal vaccination was well tolerated. The incidence of airway infections was reduced after priming with PCV (10/year for group A versus 15/year for group B). Following a PPV booster, even patients primarily not responding to PPV showed a rapid and more pronounced memory response after priming with PCV. [Abstract/Link to Full Text]

Kummrow M, Meli ML, Haessig M, Goenczi E, Poland A, Pedersen NC, Hofmann-Lehmann R, Lutz H
Feline coronavirus serotypes 1 and 2: seroprevalence and association with disease in Switzerland.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1209-15.
To determine the prevalence of antibodies to feline coronavirus (FCoV) serotypes 1 and 2 in Switzerland and their association with different disease manifestations, a serological study based on immunofluorescence tests was conducted with Swiss field cats using transmissible gastroenteritis virus (TGEV), FCoV type 1 and FCoV type 2 as antigens. A total of 639 serum samples collected in the context of different studies from naturally infected cats were tested. The current study revealed that, with an apparent prevalence of 83%, FCoV serotype 1 is the most prevalent serotype in Switzerland. FCoV type 1 viruses induced higher antibody titers than FCoV type 2, and were more frequently associated with clinical signs and/or feline infectious peritonitis. The antibody development in seven cats experimentally infected with FCoV type 1 revealed that, with progressing duration of infection, antibodies to FCoV type 1 significantly increased over those to FCoV type 2. There was a significant relationship between antibody titers against TGEV, FCoV 1, and FCoV 2 and TGEV antigen detected the highest proportion of seropositive cats. We conclude that a vaccine against FCoV should be based on FCoV type 1-related antigens and that for serodiagnosis of FCoV infection TGEV should be used to attain the highest diagnostic efficiency. When serology is used in addition to clinical signs, hematology, and clinical chemistry results as an aid to diagnose clinical FIP, TGEV shows a diagnostic efficiency equal to that of a FCoV antigen. [Abstract/Link to Full Text]

Freer G, Matteucci D, Mazzetti P, Bozzacco L, Bendinelli M
Generation of feline dendritic cells derived from peripheral blood monocytes for in vivo use.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1202-8.
Dendritic cells (DCs) are professional antigen-presenting cells that can prime T cells and polarize the cellular immune response. Because Th1-type immune responses have been connected to success in combating viral infection, a promising therapeutic application of DCs would be their differentiation in vitro and injection back into the host to boost an immune response in infected animals. This study was aimed both at developing a protocol to cultivate feline DCs in the absence of exogenous proteins for their use in vivo and at investigating what might be the most appropriate stimulus to induce their maturation in vitro and finding correlates of maturation. We generated DCs from peripheral blood monocytes in the presence of feline interleukin-4 and granulocyte-macrophage colony stimulating factor, and after 5 days their maturation was induced with either lipopolysaccharide, human recombinant tumor necrosis factor alpha, poly(I:C), or activated feline platelets. After 48 h, their CD14, CD1a, major histocompatibility complex class II, and B7.1 surface expression was analyzed in parallel with their ability to uptake antigen or prime a mixed leukocyte reaction. The results presented show that feline DCs cultured in autologous plasma differentiate and are able to mature in the presence of stimuli similar to the ones currently used for other species. The present work sets the grounds for future use of DCs obtained by the protocol described for in vivo vaccination and immunotherapy of feline immunodeficiency virus-infected cats. [Abstract/Link to Full Text]

Scott JA, Mlacha Z, Nyiro J, Njenga S, Lewa P, Obiero J, Otieno H, Sampson JS, Carlone GM
Diagnosis of invasive pneumococcal disease among children in Kenya with enzyme-linked immunosorbent assay for immunoglobulin G antibodies to pneumococcal surface adhesin A.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1195-201.
Diagnostic techniques for invasive pneumococcal disease (IPD) in children are insensitive and underestimate both the burden of disease and the cost-effectiveness of pneumococcal conjugate vaccination (PCV). Consequently, there is little demand for the highly effective PCV outside the United States and Europe. In Kenya, diagnosis of pneumococcal pneumonia in adults was achieved with a sensitivity of 0.70 and a specificity of 0.98 using enzyme-linked immunosorbent assays (ELISAs) of paired plasma samples for immunoglobulin G (IgG) to pneumococcal surface adhesin A (PsaA). We aimed to validate the same technique in children. We assayed paired blood samples from 98 children with IPD, 95 age-matched children with malaria/anemia, and 97 age-matched healthy controls by using an ELISA for anti-PsaA IgG. Sensitivity and specificity were determined in IPD patients and healthy controls. Specificity (0.97; 95% confidence interval [CI], 0.91 to 0.99) and sensitivity (0.42; 95% CI, 0.32 to 0.52) were optimized at a 2.7-fold rise in anti-PsaA antibody concentration. Sensitivity was improved to a maximum of 0.50 by restricting testing to children of <2 years old, by excluding IPD patients who were not sampled on the first day of presentation, and by incorporating high existing antibody concentrations in the analysis. Assay performance was independent of nasopharyngeal carriage of pneumococci at recruitment. This assay improves on existing diagnostic tools for IPD in children but would still leave over half of all cases undetected in epidemiological studies. Effective diagnosis of pneumococcal disease in children is urgently required but poorly served by existing technology. [Abstract/Link to Full Text]

Singh R, Subba Raju BV, Jain RK, Salotra P
Potential of direct agglutination test based on promastigote and amastigote antigens for serodiagnosis of post-kala-azar dermal leishmaniasis.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1191-4.
Post-kala-azar dermal leishmaniasis (PKDL) is a dermal complication, a sequel to kala-azar. Diagnosis of PKDL presents a challenge due to the low parasite burden in the lesions. The direct agglutination test (DAT) based on promastigote and amastigote antigens of Leishmania donovani of indigenous isolates was developed to diagnose PKDL, and the results were compared with those of the rk39 strip test. The sensitivities of DAT for antileishmanial antibody detection, based on promastigote and amastigote antigens at a cutoff titer of 1:800 were 98.5% and 100%, respectively, with corresponding specificities of 96.5% and 100%. DAT could correctly detect 100% polymorphic cases and 95.4% macular PKDL cases. In comparison, the rk39 strip test was able to correctly diagnose 95.6% of polymorphic and 86.0% macular PKDL cases. DAT based on axenic amastigote antigen provided 100% sensitivity and specificity, making it particularly useful for macular PKDL cases, which are often missed by the rk39 strip test. Thus, DAT provides a simple, reliable, and inexpensive test for PKDL diagnosis with potential applicability in field conditions. [Abstract/Link to Full Text]

Kirchherr JL, Bowden GH, Richmond DA, Sheridan MJ, Wirth KA, Cole MF
Clonal diversity and turnover of Streptococcus mitis bv. 1 on shedding and nonshedding oral surfaces of human infants during the first year of life.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1184-90.
Streptococcus mitis bv. 1 is a pioneer colonizer of the human oral cavity. Studies of its population dynamics within parents and their infants and within neonates have shown extensive diversity within and between subjects. We examined the genetic diversity and clonal turnover of S. mitis bv. 1 isolated from the cheeks, tongue, and primary incisors of four infants from birth to 1 year of age. In addition, we compared the clonotypes of S. mitis bv. 1 isolated from their mothers' saliva collected in parallel to determine whether the mother was the origin of the clones colonizing her infant. Of 859 isolates obtained from the infants, 568 were unique clones. Each of the surfaces examined, whether shedding or nonshedding, displayed the same degree of diversity. Among the four infants it was rare to detect the same clone colonizing more than one surface at a given visit. There was little evidence for persistence of clones, but when clones were isolated on multiple visits they were not always found on the same surface. A similar degree of clonal diversity of S. mitis bv. 1 was observed in the mothers' saliva as in their infants' mouths. Clones common to both infant and mothers' saliva were found infrequently suggesting that this is not the origin of the infants' clones. It is unclear whether mucosal immunity exerts the environmental pressure driving the genetic diversity and clonal turnover of S. mitis bv. 1, which may be mechanisms employed by this bacterium to evade immune elimination. [Abstract/Link to Full Text]

Dreher UM, de la Fuente J, Hofmann-Lehmann R, Meli ML, Pusterla N, Kocan KM, Woldehiwet Z, Braun U, Regula G, Staerk KD, Lutz H
Serologic cross-reactivity between Anaplasma marginale and Anaplasma phagocytophilum.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1177-83.
In the context of a serosurvey conducted on the Anaplasma marginale prevalence in Swiss cattle, we suspected that a serological cross-reactivity between A. marginale and A. phagocytophilum might exist. In the present study we demonstrate that cattle, sheep and horses experimentally infected with A. phagocytophilum not only develop antibodies to A. phagocytophilum (detected by immunofluorescent-antibody assay) but also to A. marginale (detected by a competitive enzyme-linked immunosorbent assay). Conversely, calves experimentally infected with A. marginale also developed antibodies to A. phagocytophilum using the same serological tests. The identity of 63% determined in silico within a 209-amino-acid sequence of major surface protein 5 of an isolate of A. marginale and one of A. phagocytophilum supported the observed immunological cross-reactivity. These observations have important consequences for the serotesting of both, A. marginale and A. phagocytophilum infection of several animal species. In view of these new findings, tests that have been considered specific for either infection must be interpreted carefully. [Abstract/Link to Full Text]

Subramaniam K, French N, Pirofski LA
Cryptococcus neoformans-reactive and total immunoglobulin profiles of human immunodeficiency virus-infected and uninfected Ugandans.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1168-76.
We determined total and Cryptococcus neoformans glucuronoxylomannan (GXM)-reactive antibody repertoires of human immunodeficiency virus (HIV)-infected and HIV-uninfected Ugandans in a retrospective, case-control study of participants in a randomized controlled trial of pneumococcal vaccination. The study included 192 adults: 48 who subsequently developed cryptococcal meningitis (CM); (HIV+ CM+); 2 individuals who matched them in CD4+ T-cell level, stage of HIV disease, and age but did not develop CM (HIV+ CM-); and 48 HIV-uninfected individuals. Total serum immunoglobulin concentrations and titers of immunoglobulin M (IgM), IgG, and IgA to GXM, pneumococcal polysaccharides, and antibodies expressing certain V(H)3 idiotypes were determined with banked sera obtained before the development of cryptococcosis for HIV+ CM+ subjects. The results showed that HIV-infected subjects had significantly lower levels of IgM to GXM but higher levels of total immunoglobulin and IgG and IgA to GXM than those of HIV-uninfected subjects. HIV-infected subjects with a history of pneumonia had higher levels, and those with a history of herpes zoster had lower levels of GXM-binding antibodies than subjects with no history of either disease. Minimal to no cross-reactivity was demonstrated between antibodies to GXM and polysaccharides in a pneumococcal vaccine. No significant differences between the antibody repertoires of HIV+ CM+ and HIV+ CM- subjects were identified, but among subjects without a history of pneumonia, there was a trend towards lower V(H)3-positive antibody levels among HIV+ CM+ than among HIV+ CM- subjects. Our findings demonstrate an association between previous infectious diseases and differences in the total and GXM-reactive antibody repertoires of HIV-infected subjects and suggest the question of whether certain microbes modulate subsequent antibody responses to GXM deserves further study. [Abstract/Link to Full Text]

Passos S, Carvalho LP, Orge G, Jerônimo SM, Bezerra G, Soto M, Alonso C, Carvalho EM
Recombinant leishmania antigens for serodiagnosis of visceral leishmaniasis.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1164-7.
Serological tests with crude or recombinant Leishmania antigens are important tools for the diagnosis of leishmania infection. However, these tests are not markers of active visceral leishmaniasis (VL), since antibodies to these markers are often observed in individuals with subclinical L. chagasi infection and they do not fall shortly after therapy. In this study, levels of immunoglobulin G (IgG) against three recombinant Leishmania antigens (rH2A, KMP11, and the "Q" protein) were evaluated in sera from individuals with subclinical L. chagasi infection and in patients with VL pre- and posttherapy. The sensitivity of the serological test for diagnosis of VL was 100% with all three antigens. The titers of IgG fell significantly after therapy. While most of the individuals with subclinical L. chagasi infection had antibodies to rH2A and the "Q" protein, only 1 out of 15 individuals had antibodies to KMP11. These data indicate that KMP11 may be used to discriminate L. chagasi infection from active VL and may serve as a marker of response to therapy. [Abstract/Link to Full Text]

Vizzi E, Calvińo E, González R, Pérez-Schael I, Ciarlet M, Kang G, Estes MK, Liprandi F, Ludert JE
Evaluation of serum antibody responses against the rotavirus nonstructural protein NSP4 in children after rhesus rotavirus tetravalent vaccination or natural infection.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1157-63.
The immune response elicited by the rotavirus nonstructural protein NSP4 and its potential role in protection against rotavirus disease are not well understood. We investigated the serological response to NSP4 and its correlation with disease protection in sera from 110 children suffering acute diarrhea, associated or not with rotavirus, and from 26 children who were recipients of the rhesus rotavirus tetravalent (RRV-TV) vaccine. We used, as antigens in an enzyme-linked immunosorbent assay (ELISA), affinity-purified recombinant NSP4 (residues 85 to 175) from strains SA11, Wa, and RRV (genotypes A, B, and C, respectively) fused to glutathione S-transferase. Seroconversion to NSP4 was observed in 54% (42/78) of the children who suffered from natural rotavirus infection and in 8% (2/26) of the RRV-TV vaccine recipients. Our findings indicate that NSP4 evokes significantly (P < 0.05) higher seroconversion rates after natural infection than after RRV-TV vaccination. The serum antibody levels to NSP4 were modest (titers of < or = 200) in most of the infected and vaccinated children. A heterotypic NSP4 response was detected in 48% of the naturally rotavirus-infected children with a detectable response to NSP4. Following natural infection or RRV-TV vaccination, NSP4 was significantly less immunogenic than the VP6 protein when these responses were independently measured by ELISA. A significant (P < 0.05) proportion of children who did not develop diarrhea associated with rotavirus had antibodies to NSP4 in acute-phase serum, suggesting that serum antibodies against NSP4 might correlate with protection from rotavirus diarrhea. In addition, previous exposures to rotavirus did not affect the NSP4 seroconversion rate. [Abstract/Link to Full Text]

Wongkham C, Tantrawatpan C, Intapan PM, Maleewong W, Wongkham S, Nakashima K
Evaluation of immunoglobulin G subclass antibodies against recombinant Fasciola gigantica cathepsin L1 in an enzyme-linked immunosorbent assay for serodiagnosis of human fasciolosis.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1152-6.
A cystatin capture enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola gigantica cathepsin L1 antigen was developed to detect specific immunoglobulin G (IgG) subclass antibodies (IgG1, IgG2, IgG3, and IgG4) and was evaluated for its diagnostic potential for human fasciolosis. In an analysis of the sera of 13 patients infected with F. gigantica, 209 patients with other parasitic infections, 32 cholangiocarcinoma patients, and 42 healthy controls, the IgG4-ELISA gave the highest diagnostic values. The sensitivity, specificity, accuracy, and positive and negative predictive values of this method based on the detection of IgG4 antibody were 100%, 99.3%, 99.3%, 86.7%, and 100%, respectively. The results revealed that restricting the ELISA to the detection of specific IgG4 antibody enhanced the specificity and accuracy for the serodiagnosis of human fasciolosis. [Abstract/Link to Full Text]

Hsu CC, Wobus CE, Steffen EK, Riley LK, Livingston RS
Development of a microsphere-based serologic multiplexed fluorescent immunoassay and a reverse transcriptase PCR assay to detect murine norovirus 1 infection in mice.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1145-51.
Murine norovirus 1 (MNV-1) is a newly recognized pathogen of mice that causes lethal infection in mice deficient in components of the innate immune response but not in wild-type 129 mice. In this study, in vitro-propagated MNV-1 was used as antigen to develop a multiplexed fluorescent immunoassay (MFI) to detect antibodies to MNV-1 in infected mice. The MNV-1 MFI was 100% specific and 100% sensitive in detecting anti-MNV-1 antibody in sera from experimentally infected mice. Testing of a large number of mouse serum samples (n = 12,639) submitted from contemporary laboratory mouse colonies in the United States and Canada revealed that 22.1% of these sera contained antibodies to MNV-1, indicating infection with MNV-1 is widespread in research mice. In addition, a reverse transcriptase PCR primer pair with a sensitivity of 25 virus copies was developed and used to demonstrate that MNV-1 RNA could be detected in the spleen, mesenteric lymph node, and jejunum from some experimentally infected mice 5 weeks postinoculation. These diagnostic assays provide the necessary tools to define the MNV-1 infection status of research mice and to aid in the establishment of laboratory mouse colonies free of MNV-1 infection. [Abstract/Link to Full Text]

Singh I, Sheoran AS, Zhang Q, Carville A, Tzipori S
Sensitivity and specificity of a monoclonal antibody-based fluorescence assay for detecting Enterocytozoon bieneusi spores in feces of simian immunodeficiency virus-infected macaques.
Clin Diagn Lab Immunol. 2005 Oct;12(10):1141-4.
Enterocytozoon bieneusi is clinically the most significant among the microsporidia causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. Microscopy with either calcofluor or modified trichrome stains is the standard diagnostic test for microsporidiosis and does not allow species identification. Detection of E. bieneusi infection based on PCR is limited to a few reference laboratories, and thus it is not the standard diagnostic assay. We have recently reported the development and characterization of a panel of monoclonal antibodies against E. bieneusi, and in this publication we evaluated the specificity and sensitivity of an immunofluorescence assay (IFA), compared with PCR, in simian immunodeficiency virus-infected macaques. The IFA, which correlated with the primary PCR method, with a detection limit of 1.5 x 10(5) spores per gram of feces, will simplify considerably the detection of E. bieneusi spores in clinical and environmental specimens and in laboratory and epidemiological investigations. [Abstract/Link to Full Text]

Zanetti S, Bua A, Delogu G, Pusceddu C, Mura M, Saba F, Pirina P, Garzelli C, Vertuccio C, Sechi LA, Fadda G
Patients with pulmonary tuberculosis develop a strong humoral response against methylated heparin-binding hemagglutinin.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1135-8.
Reactivities of human sera against selected recombinant Mycobacterium tuberculosis antigens were assessed by enzyme-linked immunosorbent assay. The results obtained indicate that patients with tuberculosis (TB) do not develop a strong humoral response against PE_PGRS and PPE proteins or against the Ag85B and heparin-binding hemagglutinin (HBHA) recombinant antigens. Conversely, purified methylated HBHA was strongly recognized by sera obtained from TB patients compared to controls. [Abstract/Link to Full Text]

Harimaya A, Tarkkanen J, Mattila P, Fujii N, Ylikoski J, Himi T
Difference in cytokine production and cell activation between adenoidal lymphocytes and peripheral blood lymphocytes of children with otitis media.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1130-4.
We evaluated the immunological potential of adenoidal lymphocytes from children with recurrent otitis media. Interleukin-4 release and CD69 expression were lower in adenoidal lymphocytes than in peripheral blood lymphocytes (PBL). Our results suggest that there may be a difference between the immunological potential of adenoidal lymphocytes and that of PBL in children with otitis. [Abstract/Link to Full Text]


Recent Articles in Clinical and Molecular Allergy

Chinoy B, Yee E, Bahna SL
Skin testing versus radioallergosorbent testing for indoor allergens.
Clin Mol Allergy. 2005 Apr 15;3(1):4.
BACKGROUND: Skin testing (ST) is the most common screening method for allergy evaluation. Measurement of serum specific IgE is also commonly used, but less so by allergists than by other practitioners. The sensitivity and specificity of these testing methods may vary by type of causative allergen and type of allergic manifestation. We compared ST reactivity with serum specific IgE antibodies to common indoor allergens in patients with respiratory allergies. METHODS: 118 patients (3 mo-58 yr, mean 12 yr) with allergic rhinitis and/or bronchial asthma had percutaneous skin testing (PST) supplemented by intradermal testing (ID) with those allergens suspected by history but showed negative PST. The sera were tested blindly for specific IgE antibodies by the radioallergosorbent test (Phadebas RAST). The allergens were D. farinae (118), cockroach (60), cat epithelium (90), and dog epidermal (90). Test results were scored 0-4; ST >/= 2 + and RAST >/= 1 + were considered positive. RESULTS: The two tests were in agreement (i.e., either both positive or both negative) in 52.2% (dog epidermal) to 62.2% (cat epithelium). When RAST was positive, ST was positive in 80% (dog epidermal) to 100% (cockroach mix). When ST was positive, RAST was positive in 16.3% (dog epidermal) to 50.0% (D. farinae). When RAST was negative, ST was positive in 48.5% (cat epithelium) to 69.6% (D. farinae). When ST was negative, RAST was positive in 0% (cockroach) to 5.6% (cat epithelium). The scores of ST and RAST showed weak to moderate correlation (r = 0.24 to 0.54). Regardless of history of symptoms on exposure, ST was superior to RAST in detecting sensitization to cat epithelium and dog epidermal. CONCLUSION: For all four indoor allergens tested, ST was more sensitive than RAST. When both tests were positive, their scores showed poor correlation. Sensitizations to cat epithelium and dog epidermal are common, even in subjects who claimed no direct exposure. [Abstract/Link to Full Text]

Haye R, Hřye K, Berg O, Frřnes S, Odegĺrd T
Morning versus evening dosing of desloratadine in seasonal allergic rhinitis: a randomized controlled study [ISRCTN23032971].
Clin Mol Allergy. 2005 Feb 2;3(1):3.
BACKGROUND: A circadian rhythm of symptoms has been reported in allergic rhinitis and some studies have shown the dosing time of antihistamines to be of importance for optimizing symptom relief in this disease. The objective of this study was to examine the efficacy of morning vs. evening dosing of the antihistamine desloratadine at different time points during the day. METHODS: Patients >/= 18 years, with seasonal allergic rhinitis received desloratadine 5 mg orally once daily in the morning (AM-group) or evening (PM-group) for two weeks. Rhinorrhea, nasal congestion, sneezing and eye symptoms were scored morning and evening. Wilcoxon rank sum and 2-way ANOVA test were used. RESULTS: Six-hundred and sixty-three patients were randomized; 336 in the AM-group; 327 in the PM-group. No statistically significant differences were seen between the AM and PM group at any time points. In the sub-groups with higher morning or evening total symptom score no difference in treatment efficacy was seen whether the dose was taken 12 or 24 hours before the higher score time. There was a circadian variation in baseline total symptom score; highest during daytime and lowest at night. The circadian variation in symptoms was reduced during treatment. This reduction was highest for daytime symptoms. CONCLUSIONS: A circadian rhythm was seen for most symptoms being more pronounced during daytime. This was less apparent after treatment with desloratadine. No statistically significant difference in efficacy was seen whether desloratadine was given in the morning or in the evening. This gives the patients more flexibility in choosing dosing time. [Abstract/Link to Full Text]

Nordness ME, Lynn J, Zacharisen MC, Scott PJ, Kelly KJ
Asthma is a risk factor for acute chest syndrome and cerebral vascular accidents in children with sickle cell disease.
Clin Mol Allergy. 2005 Jan 21;3(1):2.
BACKGROUND: Asthma and sickle cell disease are common conditions that both may result in pulmonary complications. We hypothesized that children with sickle cell disease with concomitant asthma have an increased incidence of vaso-occlusive crises that are complicated by episodes of acute chest syndrome. METHODS: A 5-year retrospective chart analysis was performed investigating 48 children ages 3-18 years with asthma and sickle cell disease and 48 children with sickle cell disease alone. Children were matched for age, gender, and type of sickle cell defect. Hospital admissions were recorded for acute chest syndrome, cerebral vascular accident, vaso-occlusive pain crises, and blood transfusions (total, exchange and chronic). Mann-Whitney test and Chi square analysis were used to assess differences between the groups. RESULTS: Children with sickle cell disease and asthma had significantly more episodes of acute chest syndrome (p = 0.03) and cerebral vascular accidents (p = 0.05) compared to children with sickle cell disease without asthma. As expected, these children received more total blood transfusions (p = 0.01) and chronic transfusions (p = 0.04). Admissions for vasoocclusive pain crises and exchange transfusions were not statistically different between cases and controls. SS disease is more severe than SC disease. CONCLUSIONS: Children with concomitant asthma and sickle cell disease have increased episodes of acute chest syndrome, cerebral vascular accidents and the need for blood transfusions. Whether aggressive asthma therapy can reduce these complications in this subset of children is unknown and requires further studies. [Abstract/Link to Full Text]

Westwood GS, Huang SW, Keyhani NO
Allergens of the entomopathogenic fungus Beauveria bassiana.
Clin Mol Allergy. 2005 Jan 11;3(1):1.
BACKGROUND: Beauveria bassiana is an important entomopathogenic fungus currently under development as a bio-control agent for a variety of insect pests. Although reported to be non-toxic to vertebrates, the potential allergenicity of Beauveria species has not been widely studied. METHODS: IgE-reactivity studies were performed using sera from patients displaying mould hypersensitivity by immunoblot and immunoblot inhibition. Skin reactivity to B. bassiana extracts was measured using intradermal skin testing. RESULTS: Immunoblots of fungal extracts with pooled as well as individual sera showed a distribution of IgE reactive proteins present in B. bassiana crude extracts. Proteinase K digestion of extracts resulted in loss of IgE reactive epitopes, whereas EndoH and PNGaseF (glycosidase) treatments resulted in minor changes in IgE reactive banding patterns as determined by Western blots. Immunoblot inhibitions experiments showed complete loss of IgE-binding using self protein, and partial inhibition using extracts from common allergenic fungi including; Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Candida albicans, Epicoccum purpurascens, and Penicillium notatum. Several proteins including a strongly reactive band with an approximate molecular mass of 35 kDa was uninhibited by any of the tested extracts, and may represent B. bassiana specific allergens. Intradermal skin testing confirmed the in vitro results, demonstrating allergenic reactions in a number of individuals, including those who have had occupational exposure to B. bassiana. CONCLUSIONS: Beauveria bassiana possesses numerous IgE reactive proteins, some of which are cross-reactive among allergens from other fungi. A strongly reactive potential B. bassiana specific allergen (35 kDa) was identified. Intradermal skin testing confirmed the allergenic potential of B. bassiana. [Abstract/Link to Full Text]

Sills ES, Conway SC, Kaplan CR, Perloe M, Tucker MJ
First successful case of in vitro fertilization-embryo transfer with venom immunotherapy for hymenoptera sting allergy.
Clin Mol Allergy. 2004 10 19;2(1):11.
BACKGROUND: To describe immune and endocrine responses in severe hymenoptera hypersensitivity requiring venom immunotherapy (VIT) during in vitro fertilization (IVF). CASE PRESENTATION: A 39-year old patient was referred for history of multiple miscarriage and a history of insect sting allergy. Four years earlier, she began subcutaneous injection of 100 mcg mixed vespid hymenoptera venom/venom protein every 5-6 weeks. The patient had one livebirth and three first trimester miscarriages. Allergy treatment was maintained for all pregnancies ending in miscarriage, although allergy therapy was discontinued for the pregnancy that resulted in delivery. At our institution ovulation induction incorporated venom immunotherapy (VIT) during IVF, with a reduced VIT dose when pregnancy was first identified. Serum IgE was monitored with estradiol during ovulation induction and early pregnancy. Response to controlled ovarian hyperstimulation was favorable while VIT was continued, with retrieval of 12 oocytes. Serum RAST (yellow jacket) IgE levels fluctuated in a nonlinear fashion (range 36-54%) during gonadotropin therapy and declined after hCG administration. A healthy female infant was delivered at 35 weeks gestation. The patient experienced no untoward effects from any medications during therapy. CONCLUSION: Our case confirms the safety of VIT in pregnancy, and demonstrates RAST IgE can remain <60% during IVF. With proper monitoring, VIT during IVF can be safe and appropriate for selected patients and does not appear to adversely affect blastocyst implantation, early embryo development or perinatal outcome. Further studies will be needed to develop VIT guidelines specifically applicable to IVF. [Abstract/Link to Full Text]

Baptist AP, Baldwin JL
Autoimmune progesterone dermatitis in a patient with endometriosis: case report and review of the literature.
Clin Mol Allergy. 2004 Aug 2;2(1):10.
Autoimmune progesterone dermatitis (APD) is a condition in which the menstrual cycle is associated with a number of skin findings such as urticaria, eczema, angioedema, and others. In affected women, it occurs 3-10 days prior to the onset of menstrual flow, and resolves 2 days into menses. Women with irregular menses may not have this clear correlation, and therefore may be missed. We present a case of APD in a woman with irregular menses and urticaria/angioedema for over 20 years, who had not been diagnosed or correctly treated due to the variable timing of skin manifestations and menses. In addition, we review the medical literature in regards to clinical features, pathogenesis, diagnosis, and treatment options. [Abstract/Link to Full Text]

Lewiecki EM
Management of osteoporosis.
Clin Mol Allergy. 2004 Jul 14;2(1):9.
Osteoporosis or osteopenia occurs in about 44 million Americans, resulting in 1.5 million fragility fractures per year. The consequences of these fractures include pain, disability, depression, loss of independence, and increased mortality. The burden to the healthcare system, in terms of cost and resources, is tremendous, with an estimated direct annual USA healthcare expenditure of about $17 billion. With longer life expectancy and the aging of the baby-boomer generation, the number of men and women with osteoporosis or low bone density is expected to rise to over 61 million by 2020. Osteoporosis is a silent disease that causes no symptoms until a fracture occurs. Any fragility fracture greatly increases the risk of future fractures. Most patients with osteoporosis are not being diagnosed or treated. Even those with previous fractures, who are at extremely high risk of future fractures, are often not being treated. It is preferable to diagnose osteoporosis by bone density testing of high risk individuals before the first fracture occurs. If osteoporosis or low bone density is identified, evaluation for contributing factors should be considered. Patients on long-term glucocorticoid therapy are at especially high risk for developing osteoporosis, and may sustain fractures at a lower bone density than those not taking glucocorticoids. All patients should be counseled on the importance of regular weight-bearing exercise and adequate daily intake of calcium and vitamin D. Exposure to medications that cause drowsiness or hypotension should be minimized. Non-pharmacologic therapy to reduce the non-skeletal risk factors for fracture should be considered. These include fall prevention through balance training and muscle strengthening, removal of fall hazards at home, and wearing hip protectors if the risk of falling remains high. Pharmacologic therapy can stabilize or increase bone density in most patients, and reduce fracture risk by about 50%. By selecting high risk patients for bone density testing it is possible to diagnose this disease before the first fracture occurs, and initiate appropriate treatment to reduce the risk of future fractures. [Abstract/Link to Full Text]

Killian S, McMichael J
The human allergens of mesquite (Prosopis juliflora).
Clin Mol Allergy. 2004 Jul 5;2(1):8.
BACKGROUND: A computerized statistical analysis of allergy skin test results correlating patient reactivities initiated our interest in the cross-reactive allergens of mesquite tree pollen. In-vitro testing with mesquite-sensitized rabbits and a variety of deciduous tree pollens revealed so many cross-reactivities that it became apparent there could be more allergens in mesquite than previously described in the world literature. Our purpose was to examine the allergens of mesquite tree pollen (Prosopis juliflora) which elicit an IgE response in allergic humans so that future research could determine if these human allergens cross-react with various tree pollens in the same manner as did the mesquite antiserum from sensitized rabbits. METHODS: Proteins from commercial mesquite tree pollen were separated by polyacrylamide gel electrophoresis in the presence of sodium-dodecyl-sulphate. These mesquite proteins were subjected to Western blotting using pooled sera from ten mesquite-sensitive patients and goat anti-human IgE. The allergens were detected using an Amplified Opti-4-CN kit, scanned, and then interpreted by Gel-Pro software. RESULTS: Thirteen human allergens of mesquite pollen were detected in this study. CONCLUSION: The number of allergens in this study of mesquite exceeded the number identified previously in the literature. With the increased exposure to mesquite through its use in "greening the desert", increased travel to desert areas and exposure to mesquite in cooking smoke, the possible clinical significance of these allergens and their suggested cross-reactivity with other tree pollens merit further study. [Abstract/Link to Full Text]

Copeland BH, Aramide OO, Wehbe SA, Fitzgerald SM, Krishnaswamy G
Eosinophilia in a patient with cyclical vomiting: a case report.
Clin Mol Allergy. 2004 May 14;2(1):7.
BACKGROUND: Eosinophilic gastritis is related to eosinophilic gastroenteritis, varying only in regards to the extent of disease and small bowel involvement. Common symptoms reported are similar to our patient's including: abdominal pain, epigastric pain, anorexia, bloating, weight loss, diarrhea, ankle edema, dysphagia, melaena and postprandial nausea and vomiting. Microscopic features of eosinophilic infiltration usually occur in the lamina propria or submucosa with perivascular aggregates. The disease is likely mediated by eosinophils activated by various cytokines and chemokines. Therapy centers around the use of immunosuppressive agents and dietary therapy if food allergy is a factor. CASE PRESENTATION: The patient is a 31 year old Caucasian female with a past medical history significant for ulcerative colitis. She presented with recurrent bouts of vomiting, abdominal pain and chest discomfort of 11 months duration. The bouts of vomiting had been reoccurring every 7-10 days, with each episode lasting for 1-3 days. This was associated with extreme weakness and cachexia. Gastric biopsies revealed intense eosinophilic infiltration. The patient responded to glucocorticoids and azathioprine. The differential diagnosis and molecular pathogenesis of eosinophilic gastritis as well as the molecular effects of glucocorticoids in eosinophilic disorders are discussed. CONCLUSIONS: The patient responded to a combination of glucocorticosteroids and azathioprine with decreased eosinophilia and symptoms. It is likely that eosinophil-active cytokines such as interleukin-3 (IL-3), granulocyte macrophage colony stimulating factor (GM-CSF) and IL-5 play pivotal roles in this disease. Chemokines such as eotaxin may be involved in eosinophil recruitment. These mediators are downregulated or inhibited by the use of immunosuppressive medications. [Abstract/Link to Full Text]

Abdelnoor AM, Kobeissy F, Farhat D, Hadi U
Circulating immune complexes and complement C3 and C4 levels in a selected group of patients with rhinitis in Lebanon.
Clin Mol Allergy. 2004 Apr 22;2(1):6.
BACKGROUND: A number of reports indicate that circulating immune complexes (CIC) and activation of the complement system contribute to the pathogenesis of Type I allergy. The aim of this study was to investigate the status of CIC in 113 patients with rhinitis in Lebanon and determine complement components C3 and C4 serum levels in the CIC-positive patients. Serum specific IgE antibodies were previously detected and reported in 74 of the 113 patients. METHODS: CIC were detected by polyethylene glycol precipitation and serum C3 and C4 levels quantified by radial immunodiffusion. RESULTS: CIC was positive in 20 of the specific IgE-positive and 13 of the specific IgE-negative patients. C3 and C4 levels were within the normal range in all the 33 CIC-positive patients. CONCLUSIONS: The antibody class that constitutes the complexes does not seem to be IgG or IgM. Moreover, complement activation does not seem to be involved in the allergic reaction since both C3 and C4 levels were normal in all patients. The role of these complexes, if any, in the pathogenesis of rhinitis is yet to be determined. [Abstract/Link to Full Text]

Al Hasan M, Fitzgerald SM, Saoudian M, Krishnaswamy G
Dermatology for the practicing allergist: Tinea pedis and its complications.
Clin Mol Allergy. 2004 Mar 29;2(1):5.
Tinea pedis is a chronic fungal infection of the feet, very often observed in patients who are immuno-suppressed or have diabetes mellitus. The practicing allergist may be called upon to treat this disease for various reasons. Sometimes tinea infection may be mistaken for atopic dermatitis or allergic eczema. In other patients, tinea pedis may complicate allergy and asthma and may contribute to refractory atopic disease. Patients with recurrent cellulitis may be referred to the allergist/immunologist for an immune evaluation and discovered to have tinea pedis as a predisposing factor. From a molecular standpoint, superficial fungal infections may induce a type2 T helper cell response (Th2) that can aggravate atopy. Th2 cytokines may induce eosinophil recruitment and immunoglobulin E (IgE) class switching by B cells, thereby leading to exacerbation of atopic conditions. Three groups of fungal pathogens, referred to as dermatophytes, have been shown to cause tinea pedis: Trychophyton sp, Epidermophyton sp, and Microsporum sp. The disease manifests as a pruritic, erythematous, scaly eruption on the foot and depending on its location, three variants have been described: interdigital type, moccasin type, and vesiculobullous type. Tinea pedis may be associated with recurrent cellulitis, as the fungal pathogens provide a portal for bacterial invasion of subcutaneous tissues. In some cases of refractory asthma, treatment of the associated tinea pedis infection may induce remission in airway disease. Very often, protracted topical and/or oral antifungal agents are required to treat this often frustrating and morbid disease. An evaluation for underlying immuno-suppression or diabetes may be indicated in patients with refractory disease. [Abstract/Link to Full Text]

Hatipoglu U, Rubinstein I
Low-dose, long-term macrolide therapy in asthma: An overview.
Clin Mol Allergy. 2004 Mar 16;2(1):4.
Macrolides, a class of antimicrobials isolated from Streptomycetes more than 50 years ago, are used extensively to treat sinopulmonary infections in humans. In addition, a growing body of experimental and clinical evidence indicates that long-term (years), low (sub-antimicrobial)-dose 14- and 15-membered ring macrolide antibiotics, such as erythromycin, clarithromycin, roxithromycin and azithromycin, express immunomodulatory and tissue reparative effects that are distinct from their anti-infective properties. These salutary effects are operative in various lung disorders, including diffuse panbronchiolitis, cystic fibrosis, persistent chronic rhinosinusitis, nasal polyposis, bronchiectasis, asthma and cryptogenic organizing pneumonia.The purpose of this overview is to outline the immunomodulatory effects of macrolide antibiotics in patients with asthma. [Abstract/Link to Full Text]

Ciprandi G, Cirillo I, Tosca MA, Vizzaccaro A
Bronchial hyperreactivity and spirometric impairment in polysensitized patients with allergic rhinitis.
Clin Mol Allergy. 2004 Mar 14;2(1):3.
BACKGROUND: We previously demonstrated in a group of patients with perennial allergic rhinitis alone impairment of spirometric parameters and high percentage of subjects with bronchial hyperreactivity (BHR). The present study aimed at evaluating a group of polysensitized subjects suffering from allergic rhinitis alone to investigate the presence of spirometric impairment and BHR during the pollen season. METHODS: One hundred rhinitics sensitized both to pollen and perennial allergens were evaluated during the pollen season. Spirometry and methacholine bronchial challenge were performed. RESULTS: Six rhinitics showed impaired values of FEV1 without referred symptoms of asthma. FEF 25-75 values were impaired in 28 rhinitics. Sixty-six patients showed positive methacholine bronchial challenge. FEF 25-75 values were impaired only in BHR positive patients (p < 0.001). A significant difference was observed both for FEV1 (p < 0.05) and FEF 25-75 (p < 0.001) considering BHR severity. CONCLUSIONS: This study evidences that an impairment of spirometric parameters may be observed in polysensitized patients with allergic rhinitis alone during the pollen season. A high percentage of these patients had BHR. A close relationship between upper and lower airways is confirmed. [Abstract/Link to Full Text]

Matheu V, Baeza ML, Zubeldia JM, Barrios Y
Allergy to lingonberry: A case report.
Clin Mol Allergy. 2004 Mar 1;2(1):2.
Past few years cranberry/lingonberry products have been incorporated as healthy products to the US and European market as prophylaxis of recurrent urinary tract infections in young women as well as in chronic infections in elderly which because of there are many biological activities attributed to the that fruit is a very popular additive to the new diets. To the best of our knowledge, this is the first case of allergy to lingonberry. We speculate that previous exposure to lingonberry products could be sensitising. The symptoms, timing of the episode, positive skin test, IgE-ELISA and western-blot strongly support the role of lingonberry as the causative agent. [Abstract/Link to Full Text]

Krishnaswamy G, Chi DS
Clinical and Molecular Allergy: a new open access journal that addresses rapidly evolving information in the field of allergy and immunology.
Clin Mol Allergy. 2004 Feb 4;2(1):1. [Abstract/Link to Full Text]

Kuderer NM, San-Juan-Vergara HG, Kong X, Esch R, Lockey RF, Mohapatra SS
Mite and cockroach proteases activate p44/p42 MAP kinases in human lung epithelial cells.
Clin Mol Allergy. 2003 Oct 30;1(1):1.
BACKGROUND: The mechanisms underlying epithelial cell activation by indoor inhaled antigens are poorly understood. METHODS: In this study, we investigated the role of mitogen-activated protein kinases (MAPKs) in A549 epithelial cells upon exposure to antigens of house dust mite (HDMA), German cockroach (GCA), and American cockroach (ACA). RESULTS: Each of these antigens induced a significant increase in IL-8 levels compared to the medium control. Exposure of A549 cells to these antigens induced the phosphorylation of p44/42 MAPKs within 5 minutes, which reached a peak at 25 minutes later and reached baseline levels at 1 hour after exposure. PD98059, a MEK1 inhibitor, significantly decreased phosphorylation of p44/p42 MAPKs and IL-8 production. Exposure of A549 cells with antigens, which had been preincubated with different protease inhibitors, also resulted in a reduction of both MAPK phosphorylation and IL-8 production. CONCLUSION: Thus, proteolytic antigens present in HDMA, GCA and ACA activate the p44/42 MAPKs airway epithelial cells, which lead to elevated IL-8 production and initiation of the inflammatory cascade. [Abstract/Link to Full Text]


Recent Articles in Clinical Microbiology Reviews

Fujinami RS, von Herrath MG, Christen U, Whitton JL
Molecular mimicry, bystander activation, or viral persistence: infections and autoimmune disease.
Clin Microbiol Rev. 2006 Jan;19(1):80-94.
Virus infections and autoimmune disease have long been linked. These infections often precede the occurrence of inflammation in the target organ. Several mechanisms often used to explain the association of autoimmunity and virus infection are molecular mimicry, bystander activation (with or without epitope spreading), and viral persistence. These mechanisms have been used separately or in various combinations to account for the immunopathology observed at the site of infection and/or sites of autoimmune disease, such as the brain, heart, and pancreas. These mechanisms are discussed in the context of multiple sclerosis, myocarditis, and diabetes, three immune-medicated diseases often linked with virus infections. [Abstract/Link to Full Text]

Nainan OV, Xia G, Vaughan G, Margolis HS
Diagnosis of hepatitis a virus infection: a molecular approach.
Clin Microbiol Rev. 2006 Jan;19(1):63-79.
Current serologic tests provide the foundation for diagnosis of hepatitis A and hepatitis A virus (HAV) infection. Recent advances in methods to identify and characterize nucleic acid markers of viral infections have provided the foundation for the field of molecular epidemiology and increased our knowledge of the molecular biology and epidemiology of HAV. Although HAV is primarily shed in feces, there is a strong viremic phase during infection which has allowed easy access to virus isolates and the use of molecular markers to determine their genetic relatedness. Molecular epidemiologic studies have provided new information on the types and extent of HAV infection and transmission in the United States. In addition, these new diagnostic methods have provided tools for the rapid detection of food-borne HAV transmission and identification of the potential source of the food contamination. [Abstract/Link to Full Text]

Carson CF, Hammer KA, Riley TV
Melaleuca alternifolia (Tea Tree) oil: a review of antimicrobial and other medicinal properties.
Clin Microbiol Rev. 2006 Jan;19(1):50-62.
Complementary and alternative medicines such as tea tree (melaleuca) oil have become increasingly popular in recent decades. This essential oil has been used for almost 100 years in Australia but is now available worldwide both as neat oil and as an active component in an array of products. The primary uses of tea tree oil have historically capitalized on the antiseptic and anti-inflammatory actions of the oil. This review summarizes recent developments in our understanding of the antimicrobial and anti-inflammatory activities of the oil and its components, as well as clinical efficacy. Specific mechanisms of antimicrobial and anti-inflammatory action are reviewed, and the toxicity of the oil is briefly discussed. [Abstract/Link to Full Text]

Lafond RE, Lukehart SA
Biological basis for syphilis.
Clin Microbiol Rev. 2006 Jan;19(1):29-49.
Syphilis is a chronic sexually transmitted disease caused by Treponema pallidum subsp. pallidum. Clinical manifestations separate the disease into stages; late stages of disease are now uncommon compared to the preantibiotic era. T. pallidum has an unusually small genome and lacks genes that encode many metabolic functions and classical virulence factors. The organism is extremely sensitive to environmental conditions and has not been continuously cultivated in vitro. Nonetheless, T. pallidum is highly infectious and survives for decades in the untreated host. Early syphilis lesions result from the host's immune response to the treponemes. Bacterial clearance and resolution of early lesions results from a delayed hypersensitivity response, although some organisms escape to cause persistent infection. One factor contributing to T. pallidum's chronicity is the paucity of integral outer membrane proteins, rendering intact organisms virtually invisible to the immune system. Antigenic variation of TprK, a putative surface-exposed protein, is likely to contribute to immune evasion. T. pallidum remains exquisitely sensitive to penicillin, but macrolide resistance has recently been identified in a number of geographic regions. The development of a syphilis vaccine, thus far elusive, would have a significant positive impact on global health. [Abstract/Link to Full Text]

Janda JM, Abbott SL
The genus Hafnia: from soup to nuts.
Clin Microbiol Rev. 2006 Jan;19(1):12-8.
The genus Hafnia, a member of the family Enterobacteriaceae, consists of gram-negative bacteria that are occasionally implicated in both intestinal and extraintestinal infections in humans. Despite the fact that the genus currently contains only a single species (H. alvei), more extensive phylogenetic depth (two or more species) is apparent based upon DNA relatedness and 16S rRNA gene sequencing studies. Hafnia causes a variety of systemic infections, including septicemia and pneumonia; however, its role as a gastrointestinal pathogen is controversial. Many of the data supporting a role for hafniae as enteric pathogens were incorrectly attributed to this genus rather than to the actual pathogen, Escherichia albertii. There are numerous gaps in our understanding of this genus, including ecologic habitats and population genetics, disease-producing role in animals, phenetic and genetic methods useful in distinguishing genomospecies within the H. alvei complex, and bona fide pathogenicity factors. [Abstract/Link to Full Text]

Waites KB, Katz B, Schelonka RL
Mycoplasmas and ureaplasmas as neonatal pathogens.
Clin Microbiol Rev. 2005 Oct;18(4):757-89.
The genital mycoplasmas represent a complex and unique group of microorganisms that have been associated with a wide array of infectious diseases in adults and infants. The lack of conclusive knowledge regarding the pathogenic potential of Mycoplasma and Ureaplasma spp. in many conditions is due to a general unfamiliarity of physicians and microbiology laboratories with their fastidious growth requirements, leading to difficulty in their detection; their high prevalence in healthy persons; the poor design of research studies attempting to base association with disease on the mere presence of the organisms in the lower urogenital tract; the failure to consider multifactorial aspects of diseases; and considering these genital mycoplasmas only as a last resort. The situation is now changing because of a greater appreciation of the genital mycoplasmas as perinatal pathogens and improvements in laboratory detection, particularly with regard to the development of powerful molecular nucleic acid amplification tests. This review summarizes the epidemiology of genital mycoplasmas as causes of neonatal infections and premature birth; evidence linking ureaplasmas with bronchopulmonary dysplasia; recent changes in the taxonomy of the genus Ureaplasma; the neonatal host response to mycoplasma and ureaplasma infections; advances in laboratory detection, including molecular methods; and therapeutic considerations for treatment of systemic diseases. [Abstract/Link to Full Text]

Parola P, Paddock CD, Raoult D
Tick-borne rickettsioses around the world: emerging diseases challenging old concepts.
Clin Microbiol Rev. 2005 Oct;18(4):719-56.
During most of the 20th century, the epidemiology of tick-borne rickettsioses could be summarized as the occurrence of a single pathogenic rickettsia on each continent. An element of this paradigm suggested that the many other characterized and noncharacterized rickettsiae isolated from ticks were not pathogenic to humans. In this context, it was considered that relatively few tick-borne rickettsiae caused human disease. This concept was modified extensively from 1984 through 2005 by the identification of at least 11 additional rickettsial species or subspecies that cause tick-borne rickettsioses around the world. Of these agents, seven were initially isolated from ticks, often years or decades before a definitive association with human disease was established. We present here the tick-borne rickettsioses described through 2005 and focus on the epidemiological circumstances that have played a role in the emergence of the newly recognized diseases. [Abstract/Link to Full Text]

Gavin PJ, Kazacos KR, Shulman ST
Baylisascariasis.
Clin Microbiol Rev. 2005 Oct;18(4):703-18.
The raccoon roundworm, Baylisascaris procyonis, is the most common and widespread cause of clinical larva migrans in animals. In addition, it is increasingly recognized as a cause of devastating or fatal neural larva migrans in infants and young children and ocular larva migrans in adults. Humans become infected by accidentally ingesting infective B. procyonis eggs from raccoon latrines or articles contaminated with their feces. Two features distinguish B. procyonis from other helminthes that cause larva migrans: (i) its aggressive somatic migration and invasion of the central nervous system and (ii) the continued growth of larvae to a large size within the central nervous system. Typically, B. procyonis neural larva migrans presents as acute fulminant eosinophilic meningoencephalitis. Once invasion of the central nervous system has occurred, the prognosis is grave with or without treatment. To date, despite anthelmintic treatment of cases of B. procyonis neural larva migrans, there are no documented neurologically intact survivors. Epidemiologic study of human cases of neural larva migrans demonstrate that contact with raccoon feces or an environment contaminated by infective eggs and geophagia or pica are the most important risk factors for infection. In many regions of the United States, increasingly large populations of raccoons, with high rates of B. procyonis infection, live in close proximity to humans. Although documented cases of human baylisascariasis remain relatively uncommon, widespread contamination of the domestic environment by infected raccoons suggests that the risk of exposure and human infection is probably substantial. In the absence of early diagnosis or effective treatment, prevention of infection is the most important public health measure. [Abstract/Link to Full Text]

Doherty TM, Andersen P
Vaccines for tuberculosis: novel concepts and recent progress.
Clin Microbiol Rev. 2005 Oct;18(4):687-702.
Three-quarters of a century after the introduction of Mycobacterium bovis BCG, the first tuberculosis vaccine, new vaccines for tuberculosis are finally entering clinical trials. This breakthrough is based not only on advances in proteomics and genomics which have made the construction of new vaccines possible, but also on a greatly expanded knowledge of the immunology of tuberculosis. Here we review our current understanding of how Mycobacterium tuberculosis subverts or survives the host's immune response to cause disease and why the current vaccination strategy, which relies on BCG, is only partially successful in countering the pathogen. This provides a background for describing the new generation of vaccines designed to supplement or replace the current vaccine and the different approaches they take to stimulate immunity against M. tuberculosis. [Abstract/Link to Full Text]

Paterson DL, Bonomo RA
Extended-spectrum beta-lactamases: a clinical update.
Clin Microbiol Rev. 2005 Oct;18(4):657-86.
Extended-spectrum beta-lactamases (ESBLs) are a rapidly evolving group of beta-lactamases which share the ability to hydrolyze third-generation cephalosporins and aztreonam yet are inhibited by clavulanic acid. Typically, they derive from genes for TEM-1, TEM-2, or SHV-1 by mutations that alter the amino acid configuration around the active site of these beta-lactamases. This extends the spectrum of beta-lactam antibiotics susceptible to hydrolysis by these enzymes. An increasing number of ESBLs not of TEM or SHV lineage have recently been described. The presence of ESBLs carries tremendous clinical significance. The ESBLs are frequently plasmid encoded. Plasmids responsible for ESBL production frequently carry genes encoding resistance to other drug classes (for example, aminoglycosides). Therefore, antibiotic options in the treatment of ESBL-producing organisms are extremely limited. Carbapenems are the treatment of choice for serious infections due to ESBL-producing organisms, yet carbapenem-resistant isolates have recently been reported. ESBL-producing organisms may appear susceptible to some extended-spectrum cephalosporins. However, treatment with such antibiotics has been associated with high failure rates. There is substantial debate as to the optimal method to prevent this occurrence. It has been proposed that cephalosporin breakpoints for the Enterobacteriaceae should be altered so that the need for ESBL detection would be obviated. At present, however, organizations such as the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) provide guidelines for the detection of ESBLs in klebsiellae and Escherichia coli. In common to all ESBL detection methods is the general principle that the activity of extended-spectrum cephalosporins against ESBL-producing organisms will be enhanced by the presence of clavulanic acid. ESBLs represent an impressive example of the ability of gram-negative bacteria to develop new antibiotic resistance mechanisms in the face of the introduction of new antimicrobial agents. [Abstract/Link to Full Text]

MacDougall C, Polk RE
Antimicrobial stewardship programs in health care systems.
Clin Microbiol Rev. 2005 Oct;18(4):638-56.
Antimicrobial stewardship programs in hospitals seek to optimize antimicrobial prescribing in order to improve individual patient care as well as reduce hospital costs and slow the spread of antimicrobial resistance. With antimicrobial resistance on the rise worldwide and few new agents in development, antimicrobial stewardship programs are more important than ever in ensuring the continued efficacy of available antimicrobials. The design of antimicrobial management programs should be based on the best current understanding of the relationship between antimicrobial use and resistance. Such programs should be administered by multidisciplinary teams composed of infectious diseases physicians, clinical pharmacists, clinical microbiologists, and infection control practitioners and should be actively supported by hospital administrators. Strategies for changing antimicrobial prescribing behavior include education of prescribers regarding proper antimicrobial usage, creation of an antimicrobial formulary with restricted prescribing of targeted agents, and review of antimicrobial prescribing with feedback to prescribers. Clinical computer systems can aid in the implementation of each of these strategies, especially as expert systems able to provide patient-specific data and suggestions at the point of care. Antibiotic rotation strategies control the prescribing process by scheduled changes of antimicrobial classes used for empirical therapy. When instituting an antimicrobial stewardship program, a hospital should tailor its choice of strategies to its needs and available resources. [Abstract/Link to Full Text]

Kuno G, Chang GJ
Biological transmission of arboviruses: reexamination of and new insights into components, mechanisms, and unique traits as well as their evolutionary trends.
Clin Microbiol Rev. 2005 Oct;18(4):608-37.
Among animal viruses, arboviruses are unique in that they depend on arthropod vectors for transmission. Field research and laboratory investigations related to the three components of this unique mode of transmission, virus, vector, and vertebrate host, have produced an enormous amount of valuable information that may be found in numerous publications. However, despite many reviews on specific viruses, diseases, or interests, a systematic approach to organizing the available information on all facets of biological transmission and then to interpret it in the context of the evolutionary process has not been attempted before. Such an attempt in this review clearly demonstrates tremendous progress made worldwide to characterize the viruses, to comprehend disease transmission and pathogenesis, and to understand the biology of vectors and their role in transmission. The rapid progress in molecular biologic techniques also helped resolve many virologic puzzles and yielded highly valuable data hitherto unavailable, such as characterization of virus receptors, the genetic basis of vertebrate resistance to viral infection, and phylogenetic evidence of the history of host range shifts in arboviruses. However, glaring gaps in knowledge of many critical subjects, such as the mechanism of viral persistence and the existence of vertebrate reservoirs, are still evident. Furthermore, with the accumulated data, new questions were raised, such as evolutionary directions of virus virulence and of host range. Although many fundamental questions on the evolution of this unique mode of transmission remained unresolved in the absence of a fossil record, available observations for arboviruses and the information derived from studies in other fields of the biological sciences suggested convergent evolution as a plausible process. Overall, discussion of the diverse range of theories proposed and observations made by many investigators was found to be highly valuable for sorting out the possible mechanism(s) of the emergence of arboviral diseases. [Abstract/Link to Full Text]

Lim DV, Simpson JM, Kearns EA, Kramer MF
Current and developing technologies for monitoring agents of bioterrorism and biowarfare.
Clin Microbiol Rev. 2005 Oct;18(4):583-607.
Recent events have made public health officials acutely aware of the importance of rapidly and accurately detecting acts of bioterrorism. Because bioterrorism is difficult to predict or prevent, reliable platforms to rapidly detect and identify biothreat agents are important to minimize the spread of these agents and to protect the public health. These platforms must not only be sensitive and specific, but must also be able to accurately detect a variety of pathogens, including modified or previously uncharacterized agents, directly from complex sample matrices. Various commercial tests utilizing biochemical, immunological, nucleic acid, and bioluminescence procedures are currently available to identify biological threat agents. Newer tests have also been developed to identify such agents using aptamers, biochips, evanescent wave biosensors, cantilevers, living cells, and other innovative technologies. This review describes these current and developing technologies and considers challenges to rapid, accurate detection of biothreat agents. Although there is no ideal platform, many of these technologies have proved invaluable for the detection and identification of biothreat agents. [Abstract/Link to Full Text]

Collins WE, Jeffery GM
Plasmodium ovale: parasite and disease.
Clin Microbiol Rev. 2005 Jul;18(3):570-81.
Humans are infected by four recognized species of malaria parasites. The last of these to be recognized and described is Plasmodium ovale. Like the other malaria parasites of primates, this parasite is only transmitted via the bites of infected Anopheles mosquitoes. The prepatent period in the human ranges from 12 to 20 days. Some forms in the liver have delayed development, and relapse may occur after periods of up to 4 years after infection. The developmental cycle in the blood lasts approximately 49 h. An examination of records from induced infections indicated that there were an average of 10.3 fever episodes of > or = 101 degrees F and 4.5 fever episodes of > or = 104 degrees F. Mean maximum parasite levels were 6,944/microl for sporozoite-induced infections and 7,310/microl for trophozoite-induced infections. Exoerythrocytic stages have been demonstrated in the liver of humans, chimpanzees, and Saimiri monkeys following injection of sporozoites. Many different Anopheles species have been shown to be susceptible to infection with P. ovale, including A. gambiae, A. atroparvus, A. dirus, A. freeborni, A. albimanus, A. quadrimaculatus, A. stephensi, A. maculatus, A. subpictus, and A. farauti. An enzyme-linked immunosorbent assay has been developed to detect mosquitoes infected with P. ovale using a monoclonal antibody directed against the circumsporozoite protein. Plasmodium ovale is primarily distributed throughout sub-Saharan Africa. It has also been reported from numerous islands in the western Pacific. In more recent years, there have been reports of its distribution on the Asian mainland. Whether or not it will become a major public health problem there remains to be seen. The diagnosis of P. ovale is based primarily on the characteristics of the blood stages and its differentiation from P. vivax. The sometimes elliptical shape of the infected erythrocyte is often diagnostic when combined with other, subtler differences in morphology. The advent of molecular techniques, primarily PCR, has made diagnostic confirmation possible. The development of techniques for the long-term frozen preservation of malaria parasites has allowed the development diagnostic reference standards for P. ovale. Infections in chimpanzees are used to provide reference and diagnostic material for serologic and molecular studies because this parasite has not been shown to develop in other nonhuman primates, nor has it adapted to in vitro culture. There is no evidence to suggest that P. ovale is closely related phylogenetically to any other of the primate malaria parasites that have been examined. [Abstract/Link to Full Text]

Spellberg B, Edwards J, Ibrahim A
Novel perspectives on mucormycosis: pathophysiology, presentation, and management.
Clin Microbiol Rev. 2005 Jul;18(3):556-69.
Mucormycosis is a life-threatening fungal infection that occurs in immunocompromised patients. These infections are becoming increasingly common, yet survival remains very poor. A greater understanding of the pathogenesis of the disease may lead to future therapies. For example, it is now clear that iron metabolism plays a central role in regulating mucormycosis infections and that deferoxamine predisposes patients to mucormycosis by inappropriately supplying the fungus with iron. These findings raise the possibility that iron chelator therapy may be useful to treat the infection as long as the chelator does not inappropriately supply the fungus with iron. Recent data support the concept that high-dose liposomal amphotericin is the preferred monotherapy for mucormycosis. However, several novel therapeutic strategies are available. These options include combination therapy using lipid-based amphotericin with an echinocandin or with an azole (largely itraconazole or posaconazole) or with all three. The underlying principles of therapy for this disease remain rapid diagnosis, reversal of underlying predisposition, and urgent surgical debridement. [Abstract/Link to Full Text]

Openshaw PJ, Tregoning JS
Immune responses and disease enhancement during respiratory syncytial virus infection.
Clin Microbiol Rev. 2005 Jul;18(3):541-55.
Respiratory syncytial virus (RSV) is one of the commonest and most troublesome viruses of infancy. It causes most cases of bronchiolitis, which is associated with wheezing in later childhood. In primary infection, the peak of disease typically coincides with the development of specific T- and B-cell responses, which seem, in large part, to be responsible for disease. Animal models clearly show that a range of immune responses can enhance disease severity, particularly after vaccination with formalin-inactivated RSV. Prior immune sensitization leads to exuberant chemokine production, an excessive cellular influx, and an overabundance of cytokines during RSV challenge. Under different circumstances, specific mediators and T-cell subsets and antibody-antigen immune complex deposition are incriminated as major factors in disease. Animal models of immune enhancement permit a deep understanding of the role of specific immune responses in RSV disease, assist in vaccine design, and indicate which immunomodulatory therapy might be beneficial to children with bronchiolitis. [Abstract/Link to Full Text]

Fournier B, Philpott DJ
Recognition of Staphylococcus aureus by the innate immune system.
Clin Microbiol Rev. 2005 Jul;18(3):521-40.
The gram-positive bacterium Staphylococcus aureus is a major pathogen responsible for a variety of diseases ranging from minor skin infections to life-threatening conditions such as sepsis. Cell wall-associated and secreted proteins (e.g., protein A, hemolysins, and phenol-soluble modulin) and cell wall components (e.g., peptidoglycan and alanylated lipoteichoic acid) have been shown to be inflammatory, and these staphylococcal components may contribute to sepsis. On the host side, many host factors have been implicated in the innate detection of staphylococcal components. One class of pattern recognition molecules, Toll-like receptor 2, has been shown to function as the transmembrane component involved in the detection of staphylococcal lipoteichoic acid and phenol-soluble modulin and is involved in the synthesis of inflammatory cytokines by monocytes/macrophages in response to these components. Nod2 (nucleotide-binding oligomerization domain 2) is the intracellular sensor for muramyl dipeptide, the minimal bioactive structure of peptidoglycan, and it may contribute to the innate immune defense against S. aureus. The staphylococcal virulence factor protein A was recently shown to interact directly with tumor necrosis factor receptor 1 in airway epithelium and to reproduce the effects of tumor necrosis factor alpha. Finally, peptidoglycan recognition protein L is an amidase that inactivates the proinflammatory activities of peptidoglycan. However, peptidoglycan recognition protein L probably plays a minor role in the innate immune response to S. aureus. Thus, several innate immunity receptors may be implicated in host defense against S. aureus. [Abstract/Link to Full Text]

Murray TS, Groth ME, Weitzman C, Cappello M
Epidemiology and management of infectious diseases in international adoptees.
Clin Microbiol Rev. 2005 Jul;18(3):510-20.
International adoptees represent a group of children with unique health care needs. Data from published studies, along with the recent experience of the Yale International Adoption Clinic, suggest that the risk of serious infections in adoptees is low, although infections associated with institutionalization still occur commonly. Interpretation of these data must be undertaken with caution, however, since many, if not most, international adoptees are not evaluated in specialty clinics. Thus, prospective studies designed to minimize selection and referral bias are needed in order to accurately define the risk of infectious and noninfectious diseases in all international adoptees. [Abstract/Link to Full Text]

Aguero-Rosenfeld ME, Wang G, Schwartz I, Wormser GP
Diagnosis of lyme borreliosis.
Clin Microbiol Rev. 2005 Jul;18(3):484-509.
A large amount of knowledge has been acquired since the original descriptions of Lyme borreliosis (LB) and of its causative agent, Borrelia burgdorferi sensu stricto. The complexity of the organism and the variations in the clinical manifestations of LB caused by the different B. burgdorferi sensu lato species were not then anticipated. Considerable improvement has been achieved in detection of B. burgdorferi sensu lato by culture, particularly of blood specimens during early stages of disease. Culturing plasma and increasing the volume of material cultured have accomplished this. Further improvements might be obtained if molecular methods are used for detection of growth in culture and if culture methods are automated. Unfortunately, culture is insensitive in extracutaneous manifestations of LB. PCR and culture have high sensitivity on skin samples of patients with EM whose diagnosis is based mostly on clinical recognition of the lesion. PCR on material obtained from extracutaneous sites is in general of low sensitivity, with the exception of synovial fluid. PCR on synovial fluid has shown a sensitivity of up to >90% (when using four different primer sets) in patients with untreated or partially treated Lyme arthritis, making it a helpful confirmatory test in these patients. Currently, the best use of PCR is for confirmation of the clinical diagnosis of suspected Lyme arthritis in patients who are IgG immunoblot positive. PCR should not be used as the sole laboratory modality to support a clinical diagnosis of extracutaneous LB. PCR positivity in seronegative patients suspected of having late manifestations of LB most likely represents a false-positive result. Because of difficulties in direct methods of detection, laboratory tests currently in use are mainly those detecting antibodies to B. burgdorferi sensu lato. Tests used to detect antibodies to B. burgdorferi sensu lato have evolved from the initial formats as more knowledge on the immunodominant antigens has been collected. The recommendation for two-tier testing was an attempt to standardize testing and improve specificity in the United States. First-tier assays using whole-cell sonicates of B. burgdorferi sensu lato need to be standardized in terms of antigen composition and detection threshold of specific immunoglobulin classes. The search for improved serologic tests has stimulated the development of recombinant protein antigens and the synthesis of specific peptides from immunodominant antigens. The use of these materials alone or in combination as the source of antigen in a single-tier immunoassay may someday replace the currently recommended two-tier testing strategy. Evaluation of these assays is currently being done, and there is evidence that certain of these antigens may be broadly cross-reactive with the B. burgdorferi sensu lato species causing LB in Europe. [Abstract/Link to Full Text]

Qadri F, Svennerholm AM, Faruque AS, Sack RB
Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention.
Clin Microbiol Rev. 2005 Jul;18(3):465-83.
ETEC is an underrecognized but extremely important cause of diarrhea in the developing world where there is inadequate clean water and poor sanitation. It is the most frequent bacterial cause of diarrhea in children and adults living in these areas and also the most common cause of traveler's diarrhea. ETEC diarrhea is most frequently seen in children, suggesting that a protective immune response occurs with age. The pathogenesis of ETEC-induced diarrhea is similar to that of cholera and includes the production of enterotoxins and colonization factors. The clinical symptoms of ETEC infection can range from mild diarrhea to a severe cholera-like syndrome. The effective treatment of ETEC diarrhea by rehydration is similar to treatment for cholera, but antibiotics are not used routinely for treatment except in traveler's diarrhea. The frequency and characterization of ETEC on a worldwide scale are inadequate because of the difficulty in recognizing the organisms; no simple diagnostic tests are presently available. Protection strategies, as for other enteric infections, include improvements in hygiene and development of effective vaccines. Increases in antimicrobial resistance will dictate the drugs used for the treatment of traveler's diarrhea. Efforts need to be made to improve our understanding of the worldwide importance of ETEC. [Abstract/Link to Full Text]

Villamor E, Fawzi WW
Effects of vitamin a supplementation on immune responses and correlation with clinical outcomes.
Clin Microbiol Rev. 2005 Jul;18(3):446-64.
Vitamin A supplementation to preschool children is known to decrease the risks of mortality and morbidity from some forms of diarrhea, measles, human immunodeficiency virus (HIV) infection, and malaria. These effects are likely to be the result of the actions of vitamin A on immunity. Some of the immunomodulatory mechanisms of vitamin A have been described in clinical trials and can be correlated with clinical outcomes of supplementation. The effects on morbidity from measles are related to enhanced antibody production and lymphocyte proliferation. Benefits for severe diarrhea could be attributable to the functions of vitamin A in sustaining the integrity of mucosal epithelia in the gut, whereas positive effects among HIV-infected children could also be related to increased T-cell lymphopoiesis. There is no conclusive evidence for a direct effect of vitamin A supplementation on cytokine production or lymphocyte activation. Under certain circumstances, vitamin A supplementation to infants has the potential to improve the antibody response to some vaccines, including tetanus and diphtheria toxoids and measles. There is limited research on the effects of vitamin A supplementation to adults and the elderly on their immune function; currently available data provide no consistent evidence for beneficial effects. Additional studies with these age groups are needed. [Abstract/Link to Full Text]

Mathis A, Weber R, Deplazes P
Zoonotic potential of the microsporidia.
Clin Microbiol Rev. 2005 Jul;18(3):423-45.
Microsporidia are long-known parasitic organisms of almost every animal group, including invertebrates and vertebrates. Microsporidia emerged as important opportunistic pathogens in humans when AIDS became pandemic and, more recently, have also increasingly been detected in otherwise immunocompromised patients, including organ transplant recipients, and in immunocompetent persons with corneal infection or diarrhea. Two species causing rare infections in humans, Encephalitozoon cuniculi and Brachiola vesicularum, had previously been described from animal hosts (vertebrates and insects, respectively). However, several new microsporidial species, including Enterocytozoon bieneusi, the most prevalent human microsporidian causing human immunodeficiency virus-associated diarrhea, have been discovered in humans, raising the question of their natural origin. Vertebrate hosts are now identified for all four major microsporidial species infecting humans (E. bieneusi and the three Encephalitozoon spp.), implying a zoonotic nature of these parasites. Molecular studies have identified phenotypic and/or genetic variability within these species, indicating that they are not uniform, and have allowed the question of their zoonotic potential to be addressed. The focus of this review is the zoonotic potential of the various microsporidia and a brief update on other microsporidia which have no known host or an invertebrate host and which cause rare infections in humans. [Abstract/Link to Full Text]

Zorc JJ, Kiddoo DA, Shaw KN
Diagnosis and management of pediatric urinary tract infections.
Clin Microbiol Rev. 2005 Apr;18(2):417-22.
Urinary tract infection (UTI) is among the most commonly diagnosed bacterial infections of childhood. Although frequently encountered and well researched, diagnosis and management of UTI continue to be a controversial issue with many challenges for the clinician. Prevalence studies have shown that UTI may often be missed on history and physical examination, and the decision to screen for UTI must balance the risk for missed infections with the cost and inconvenience of testing. Interpretation of rapid diagnostic tests and culture is complicated by issues of contamination, false test results, and asymptomatic colonization of the urinary tract with nonpathogenic bacteria. The appropriate treatment of UTI has been controversial and has become more complex with the emergence of resistance to commonly used antibiotics. Finally, the anatomic evaluation and long-term management of a child after a UTI have been based on limited evidence, and newer studies question some of the tenets of prior recommendations. The goal of this review is to provide an up-to-date summary of the literature with particular attention to practical questions about diagnosis and management for the clinician. [Abstract/Link to Full Text]

Cheng AC, Currie BJ
Melioidosis: epidemiology, pathophysiology, and management.
Clin Microbiol Rev. 2005 Apr;18(2):383-416.
Melioidosis, caused by the gram-negative saprophyte Burkholderia pseudomallei, is a disease of public health importance in southeast Asia and northern Australia that is associated with high case-fatality rates in animals and humans. It has the potential for epidemic spread to areas where it is not endemic, and sporadic case reports elsewhere in the world suggest that as-yet-unrecognized foci of infection may exist. Environmental determinants of this infection, apart from a close association with rainfall, are yet to be elucidated. The sequencing of the genome of a strain of B. pseudomallei has recently been completed and will help in the further identification of virulence factors. The presence of specific risk factors for infection, such as diabetes, suggests that functional neutrophil defects are important in the pathogenesis of melioidosis; other studies have defined virulence factors (including a type III secretion system) that allow evasion of killing mechanisms by phagocytes. There is a possible role for cell-mediated immunity, but repeated environmental exposure does not elicit protective humoral or cellular immunity. A vaccine is under development, but economic constraints may make vaccination an unrealistic option for many regions of endemicity. Disease manifestations are protean, and no inexpensive, practical, and accurate rapid diagnostic tests are commercially available; diagnosis relies on culture of the organism. Despite the introduction of ceftazidime- and carbapenem-based intravenous treatments, melioidosis is still associated with a significant mortality attributable to severe sepsis and its complications. A long course of oral eradication therapy is required to prevent relapse. Studies exploring the role of preventative measures, earlier clinical identification, and better management of severe sepsis are required to reduce the burden of this disease. [Abstract/Link to Full Text]

Mattoo S, Cherry JD
Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies.
Clin Microbiol Rev. 2005 Apr;18(2):326-82.
Bordetella respiratory infections are common in people (B. pertussis) and in animals (B. bronchiseptica). During the last two decades, much has been learned about the virulence determinants, pathogenesis, and immunity of Bordetella. Clinically, the full spectrum of disease due to B. pertussis infection is now understood, and infections in adolescents and adults are recognized as the reservoir for cyclic outbreaks of disease. DTaP vaccines, which are less reactogenic than DTP vaccines, are now in general use in many developed countries, and it is expected that the expansion of their use to adolescents and adults will have a significant impact on reducing pertussis and perhaps decrease the circulation of B. pertussis. Future studies should seek to determine the cause of the unique cough which is associated with Bordetella respiratory infections. It is also hoped that data gathered from molecular Bordetella research will lead to a new generation of DTaP vaccines which provide greater efficacy than is provided by today's vaccines. [Abstract/Link to Full Text]

Walsh TR, Toleman MA, Poirel L, Nordmann P
Metallo-beta-lactamases: the quiet before the storm?
Clin Microbiol Rev. 2005 Apr;18(2):306-25.
The ascendancy of metallo-beta-lactamases within the clinical sector, while not ubiquitous, has nonetheless been dramatic; some reports indicate that nearly 30% of imipenem-resistant Pseudomonas aeruginosa strains possess a metallo-beta-lactamase. Acquisition of a metallo-beta-lactamase gene will invariably mediate broad-spectrum beta-lactam resistance in P. aeruginosa, but the level of in vitro resistance in Acinetobacter spp. and Enterobacteriaceae is less dependable. Their clinical significance is further embellished by their ability to hydrolyze all beta-lactams and by the fact that there is currently no clinical inhibitor, nor is there likely to be for the foreseeable future. The genes encoding metallo-beta-lactamases are often procured by class 1 (sometimes class 3) integrons, which, in turn, are embedded in transposons, resulting in a highly transmissible genetic apparatus. Moreover, other gene cassettes within the integrons often confer resistance to aminoglycosides, precluding their use as an alternative treatment. Thus far, the metallo-beta-lactamases encoded on transferable genes include IMP, VIM, SPM, and GIM and have been reported from 28 countries. Their rapid dissemination is worrisome and necessitates the implementation of not just surveillance studies but also metallo-beta-lactamase inhibitor studies securing the longevity of important anti-infectives. [Abstract/Link to Full Text]

Olivier M, Gregory DJ, Forget G
Subversion mechanisms by which Leishmania parasites can escape the host immune response: a signaling point of view.
Clin Microbiol Rev. 2005 Apr;18(2):293-305.
The obligate intracellular parasite Leishmania must survive the antimicrobial activities of its host cell, the macrophage, and prevent activation of an effective immune response. In order to do this, it has developed numerous highly successful strategies for manipulating activities, including antigen presentation, nitric oxide and oxygen radical generation, and cytokine production. This is generally the result of interactions between Leishmania cell surface molecules, particularly gp63 and LPG, and less well identified macrophage surface receptors, causing the distortion of specific intracellular signaling cascades. We describe some of the signaling pathways and intermediates that are repressed in infected cells, including JAK/STAT, Ca(2+)-dependent protein kinase C (PKC) isoforms, and mitogen-activated protein kinases (especially ERK1/2), and proteasome-mediated transcription factor degradation. We also discuss protein tyrosine phosphatases (particularly SHP-1), intracellular Ca2+, Ca(2+)-independent PKC, ceramide, and the suppressors of cytokine signaling family of repressors, which are all reported to be activated following infection, and the role of parasite-secreted cysteine proteases. [Abstract/Link to Full Text]

Servin AL
Pathogenesis of Afa/Dr diffusely adhering Escherichia coli.
Clin Microbiol Rev. 2005 Apr;18(2):264-92.
Over the last few years, dramatic increases in our knowledge about diffusely adhering Escherichia coli (DAEC) pathogenesis have taken place. The typical class of DAEC includes E. coli strains harboring AfaE-I, AfaE-II, AfaE-III, AfaE-V, Dr, Dr-II, F1845, and NFA-I adhesins (Afa/Dr DAEC); these strains (i) have an identical genetic organization and (ii) allow binding to human decay-accelerating factor (DAF) (Afa/Dr(DAF) subclass) or carcinoembryonic antigen (CEA) (Afa/Dr(CEA) subclass). The atypical class of DAEC includes two subclasses of strains; the atypical subclass 1 includes E. coli strains that express AfaE-VII, AfaE-VIII, AAF-I, AAF-II, and AAF-III adhesins, which (i) have an identical genetic organization and (ii) do not bind to human DAF, and the atypical subclass 2 includes E. coli strains that harbor Afa/Dr adhesins or others adhesins promoting diffuse adhesion, together with pathogenicity islands such as the LEE pathogenicity island (DA-EPEC). In this review, the focus is on Afa/Dr DAEC strains that have been found to be associated with urinary tract infections and with enteric infection. The review aims to provide a broad overview and update of the virulence aspects of these intriguing pathogens. Epidemiological studies, diagnostic techniques, characteristic molecular features of Afa/Dr operons, and the respective role of Afa/Dr adhesins and invasins in pathogenesis are described. Following the recognition of membrane-bound receptors, including type IV collagen, DAF, CEACAM1, CEA, and CEACAM6, by Afa/Dr adhesins, activation of signal transduction pathways leads to structural and functional injuries at brush border and junctional domains and to proinflammatory responses in polarized intestinal cells. In addition, uropathogenic Afa/Dr DAEC strains, following recognition of beta(1) integrin as a receptor, enter epithelial cells by a zipper-like, raft- and microtubule-dependent mechanism. Finally, the presence of other, unknown virulence factors and the way that an Afa/Dr DAEC strain emerges from the human intestinal microbiota as a "silent pathogen" are discussed. [Abstract/Link to Full Text]

Voth DE, Ballard JD
Clostridium difficile toxins: mechanism of action and role in disease.
Clin Microbiol Rev. 2005 Apr;18(2):247-63.
As the leading cause of hospital-acquired diarrhea, Clostridium difficile colonizes the large bowel of patients undergoing antibiotic therapy and produces two toxins, which cause notable disease pathologies. These two toxins, TcdA and TcdB, are encoded on a pathogenicity locus along with negative and positive regulators of their expression. Following expression and release from the bacterium, TcdA and TcdB translocate to the cytosol of target cells and inactivate small GTP-binding proteins, which include Rho, Rac, and Cdc42. Inactivation of these substrates occurs through monoglucosylation of a single reactive threonine, which lies within the effector-binding loop and coordinates a divalent cation critical to binding GTP. By glucosylating small GTPases, TcdA and TcdB cause actin condensation and cell rounding, which is followed by death of the cell. TcdA elicits effects primarily within the intestinal epithelium, while TcdB has a broader cell tropism. Important advances in the study of these toxins have been made in the past 15 years, and these are detailed in this review. The domains, subdomains, and residues of these toxins important for receptor binding and enzymatic activity have been elegantly studied and are highlighted herein. Furthermore, there have been major advances in defining the role of these toxins in modulating the inflammatory events involving the disruption of cell junctions, neuronal activation, cytokine production, and infiltration by polymorphonuclear cells. Collectively, the present review provides a comprehensive update on TcdA and TcdB's mechanism of action as well as the role of these toxins in disease. [Abstract/Link to Full Text]

De Bolle L, Naesens L, De Clercq E
Update on human herpesvirus 6 biology, clinical features, and therapy.
Clin Microbiol Rev. 2005 Jan;18(1):217-45.
Human herpesvirus 6 (HHV-6) is a betaherpesvirus that is closely related to human cytomegalovirus. It was discovered in 1986, and HHV-6 literature has expanded considerably in the past 10 years. We here present an up-to-date and complete overview of the recent developments concerning HHV-6 biological features, clinical associations, and therapeutic approaches. HHV-6 gene expression regulation and gene products have been systematically characterized, and the multiple interactions between HHV-6 and the host immune system have been explored. Moreover, the discovery of the cellular receptor for HHV-6, CD46, has shed a new light on HHV-6 cell tropism. Furthermore, the in vitro interactions between HHV-6 and other viruses, particularly human immunodeficiency virus, and their relevance for the in vivo situation are discussed, as well as the transactivating capacities of several HHV-6 proteins. The insight into the clinical spectrum of HHV-6 is still evolving and, apart from being recognized as a major pathogen in transplant recipients (as exemplified by the rising number of prospective clinical studies), its role in central nervous system disease has become increasingly apparent. Finally, we present an overview of therapeutic options for HHV-6 therapy (including modes of action and resistance mechanisms). [Abstract/Link to Full Text]


Recent Articles in Infection and Immunity

Tait AS, Dalton M, Geny B, D'Agnillo F, Popoff MR, Sternberg EM
The large clostridial toxins from Clostridium sordellii and C. difficile repress glucocorticoid receptor activity.
Infect Immun. 2007 Aug;75(8):3935-40.
We have previously shown that Bacillus anthracis lethal toxin represses glucocorticoid receptor (GR) transactivation. We now report that repression of GR activity also occurs with the large clostridial toxins produced by Clostridium sordellii and C. difficile. This was demonstrated using a transient transfection assay system for GR transactivation. We also report that C. sordellii lethal toxin inhibited GR function in an ex vivo assay, where toxin reduced the dexamethasone suppression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). Furthermore, the glucocorticoid antagonist RU-486 in combination with C. sordellii lethal toxin additively prevented glucocorticoid suppression of TNF-alpha. These findings corroborate the fact that GR is a target for the toxin and suggest a physiological role for toxin-associated GR repression in inflammation. Finally, we show that this repression is associated with toxins that inactivate p38 mitogen-activated protein kinase (MAPK). [Abstract/Link to Full Text]

Carlsson KE, Liu J, Edqvist PJ, Francis MS
Extracytoplasmic-stress-responsive pathways modulate type III secretion in Yersinia pseudotuberculosis.
Infect Immun. 2007 Aug;75(8):3913-24.
Three signal transduction pathways, the two-component systems CpxRA and BaeSR and the alternative sigma factor sigma(E), respond to extracytoplasmic stress that facilitates bacterial adaptation to changing environments. At least the CpxRA and sigma(E) pathways control the production of protein-folding and degradation factors that counter the effects of protein misfolding in the periplasm. This function also influences the biogenesis of multicomponent extracellular appendages that span the bacterial envelope, such as various forms of pili. Herein, we investigated whether any of these regulatory pathways in the enteropathogen Yersinia pseudotuberculosis affect the functionality of the Ysc-Yop type III secretion system. This is a multicomponent molecular syringe spanning the bacterial envelope used to inject effector proteins directly into eukaryotic cells. Disruption of individual components revealed that the Cpx and sigma(E) pathways are important for Y. pseudotuberculosis type III secretion of Yops (Yersinia outer proteins). In particular, a loss of CpxA, a sensor kinase, reduced levels of structural Ysc (Yersinia secretion) components in bacterial membranes, suggesting that these mutant bacteria are less able to assemble a functional secretion apparatus. Moreover, these bacteria were no longer capable of localizing Yops into the eukaryotic cell interior. In addition, a cpxA lcrQ double mutant engineered to overproduce and secrete Yops was still impaired in intoxicating cells. Thus, the Cpx pathway might mediate multiple influences on bacterium-target cell contact that modulate Yersinia type III secretion-dependent host cell cytotoxicity. [Abstract/Link to Full Text]

Rocha FJ, Schleicher U, Mattner J, Alber G, Bogdan C
Cytokines, signaling pathways, and effector molecules required for the control of Leishmania (Viannia) braziliensis in mice.
Infect Immun. 2007 Aug;75(8):3823-32.
Cutaneous leishmaniasis is caused by protozoan parasites of the genus Leishmania. The mechanisms of pathogen control have been established primarily in the mouse model of Leishmania major infection, but they might not hold true for other Leishmania species associated with cutaneous disease. Here, we analyzed the role of cytokines, signaling components, and effector molecules in the control of New World cutaneous leishmaniasis due to L. braziliensis. Unlike L. major, L. braziliensis caused small, nonulcerative, and self-healing skin swelling in C57BL/6 mice, as well as BALB/c mice. In contrast to the results obtained for L. mexicana, mice deficient for interleukin-12 or its key signaling molecule, signal transducer and activator of transcription 4, rapidly succumbed to severe visceral leishmaniasis. Infection of tumor necrosis factor knockout mice with L. braziliensis led to progressive, nonhealing skin lesions with erosions and hemorrhagic ulcerations, but in contrast to the results with L. major, only 20 to 30% of the mice developed fatal visceral disease. As seen with L. major, mice with a deleted inducible nitric oxide synthase gene (iNOS(-/-)) were unable to contain L. braziliensis in the skin, whereas the control of the parasite in the spleen remained unimpaired. Unlike what happens in L. major infections, NADPH oxidase had no impact on the course of disease in L. braziliensis-infected mice. These results not only define essential components of a protective immune response to L. braziliensis but also illustrate that the requirements for the control of cutaneous leishmaniasis vary between different parasite species. [Abstract/Link to Full Text]

Taubman MA, Han X, Larosa KB, Socransky SS, Smith DJ
Periodontal bacterial DNA suppresses the immune response to mutans streptococcal glucosyltransferase.
Infect Immun. 2007 Aug;75(8):4088-96.
Certain CpG motifs found in bacterial DNA enhance immune responses through Toll-like receptor 9 (TLR-9) and may also demonstrate adjuvant properties. Our objective was to determine if DNA from bacteria associated with periodontal disease could affect the immune response to other bacterial antigens in the oral cavity. Streptococcus sobrinus glucosyltransferase (GTF), an enzyme involved in dental caries pathogenesis, was used as a test antigen. Rowett rats were injected with aluminum hydroxide (alum) with buffer, alum-GTF, or alum-GTF together with either Escherichia coli DNA, Fusobacterium nucleatum DNA, or Porphyromonas gingivalis DNA. Contrary to expectation, animals receiving alum-GTF plus bacterial DNA (P. gingivalis in particular) demonstrated significantly reduced serum immunoglobulin G (IgG) antibody, salivary IgA antibody, and T-cell proliferation to GTF compared to animals immunized with alum-GTF alone. A diminished antibody response was also observed after administration of alum-GTF with the P. gingivalis DNA either together or separately, indicating that physical complexing of antigen and DNA was not responsible for the reduction in antibody. Since TLR triggering by DNA induces synthesis of prospective suppressive factors (e.g., suppressor of cytokine signaling [SOCS]), the effects of P. gingivalis DNA and GTF exposure on rat splenocyte production of SOCS family molecules and inflammatory cytokines were investigated in vitro. P. gingivalis DNA significantly up-regulated SOCS1 and SOCS5 expression and down-regulated interleukin-10 expression by cultured splenocytes. These results suggested that DNA from periodontal disease-associated bacteria did not enhance, but in fact suppressed, the immune response to a protein antigen from cariogenic streptococci, potentially through suppressive SOCS components triggered by innate mechanisms. [Abstract/Link to Full Text]

Leduc D, Beaufort N, de Bentzmann S, Rousselle JC, Namane A, Chignard M, Pidard D
The Pseudomonas aeruginosa LasB metalloproteinase regulates the human urokinase-type plasminogen activator receptor through domain-specific endoproteolysis.
Infect Immun. 2007 Aug;75(8):3848-58.
Pseudomonas aeruginosa is an opportunistic pathogen in human lungs, where its secretable LasB metalloproteinase can be a virulence factor. The urokinase-type plasminogen activator receptor (uPAR) participates in pericellular proteolysis and the adherence/migration of epithelial cells and leukocytes recruited during infection and shows functional regulation by various proteinases via limited endoproteolysis occurring within its three domains (D1 to D3). We thus examined the proteolytic activity of LasB on uPAR by using recombinant uPAR as well as uPAR-expressing, human monocytic, and bronchial epithelial cell lines. Protein immunoblotting and flow immunocytometry using a panel of domain-specific anti-uPAR antibodies showed that LasB is able to cleave uPAR both within the sequence linking D1 to D2 and at the carboxy terminus of D3. Comparison of LasB-producing and LasB-deficient bacterial strains indicated that LasB is entirely responsible for the uPAR cleavage ability of P. aeruginosa. Based on amino-terminal protein microsequencing and mass spectrometry analysis of the cleavage of peptides mimicking the uPAR sequences targeted by LasB, cleavage sites were determined to be Ala(84)-Val(85) and Thr(86)-Tyr(87) (D1-D2) and Gln(279)-Tyr(280) (D3). Such a dual cleavage of uPAR led to the removal of amino-terminal D1, the generation of a truncated D2D3 species, and the shedding of D2D3 from cells. This proteolytic processing of uPAR was found to (i) drastically reduce the capacity of cells to bind urokinase and (ii) abrogate the interaction between uPAR and the matrix adhesive protein vitronectin. The LasB proteinase is thus endowed with a high potential for the alteration of uPAR expression and functioning on inflammatory cells during infections by P. aeruginosa. [Abstract/Link to Full Text]

Cole LE, Shirey KA, Barry E, Santiago A, Rallabhandi P, Elkins KL, Puche AC, Michalek SM, Vogel SN
Toll-like receptor 2-mediated signaling requirements for Francisella tularensis live vaccine strain infection of murine macrophages.
Infect Immun. 2007 Aug;75(8):4127-37.
Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously that F. tularensis live vaccine strain (LVS) elicited strong, dose-dependent NF-kappaB reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report that F. tularensis LVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2(-/-) macrophages. F. tularensis LVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers from F. tularensis LVS-infected mice. Colocalization of intracellular F. tularensis LVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if the F. tularensis LVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis. F. tularensis LVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVSDeltaguaA, an F. tularensis LVS guanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-kappaB in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-type F. tularensis LVS. Collectively, these data indicate that the primary macrophage response to F. tularensis LVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellular F. tularensis replication. [Abstract/Link to Full Text]

Simmons WL, Dybvig K
Biofilms protect Mycoplasma pulmonis cells from lytic effects of complement and gramicidin.
Infect Immun. 2007 Aug;75(8):3696-9.
The length of the tandem repeat region of the Vsa protein of Mycoplasma pulmonis has previously been shown to modulate the susceptibility of mycoplasmas to killing by complement: cells that produce a short form of the Vsa protein are highly sensitive, and cells producing the long Vsa protein are resistant. In contrast to their differing susceptibilities to complement, the mycoplasmas were highly sensitive to gramicidin irrespective of the length of the Vsa protein produced. We show here that when encased within a biofilm, cells of M. pulmonis producing a short form of the Vsa protein were more resistant to complement and gramicidin than mycoplasmas that were dispersed. The resistance appeared to be localized to those mycoplasmas within tower structures of the biofilms. Biofilm formation may be a mechanism that protects mycoplasmas from host immunity. [Abstract/Link to Full Text]

Hara H, Kawamura I, Nomura T, Tominaga T, Tsuchiya K, Mitsuyama M
Cytolysin-dependent escape of the bacterium from the phagosome is required but not sufficient for induction of the Th1 immune response against Listeria monocytogenes infection: distinct role of Listeriolysin O determined by cytolysin gene replacement.
Infect Immun. 2007 Aug;75(8):3791-801.
Listeria monocytogenes evades the antimicrobial mechanisms of macrophages by escaping from the phagosome into the cytosolic space via a unique cytolysin that targets the phagosomal membrane, listeriolysin O (LLO), encoded by hly. Gamma interferon (IFN-gamma), which is known to play a pivotal role in the induction of Th1-dependent protective immunity in mice, appears to be produced, depending on the bacterial virulence factor. To determine whether the LLO molecule (the major virulence factor of L. monocytogenes) is indispensable or the escape of bacteria from the phagosome is sufficient to induce IFN-gamma production, we first constructed an hly-deleted mutant of L. monocytogenes and then established isogenic L. monocytogenes mutants expressing LLO or ivanolysin O (ILO), encoded by ilo from Listeria ivanovii. LLO-expressing L. monocytogenes was highly capable of inducing IFN-gamma production and Listeria-specific protective immunity, while the hly-deleted mutant was not. In contrast, the level of IFN-gamma induced by ILO-expressing L. monocytogenes was significantly lower both in vitro and in vivo, despite the ability of this strain to escape the phagosome and the intracellular multiplication at a level equivalent to that of LLO-expressing L. monocytogenes. Only a negligible level of protective immunity was induced in mice against challenge with LLO- and ILO-expressing L. monocytogenes. These results clearly show that escape of the bacterium from the phagosome is a prerequisite but is not sufficient for the IFN-gamma-dependent Th1 response against L. monocytogenes, and some distinct molecular nature of LLO is indispensable for the final induction of IFN-gamma that is essentially required to generate a Th1-dependent immune response. [Abstract/Link to Full Text]

Poly F, Ewing C, Goon S, Hickey TE, Rockabrand D, Majam G, Lee L, Phan J, Savarino NJ, Guerry P
Heterogeneity of a Campylobacter jejuni protein that is secreted through the flagellar filament.
Infect Immun. 2007 Aug;75(8):3859-67.
Cj0859c, or FspA, is a small, acidic protein of Campylobacter jejuni that is expressed by a sigma(28) promoter. Analysis of the fspA gene in 41 isolates of C. jejuni revealed two overall variants of the predicted protein, FspA1 and FspA2. Secretion of FspA occurs in broth-grown bacteria and requires a minimum flagellar structure. The addition of recombinant FspA2, but not FspA1, to INT407 cells in vitro resulted in a rapid induction of apoptosis. These data define a novel C. jejuni virulence factor, and the observed heterogeneity among fspA alleles suggests alternate virulence potential among different strains. [Abstract/Link to Full Text]

Hagan EC, Mobley HL
Uropathogenic Escherichia coli outer membrane antigens expressed during urinary tract infection.
Infect Immun. 2007 Aug;75(8):3941-9.
Uncomplicated urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) represents a prevalent and potentially severe infectious disease. In this study, we describe the application of an immunoproteomics approach to vaccine development that has been used successfully to identify vaccine targets in other pathogenic bacteria. Outer membranes were isolated from pyelonephritis strain E. coli CFT073 cultured under conditions that mimic the urinary tract environment, including iron limitation, osmotic stress, human urine, and exposure to uroepithelial cells. To identify antigens that elicit a humoral response during experimental UTI, outer membrane proteins were separated by two-dimensional gel electrophoresis and probed using pooled antisera from 20 CBA/J mice chronically infected with E. coli CFT073. In total, 23 outer membrane antigens, including a novel iron compound receptor, reacted with the antisera and were identified by mass spectrometry. These antigens also included proteins with known roles in UPEC pathogenesis, such as ChuA, IroN, IreA, Iha, IutA, and FliC. These data demonstrate that an antibody response is directed against these virulence-associated factors during UTI. We also show that the genes encoding ChuA, IroN, hypothetical protein c2482, and IutA are significantly more prevalent (P < 0.01) among UPEC strains than among fecal-commensal E. coli isolates. Thus, we suggest that the conserved outer membrane antigens identified in this study could be rational candidates for a UTI vaccine designed to elicit protective immunity against UPEC infection. [Abstract/Link to Full Text]

Leroy M, Cabral H, Figueira M, Bouchet V, Huot H, Ram S, Pelton SI, Goldstein R
Multiple consecutive lavage samplings reveal greater burden of disease and provide direct access to the nontypeable Haemophilus influenzae biofilm in experimental otitis media.
Infect Immun. 2007 Aug;75(8):4158-72.
The typically recovered quantity of nontypeable Haemophilus influenzae (NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi. [Abstract/Link to Full Text]

Ge Y, Rikihisa Y
Surface-exposed proteins of Ehrlichia chaffeensis.
Infect Immun. 2007 Aug;75(8):3833-41.
The surface proteins of Ehrlichia chaffeensis provide an important interface for pathogen-host interactions. To investigate the surface proteins of E. chaffeensis, membrane-impermeable, cleavable Sulfo-NHS-SS-Biotin was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin-agarose affinity purification. The affinity-captured proteins were separated by electrophoresis, and five relatively abundant protein bands containing immunoreactive proteins were subjected to capillary-liquid chromatography-nanospray tandem mass spectrometry analysis. Nineteen out of 22 OMP-1/P28 family proteins, including P28 (which previously was shown to be surface exposed), were detected in E. chaffeensis cultured in human monocytic leukemia THP-1 cells. For the first time, with the exception of P28 and P28-1, 17 OMP-1/P28 family proteins were demonstrated to be expressed at the protein level. The surface exposure of OMP-1A and OMP-1N was verified by immunofluorescence microscopy. OMP-1B was undetectable either by surface biotinylation or by Western blotting of the whole bacterial lysate, suggesting that it is not expressed by E. chaffeensis cultured in THP-1 cells. Additional E. chaffeensis surface proteins detected were OMP85, hypothetical protein ECH_0525 (here named Esp73), immunodominant surface protein gp47, and 11 other proteins. The identification of E. chaffeensis surface-exposed proteins provides novel insights into the E. chaffeensis surface and lays the foundation for rational studies on pathogen-host interactions and vaccine development. [Abstract/Link to Full Text]

Alteri CJ, Mobley HL
Quantitative profile of the uropathogenic Escherichia coli outer membrane proteome during growth in human urine.
Infect Immun. 2007 Jun;75(6):2679-88.
Outer membrane proteins (OMPs) of microbial pathogens are critical components that mediate direct interactions between microbes and their surrounding environment. Consequently, the study of OMPs is integral to furthering the understanding of host-pathogen interactions and to identifying key targets for development of improved antimicrobial agents and vaccines. In this study, we used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and tandem mass spectrometry to characterize the uropathogenic Escherichia coli (UPEC) outer membrane subproteome; 30 individual OMPs present on the bacterial surface during growth in human urine were identified. Fluorescence difference gel electrophoresis was used to identify quantitative changes in levels of UPEC strain CFT073 OMPs during growth in urine; six known receptors for iron compounds were induced in this environment, i.e., ChuA, IutA, FhuA, IroN, IreA, and Iha. A seventh putative iron compound receptor, encoded by CFT073 open reading frame (ORF) c2482, was also identified and found to be induced in urine. Further, the induction of these seven iron receptors in human urine and during defined iron limitation was verified by using quantitative real-time PCR (qPCR). An eighth iron receptor, fepA, displayed similar induction levels under these conditions as measured by qPCR but was not identified by 2D-PAGE. Addition of 10 microM FeCl(2) to human urine repressed the transcription of all eight iron receptor genes. A number of fecal-commensal, intestinal pathogenic, and uropathogenic E. coli strains all displayed similar growth rates in human urine, showing that the ability to grow in urine per se is not a urovirulence trait. Thus, human urine is an iron-limiting environment and UPEC enriches its outer membrane with iron receptors to contend with this iron limitation. [Abstract/Link to Full Text]

Fan W, Idnurm A, Breger J, Mylonakis E, Heitman J
Eca1, a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, is involved in stress tolerance and virulence in Cryptococcus neoformans.
Infect Immun. 2007 Jul;75(7):3394-405.
The basidiomycetous fungal pathogen Cryptococcus neoformans is adapted to survive challenges in the soil and environment and within the unique setting of the mammalian host. A C. neoformans mutant was isolated with enhanced virulence in a soil amoeba model that nevertheless exhibits dramatically reduced growth at mammalian body temperature (37 degrees C). This mutant phenotype results from an insertion in the ECA1 gene, which encodes a sarcoplasmic/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA)-type calcium pump. Infection in murine macrophages, amoebae (Acanthamoeba castellanii), nematodes (Caenorhabditis elegans), and wax moth (Galleria mellonella) larvae revealed that the eca1 mutants are virulent or hypervirulent at permissive growth temperatures but attenuated at 37 degrees C. Deletion mutants lacking the entire ECA1 gene were also hypersensitive to the calcineurin inhibitors cyclosporin and FK506 and to ER and osmotic stresses. An eca1Delta cna1Delta mutant lacking both Eca1 and the calcineurin catalytic subunit was more sensitive to high temperature and ER stresses than the single mutants and exhibited reduced survival in C. elegans and attenuated virulence towards wax moth larvae at temperatures that permit normal growth in vitro. Eca1 is likely involved in maintaining ER function, thus contributing to stress tolerance and virulence acting in parallel with Ca2+-calcineurin signaling. [Abstract/Link to Full Text]

Lin MY, Geluk A, Smith SG, Stewart AL, Friggen AH, Franken KL, Verduyn MJ, van Meijgaarden KE, Voskuil MI, Dockrell HM, Huygen K, Ottenhoff TH, Klein MR
Lack of immune responses to Mycobacterium tuberculosis DosR regulon proteins following Mycobacterium bovis BCG vaccination.
Infect Immun. 2007 Jul;75(7):3523-30.
Mycobacterium bovis BCG is widely used as a vaccine against tuberculosis (TB), despite its variable protective efficacy. Relatively little is known about the immune response profiles following BCG vaccination in relation to protection against TB. Here we tested whether BCG vaccination results in immune responses to DosR (Rv3133c) regulon-encoded proteins. These so-called TB latency antigens are targeted by the immune system during persistent Mycobacterium tuberculosis infection and have been associated with immunity against latent M. tuberculosis infection. In silico analysis of the DosR regulon in BCG and M. tuberculosis showed at least 97% amino acid sequence homology, with 41 out of 48 genes being identical. Transcriptional profiling of 14 different BCG strains, under hypoxia and nitric oxide exposure in vitro, revealed a functional DosR regulon similar to that observed in M. tuberculosis. Next, we assessed human immune responses to a series of immunodominant TB latency antigens and found that BCG vaccination fails to induce significant responses to latency antigens. Similar results were obtained with BCG-vaccinated BALB/c mice. In contrast, responses to latency antigens were observed in individuals with suspected exposure to TB (as indicated by positive gamma interferon responses to TB-specific antigens ESAT-6 and CFP-10) and in mice vaccinated with plasmid DNA encoding selected latency antigens. Since immune responses to TB latency antigens have been associated with control of latent M. tuberculosis infection, our findings support the development of vaccination strategies incorporating DosR regulon antigens to complement and improve the current BCG vaccine. [Abstract/Link to Full Text]

Taylor JM, Ziman ME, Fong J, Solnick JV, Vajdy M
Possible correlates of long-term protection against Helicobacter pylori following systemic or combinations of mucosal and systemic immunizations.
Infect Immun. 2007 Jul;75(7):3462-9.
The ability to induce long-term immunity to Helicobacter pylori is necessary for an effective vaccine. This study was designed to establish the most efficient route(s) (systemic, mucosal, or a combination) of immunization for induction of long-term immunity and to define correlates of protection. Mice were immunized orally alone (oral group), intramuscularly (i.m.) alone (i.m. group), orally followed by i.m. (oral/i.m. group), or i.m. followed by orally (i.m./oral group). Long-term protective immunity to oral H. pylori challenge was observed 3 months after immunization through the i.m. or oral/i.m. route. Protection correlated with an increase in H. pylori-specific interleukin-12 and both immunoglobulin G1 (IgG1) and IgG2a serum titers following challenge. Mice that were not protected (oral or i.m./oral) had increased levels of IgA in both sera and Peyer's patches. This study demonstrates the ability to induce long-term immunity against H. pylori, provides correlates of protection, and illustrates the crucial role of the immunization route(s). [Abstract/Link to Full Text]

Weissman SJ, Beskhlebnaya V, Chesnokova V, Chattopadhyay S, Stamm WE, Hooton TM, Sokurenko EV
Differential stability and trade-off effects of pathoadaptive mutations in the Escherichia coli FimH adhesin.
Infect Immun. 2007 Jul;75(7):3548-55.
FimH is the tip adhesin of mannose-specific type 1 fimbriae of Escherichia coli, which are critical to the pathogenesis of urinary tract infections. Point FimH mutations increasing monomannose (1M)-specific uroepithelial adhesion are commonly found in uropathogenic strains of E. coli. Here, we demonstrate the emergence of a mixed population of clonally identical E. coli strains in the urine of a patient with acute cystitis, where half of the isolates carried a glycine-to-arginine substitution at position 66 of the mature FimH. The R66 mutation induced an unusually strong 1M-binding phenotype and a 20-fold advantage in mouse bladder colonization. However, E. coli strains carrying FimH-R66, but not the parental FimH-G66, had disappeared from the patient's rectal and urine samples collected from 29 to 44 days later, demonstrating within-host instability of the R66 mutation. No FimH variants with R66 were identified in a large (>600 strains) sequence database of fimH-positive E. coli strains. However, several strains carrying genes encoding FimH with either S66 or C66 mutations appeared to be relatively stable in the E. coli population. Relative to FimH-R66, the FimH-S66 and FimH-C66 variants mediated only moderate increases in 1M binding but preserved the ability to enhance binding under flow-induced shear conditions. In contrast, FimH-R66 completely lost shear-enhanced binding properties, with bacterial adhesion being inhibited by shear forces and lacking a rolling mode of binding. These functional trade-offs may determine the natural populational instability of this mutation or other pathoadaptive FimH mutations that confer dramatic increases in 1M binding strength. [Abstract/Link to Full Text]

de Filippis I, de Andrade CF, Silva L, Prevots DR, Vicente AC
PorA variable antigenic regions VR1, VR2, and VR3 of Neisseria meningitidis serogroups B and C isolated in Brazil from 1999 to 2004.
Infect Immun. 2007 Jul;75(7):3683-5.
The high genetic diversity found among the PorA regions VR1 and VR2 of 101 Neisseria meningitidis isolates from patients with meningococcal disease and healthy carriers in Brazil contrasts with the stability found in the PorA VR3 of these isolates. The presence of VR3 epitope variant 35 or 36 on the surfaces of 87% of the strains analyzed suggests that these antigens should be considered for inclusion in new formulations of vaccines against serogroup B meningococci in Brazil. [Abstract/Link to Full Text]

Chinchilla M, Pasetti MF, Medina-Moreno S, Wang JY, Gomez-Duarte OG, Stout R, Levine MM, Galen JE
Enhanced immunity to Plasmodium falciparum circumsporozoite protein (PfCSP) by using Salmonella enterica serovar Typhi expressing PfCSP and a PfCSP-encoding DNA vaccine in a heterologous prime-boost strategy.
Infect Immun. 2007 Aug;75(8):3769-79.
Two Salmonella enterica serovar Typhi strains that express and export a truncated version of Plasmodium falciparum circumsporozoite surface protein (tCSP) fused to Salmonella serovar Typhi cytolysin A (ClyA) were constructed as a first step in the development of a preerythrocytic malaria vaccine. Synthetic codon-optimized genes (t-csp1 and t-csp2), containing immunodominant B- and T-cell epitopes present in native P. falciparum circumsporozoite surface protein (PfCSP), were fused in frame to the carboxyl terminus of the ClyA gene (clyA::t-csp) in genetically stabilized expression plasmids. Expression and export of ClyA-tCSP1 and ClyA-tCSP2 by Salmonella serovar Typhi vaccine strain CVD 908-htrA were demonstrated by immunoblotting of whole-cell lysates and culture supernatants. The immunogenicity of these constructs was evaluated using a "heterologous prime-boost" approach consisting of mucosal priming with Salmonella serovar Typhi expressing ClyA-tCSP1 and ClyA-tCSP2, followed by parenteral boosting with PfCSP DNA vaccines pVR2510 and pVR2571. Mice primed intranasally on days 0 and 28 with CVD 908-htrA(pSEC10tcsp2) and boosted intradermally on day 56 with PfCSP DNA vaccine pVR2571 induced high titers of serum NANP immunoglobulin G (IgG) (predominantly IgG2a); no serological responses to DNA vaccination were observed in the absence of Salmonella serovar Typhi-PfCSP priming. Mice primed with Salmonella serovar Typhi expressing tCSP2 and boosted with PfCSP DNA also developed high frequencies of gamma interferon-secreting cells, which surpassed those produced by PfCSP DNA in the absence of priming. A prime-boost regimen consisting of mucosal delivery of PfCSP exported from a Salmonella-based live-vector vaccine followed by a parenteral PfCSP DNA boosting is a promising strategy for the development of a live-vector-based malaria vaccine. [Abstract/Link to Full Text]

Liu M, Zhu H, Zhang J, Lei B
Active and passive immunizations with the streptococcal esterase Sse protect mice against subcutaneous infection with group A streptococci.
Infect Immun. 2007 Jul;75(7):3651-7.
The human pathogen group A Streptococcus (GAS) produces many secreted proteins that play important roles in GAS pathogenesis, including hydrolases that degrade proteins and nucleic acids. This study targets another kind of hydrolase, carboxylic esterase, with the objectives of identifying GAS esterase and determining whether it is a protective antigen. The putative esterase gene SPy1718 was cloned, and the recombinant protein (Sse) was prepared. Sse was detected in GAS culture supernatant, and patients with streptococcal pharyngitis seroconverted to Sse, indicating that Sse was produced in vivo and in vitro. Sse hydrolyzes p-nitrophenyl butyrate, and the residue (178)Ser is critical for this esterase activity. There are two Sse variant complexes according to the available genome databases, consistent with the previous finding of two antigenic Sse variants. Complex I includes serotypes M1, M2, M3, M5, M6, M12, and M18, whereas M4, M28, and M49 belong to complex II. Sse variants share >98% identity in amino acid sequence within each complex but have about 37% variation between the two groups. Active immunization with M1 Sse significantly protects mice against lethal subcutaneous infection with virulent M1 and M3 strains and inhibits GAS invasion of mouse skin tissue. Passive immunization with anti-Sse antiserum also significantly protects mice against subcutaneous GAS infection, indicating that the protection is mediated by Sse-specific antibodies. The results suggest that Sse plays an important role in tissue invasion and is an antigen protective in subcutaneous infection against GAS strains of more than one serotype. [Abstract/Link to Full Text]

Wu B, Huang C, Garcia L, Ponce de Leon A, Osornio JS, Bobadilla-del-Valle M, Ferreira L, Canizales S, Small P, Kato-Maeda M, Krensky AM, Clayberger C
Unique gene expression profiles in infants vaccinated with different strains of Mycobacterium bovis bacille Calmette-Guerin.
Infect Immun. 2007 Jul;75(7):3658-64.
Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) has variable efficacy in preventing tuberculosis. We hypothesized that some of this variation might be due to differences among BCG strains. To test this, neonates in Orizaba, Mexico, were vaccinated with one of three different BCG strains (BCG-Brazil [BBCG], BCG-Denmark [DBCG], or BCG-Japan [JBCG]). One year after vaccination, peripheral blood mononuclear cells (PBMC) were obtained and recall immune responses to culture filtrate proteins (CFP) of Mycobacterium tuberculosis were evaluated using quantitative real-time PCR. CFP-activated PBMC from BBCG- and DBCG-immunized children expressed high levels of cytokines characteristic of an adaptive immune response (gamma interferon, interleukin-2beta [IL-12beta], and IL-27), while those from children immunized with JBCG did not. In contrast, vaccination with JBCG resulted in significantly greater expression of cytokines characteristic of a proinflammatory immune response (IL-1alpha, IL-1beta, IL-6, and IL-24) in PBMC activated with CFP compared to PBMC from children vaccinated with BBCG or DBCG. Thus, different strains of BCG can activate different immune pathways, which may affect long-term vaccine efficacy. [Abstract/Link to Full Text]

Rey-Ladino J, Jiang X, Gabel BR, Shen C, Brunham RC
Survival of Chlamydia muridarum within dendritic cells.
Infect Immun. 2007 Aug;75(8):3707-14.
Immune responses to Chlamydia trachomatis underlay both immunity and immunopathology. Immunopathology in turn has been attributed to chronic persistent infection with persistence being defined as the presence of organisms in the absence of replication. We hypothesized that dendritic cells (DCs) play a central role in Chlamydia immunity and immunopathology by favoring the long-term survival of C. muridarum. This hypothesis was examined based on (i) direct staining of Chlamydia in infected DCs to evaluate the development of inclusions, (ii) titration of infected DCs on HeLa cells to determine cultivability, and (iii) transfer of Chlamydia-infected DCs to naive mice to evaluate infectivity. The results show that Chlamydia survived within DCs and developed both typical and atypical inclusions that persisted in a subpopulation of DCs for more than 9 days after infection. Since the cultivability of Chlamydia from DCs onto HeLa was lower than that estimated by the number of inclusions in DCs, this suggests that the organisms may be in state of persistence. Intranasal transfer of long-term infected DCs or DCs purified from the lungs of infected mice caused mouse lung infection, suggesting that in addition to persistent forms, infective Chlamydia organisms also developed within chronically infected DCs. Interestingly, after in vitro infection with Chlamydia, most DCs died. However, Chlamydia appeared to survive in a subpopulation of DCs that resisted infection-induced cell death. Surviving DCs efficiently presented Chlamydia antigens to Chlamydia-specific CD4+ T cells, suggesting that the bacteria are able to both direct their own survival and still allow DC antigen-presenting function. Together, these results raise the possibility that Chlamydia-infected DCs may be central to the maintenance of T-cell memory that underlies both immunity and immunopathology. [Abstract/Link to Full Text]

Pettersson J, Schrumpf ME, Raffel SJ, Porcella SF, Guyard C, Lawrence K, Gherardini FC, Schwan TG
Purine salvage pathways among Borrelia species.
Infect Immun. 2007 Aug;75(8):3877-84.
Genome sequencing projects on two relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed differences in genes involved in purine metabolism and salvage compared to those in the Lyme disease spirochete Borrelia burgdorferi. The relapsing fever spirochetes contained six open reading frames that are absent from the B. burgdorferi genome. These genes included those for hypoxanthine-guanine phosphoribosyltransferase (hpt), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), auxiliary protein (nrdI), the ribonucleotide-diphosphate reductase alpha subunit (nrdE), and the ribonucleotide-diphosphate reductase beta subunit (nrdF). Southern blot assays with multiple Borrelia species and isolates confirmed the presence of these genes in the relapsing fever group of spirochetes but not in B. burgdorferi and related species. TaqMan real-time reverse transcription-PCR demonstrated that the chromosomal genes (hpt, purA, and purB) were transcribed in vitro and in mice. Phosphoribosyltransferase assays revealed that, in general, B. hermsii exhibited significantly higher activity than did the B. burgdorferi cell lysate, and enzymatic activity was observed with adenine, hypoxanthine, and guanine as substrates. B. burgdorferi showed low but detectable phosphoribosyltransferase activity with hypoxanthine even though the genome lacks a discernible ortholog to the hpt gene in the relapsing fever spirochetes. B. hermsii incorporated radiolabeled hypoxanthine into RNA and DNA to a much greater extent than did B. burgdorferi. This complete pathway for purine salvage in the relapsing fever spirochetes may contribute, in part, to these spirochetes achieving high cell densities in blood. [Abstract/Link to Full Text]

MacEachran DP, Ye S, Bomberger JM, Hogan DA, Swiatecka-Urban A, Stanton BA, O'Toole GA
The Pseudomonas aeruginosa secreted protein PA2934 decreases apical membrane expression of the cystic fibrosis transmembrane conductance regulator.
Infect Immun. 2007 Aug;75(8):3902-12.
We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl(-) ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in alpha/beta hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe. [Abstract/Link to Full Text]

Weaver J, Kang TJ, Raines KW, Cao GL, Hibbs S, Tsai P, Baillie L, Rosen GM, Cross AS
Protective role of Bacillus anthracis exosporium in macrophage-mediated killing by nitric oxide.
Infect Immun. 2007 Aug;75(8):3894-901.
The ability of the endospore-forming, gram-positive bacterium Bacillus anthracis to survive in activated macrophages is key to its germination and survival. In a previous publication, we discovered that exposure of primary murine macrophages to B. anthracis endospores upregulated NOS 2 concomitant with an .NO-dependent bactericidal response. Since NOS 2 also generates O(2).(-), experiments were designed to determine whether NOS 2 formed peroxynitrite (ONOO(-)) from the reaction of .NO with O(2).(-) and if so, was ONOO(-) microbicidal toward B. anthracis. Our findings suggest that ONOO(-) was formed upon macrophage infection by B. anthracis endospores; however, ONOO(-) does not appear to exhibit microbicidal activity toward this bacterium. In contrast, the exosporium of B. anthracis, which exhibits arginase activity, protected B. anthracis from macrophage-mediated killing by decreasing .NO levels in the macrophage. Thus, the ability of B. anthracis to subvert .NO production has important implications in the control of B. anthracis-induced infection. [Abstract/Link to Full Text]

Hybiske K, Stephens RS
Mechanisms of Chlamydia trachomatis entry into nonphagocytic cells.
Infect Immun. 2007 Aug;75(8):3925-34.
The mechanisms of entry for the obligate intracellular bacterium C. trachomatis were examined by functional disruption of proteins essential for various modes of entry. RNA interference was used to disrupt proteins with established roles in clathrin-mediated endocytosis (clathrin heavy chain, dynamin-2, heat shock 70-kDa protein 8, Arp2, cortactin, and calmodulin), caveola-mediated endocytosis (caveolin-1, dynamin-2, Arp2, NSF, and annexin II), phagocytosis (RhoA, dynamin-2, Rac1, and Arp2), and macropinocytosis (Pak1, Rac1, and Arp2). Comparative quantitative PCR analysis was performed on small interfering RNA-transfected HeLa cells to accurately determine the extent of C. trachomatis entry after these treatments. Key structural and regulatory factors associated with clathrin-mediated endocytosis were found to be involved in Chlamydia entry, whereas those for caveola-mediated endocytosis, phagocytosis, and macropinocytosis were not. Thus, clathrin and its coordinate accessory factors were required for entry of C. trachomatis, although additional, uncharacterized mechanisms are also utilized. [Abstract/Link to Full Text]

Singh UP, Singh S, Singh R, Karls RK, Quinn FD, Potter ME, Lillard JW
Influence of Mycobacterium avium subsp. paratuberculosis on colitis development and specific immune responses during disease.
Infect Immun. 2007 Aug;75(8):3722-8.
The granulomatous and intramural inflammation observed in cases of inflammatory bowel diseases (IBD) and veterinary Johne's disease suggests that Mycobacterium avium subsp. paratuberculosis is a causative agent. However, an incomplete understanding of the immunological steps responsible for the pathologies of IBD makes this conclusion uncertain. Sera from interleukin-10-deficient (IL-10(-/-)) mice with spontaneous colitis displayed significantly higher M. avium subsp. paratuberculosis-specific immunoglobulin G2a antibody responses than did sera from similar mice without disease. Pathogen-free IL-10(-/-) mice received control vehicle or the vehicle containing heat-killed or live M. avium subsp. paratuberculosis. Mucosal CD4(+) T cells from the mice that developed colitis proliferated and secreted higher levels of gamma interferon and tumor necrosis factor alpha after ex vivo stimulation with a Vbeta11(+) T-cell receptor-restricted peptide from the MPT59 antigen (Ag85B) than those secreted from cells from mice before the onset of colitis. The data from this study provide important information regarding the mechanisms of colitis in IL-10(-/-) mice, which are driven in part by Ag85B-specific T cells. The data suggest a plausible mechanism of Ag-specific T-cell responses in colitis driven by potent Ags conserved in Mycobacterium species. [Abstract/Link to Full Text]

Coimbra VC, Yamamoto D, Khusal KG, Atayde VD, Fernandes MC, Mortara RA, Yoshida N, Alves MJ, Rabinovitch M
Enucleated L929 cells support invasion, differentiation, and multiplication of Trypanosoma cruzi parasites.
Infect Immun. 2007 Aug;75(8):3700-6.
Cell infection with Trypanosoma cruzi, the agent of Chagas' disease, begins with the uptake of infective trypomastigotes within phagosomes and their release into the cytosol, where they transform into replicating amastigotes; the latter, in turn, differentiate into cytolytically released and infective trypomastigotes. We ask here if the T. cruzi infection program can develop in enucleated host cells. Monolayers of L929 cells, enucleated by centrifugation in the presence of cytochalasin B and kept at 34 degrees C to extend the survival of cytoplasts, were infected with parasites of the CL strain. Percent infection, morphology, stage-specific markers, and numbers of parasites per cell were evaluated in nucleated and enucleated cells, both of which were present in the same preparations. Parasite uptake, differentiation and multiplication of amastigotes, development of epimastigote- and trypomastigote-like forms, and initial cytolytic release of parasites were all documented for cytoplasts and nucleated cells. Although the doubling times were similar, parasite loads at 48 and 72 h were significantly lower in the cytoplasts than in nucleated cells. Similar results were obtained with the highly virulent strain Y as well as with strains CL-14 and G, which exhibit low virulence for mice. Cytoplasts could also be infected with the CL strain 24 or 48 h after enucleation. Thus, infection of cells by T. cruzi can take place in enucleated host cells, i.e., in the absence of modulation of chromosomal and nucleolar gene transcription and of RNA modification and processing in the nucleus. [Abstract/Link to Full Text]

Gentry M, Taormina J, Pyles RB, Yeager L, Kirtley M, Popov VL, Klimpel G, Eaves-Pyles T
Role of primary human alveolar epithelial cells in host defense against Francisella tularensis infection.
Infect Immun. 2007 Aug;75(8):3969-78.
Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-kappaB activation. ATII cells pretreated with an NF-kappaB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis. [Abstract/Link to Full Text]

Klemm P, Hancock V, Schembri MA
Mellowing out: adaptation to commensalism by Escherichia coli asymptomatic bacteriuria strain 83972.
Infect Immun. 2007 Aug;75(8):3688-95. [Abstract/Link to Full Text]


Recent Articles in Journal of Bacteriology

Wilks JC, Slonczewski JL
pH of the cytoplasm and periplasm of Escherichia coli: rapid measurement by green fluorescent protein fluorimetry.
J Bacteriol. 2007 Aug;189(15):5601-7.
Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the external pH was lowered from pH 7.5 to 5.5, the cytoplasmic pH fell within 10 to 20 s to pH 5.6 to 6.5. Rapid recovery occurred until about 30 s after HCl addition and was followed by slower recovery over the next 5 min. As a control, KCl addition had no effect on fluorescence. In the presence of 5 to 10 mM acetate or benzoate, recovery from external acidification was diminished. Addition of benzoate at pH 7.0 resulted in cytoplasmic acidification with only slow recovery. Periplasmic pH was observed using TorA-GFPmut3* exported to the periplasm through the Tat system. The periplasmic location of the fusion protein was confirmed by the observation that osmotic shock greatly decreased the periplasmic fluorescence signal by loss of the protein but had no effect on the fluorescence of the cytoplasmic protein. Based on GFPmut3* fluorescence, the pH of the periplasm equaled the external pH under all conditions tested, including rapid acid shift. Benzoate addition had no effect on periplasmic pH. The cytoplasmic pH of E. coli was measured with 4-s time resolution using a method that can be applied to any strain construct, and the periplasmic pH was measured directly for the first time. [Abstract/Link to Full Text]

Punginelli C, Maldonado B, Grahl S, Jack R, Alami M, Schröder J, Berks BC, Palmer T
Cysteine scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC Component.
J Bacteriol. 2007 Aug;189(15):5482-94.
The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices, rather than the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperatures. These results are consistent with the likelihood that most of the disulfide cross-links are between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex. [Abstract/Link to Full Text]

Maiques E, Ubeda C, Tormo MA, Ferrer MD, Lasa I, Novick RP, Penadés JR
Role of staphylococcal phage and SaPI integrase in intra- and interspecies SaPI transfer.
J Bacteriol. 2007 Aug;189(15):5608-16.
SaPIbov2 is a member of the SaPI family of staphylococcal pathogenicity islands and is very closely related to SaPIbov1. Typically, certain temperate phages can induce excision and replication of one or more of these islands and can package them into special small phage-like particles commensurate with their genome sizes (referred to as the excision-replication-packaging [ERP] cycle). We have studied the phage-SaPI interaction in some depth using SaPIbov2, with special reference to the role of its integrase. We demonstrate here that SaPIbov2 can be induced to replicate by different staphylococcal phages. After replication, SaPIbov2 is efficiently encapsidated and transferred to recipient organisms, including different non-Staphylococcus aureus staphylococci, where it integrates at a SaPI-specific attachment site, att(C), by means of a self-coded integrase (Int). Phages that cannot induce the SaPIbov2 ERP cycle can transfer the island by recA-dependent classical generalized transduction and can also transfer it by a novel mechanism that requires the expression of SaPIbov2 int in the recipient but not in the donor. It is suggested that this mechanism involves the encapsidation of standard transducing fragments containing the intact island followed by int-mediated excision, circularization, and integration in the recipient. [Abstract/Link to Full Text]

Li W, Lu CD
Regulation of carbon and nitrogen utilization by CbrAB and NtrBC two-component systems in Pseudomonas aeruginosa.
J Bacteriol. 2007 Aug;189(15):5413-20.
The global effect of the CbrAB and NtrBC two-component systems on the control of carbon and nitrogen utilization in Pseudomonas aeruginosa was characterized by phenotype microarray analyses with single and double mutants and the isogenic parent strain. The tested compounds were clustered based on the growth phenotypes of these strains, and the results clearly demonstrated the pivotal roles of CbrAB and NtrBC in carbon and nitrogen utilization, respectively. Growth of the cbrAB deletion mutant on arginine, histidine, and polyamines used as the sole carbon source was abolished, while growth on the tricarboxylic acid (TCA) cycle intermediates was sustained. In this study, suppressors of the cbr mutant were selected from minimal medium containing l-arginine as the sole carbon and nitrogen source. These mutants fell into two groups according to the ability to utilize histidine. The genomic library of a histidine-positive suppressor mutant was constructed, and the corresponding suppressor gene was identified by complementation as an ntrB allele. Similar results were obtained from four additional suppressor mutants, and point mutations of these ntrB alleles resulting in the following changes in residues were identified, with implications for reduced phosphatase activities: L126W, D227A, P228L, and S229I. The Ntr systems of these ntrB mutants became constitutively active, as revealed by the activity profiles of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. As a result, these mutants not only regain the substrate-specific induction on catabolic arginine and histidine operons but are also expressed to higher levels than the wild type. While the DeltacbrAB ntrB(Con) mutant restored growth on many N-containing compounds used as the carbon sources, its capability to grow on TCA cycle intermediates and glucose was compromised when ammonium served as the sole nitrogen source, mostly due to an extreme imbalance of carbon and nitrogen regulatory systems. In summary, this study supports the notion that CbrAB and NtrBC form a network to control the C/N balance in P. aeruginosa. Possible molecular mechanisms of these two regulatory elements in the control of arginine and histidine operons used as the model systems are discussed. [Abstract/Link to Full Text]

Saumaa S, Tover A, Tark M, Tegova R, Kivisaar M
Oxidative DNA damage defense systems in avoidance of stationary-phase mutagenesis in Pseudomonas putida.
J Bacteriol. 2007 Aug;189(15):5504-14.
Oxidative damage of DNA is a source of mutation in living cells. Although all organisms have evolved mechanisms of defense against oxidative damage, little is known about these mechanisms in nonenteric bacteria, including pseudomonads. Here we have studied the involvement of oxidized guanine (GO) repair enzymes and DNA-protecting enzyme Dps in the avoidance of mutations in starving Pseudomonas putida. Additionally, we examined possible connections between the oxidative damage of DNA and involvement of the error-prone DNA polymerase (Pol)V homologue RulAB in stationary-phase mutagenesis in P. putida. Our results demonstrated that the GO repair enzymes MutY, MutM, and MutT are involved in the prevention of base substitution mutations in carbon-starved P. putida. Interestingly, the antimutator effect of MutT was dependent on the growth phase of bacteria. Although the lack of MutT caused a strong mutator phenotype under carbon starvation conditions for bacteria, only a twofold increased effect on the frequency of mutations was observed for growing bacteria. This indicates that MutT has a backup system which efficiently complements the absence of this enzyme in actively growing cells. The knockout of MutM affected only the spectrum of mutations but did not change mutation frequency. Dps is known to protect DNA from oxidative damage. We found that dps-defective P. putida cells were more sensitive to sudden exposure to hydrogen peroxide than wild-type cells. At the same time, the absence of Dps did not affect the accumulation of mutations in populations of starved bacteria. Thus, it is possible that the protective role of Dps becomes essential for genome integrity only when bacteria are exposed to exogenous agents that lead to oxidative DNA damage but not under physiological conditions. Introduction of the Y family DNA polymerase PolV homologue rulAB into P. putida increased the proportion of A-to-C and A-to-G base substitutions among mutations, which occurred under starvation conditions. Since PolV is known to perform translesion synthesis past damaged bases in DNA (e.g., some oxidized forms of adenine), our results may imply that adenine oxidation products are also an important source of mutation in starving bacteria. [Abstract/Link to Full Text]

Lasocki K, Bartosik AA, Mierzejewska J, Thomas CM, Jagura-Burdzy G
Deletion of the parA (soj) homologue in Pseudomonas aeruginosa causes ParB instability and affects growth rate, chromosome segregation, and motility.
J Bacteriol. 2007 Aug;189(15):5762-72.
The parA and parB genes of Pseudomonas aeruginosa are located approximately 8 kb anticlockwise from oriC. ParA is a cytosolic protein present at a level of around 600 molecules per cell in exponential phase, but the level drops about fivefold in stationary phase. Overproduction of full-length ParA or the N-terminal 85 amino acids severely inhibits growth of P. aeruginosa and P. putida. Both inactivation of parA and overexpression of parA in trans in P. aeruginosa also lead to accumulation of anucleate cells and changes in motility. Inactivation of parA also increases the turnover rate (degradation) of ParB. This may provide a mechanism for controlling the level of ParB in response to the growth rate and expression of the parAB operon. [Abstract/Link to Full Text]

Klein AH, Shulla A, Reimann SA, Keating DH, Wolfe AJ
The intracellular concentration of acetyl phosphate in Escherichia coli is sufficient for direct phosphorylation of two-component response regulators.
J Bacteriol. 2007 Aug;189(15):5574-81.
Acetyl phosphate, the intermediate of the AckA-Pta pathway, acts as a global signal in Escherichia coli. Although acetyl phosphate clearly signals through two-component response regulators, it remains unclear whether acetyl phosphate acts as a direct phospho donor or functions through an indirect mechanism. We used two-dimensional thin-layer chromatography to measure the relative concentrations of acetyl phosphate, acetyl coenzyme A, ATP, and GTP over the course of the entire growth curve. We estimated that the intracellular concentration of acetyl phosphate in wild-type cells reaches at least 3 mM, a concentration sufficient to activate two-component response regulators via direct phosphoryl transfer. [Abstract/Link to Full Text]

Pogoryelov D, Reichen C, Klyszejko AL, Brunisholz R, Muller DJ, Dimroth P, Meier T
The oligomeric state of c rings from cyanobacterial F-ATP synthases varies from 13 to 15.
J Bacteriol. 2007 Aug;189(16):5895-902.
We isolated the c rings of F-ATP synthases from eight cyanobacterial strains belonging to four different taxonomic classes (Chroococcales, Nostocales, Oscillatoriales, and Gloeobacteria). These c rings showed different mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), probably reflecting their molecular masses. This supposition was validated with the previously characterized c(11), c(14), and c(15) rings, which migrated on SDS-PAGE in proportion to their molecular masses. Hence, the masses of the cyanobacterial c rings can conveniently be deduced from their electrophoretic mobilities and, together with the masses of the c monomers, allow the calculation of the c ring stoichiometries. The method is a simple and fast way to determine stoichiometries of SDS-stable c rings and hence a convenient means to unambiguously determine the ion-to-ATP ratio, a parameter reflecting the bioenergetic efficacy of F-ATP synthases. AFM imaging was used to prove the accuracy of the method and confirmed that the c ring of Synechococcus elongatus SAG 89.79 is a tridecameric oligomer. Despite the high conservation of the c-subunit sequences from cyanobacterial strains from various environmental groups, the stoichiometries of their c rings varied between c(13) and c(15). This systematic study of the c-ring stoichiometries suggests that variability of c-ring sizes might represent an adaptation of the individual cyanobacterial species to their particular environmental and physiological conditions. Furthermore, the two new examples of c(15) rings underline once more that an F(1)/F(o) symmetry mismatch is not an obligatory feature of all F-ATP synthases. [Abstract/Link to Full Text]

Mukhopadhyay A, Redding AM, Joachimiak MP, Arkin AP, Borglin SE, Dehal PS, Chakraborty R, Geller JT, Hazen TC, He Q, Joyner DC, Martin VJ, Wall JD, Yang ZK, Zhou J, Keasling JD
Cell-wide responses to low-oxygen exposure in Desulfovibrio vulgaris Hildenborough.
J Bacteriol. 2007 Aug;189(16):5996-6010.
The responses of the anaerobic, sulfate-reducing organism Desulfovibrio vulgaris Hildenborough to low-oxygen exposure (0.1% O(2)) were monitored via transcriptomics and proteomics. Exposure to 0.1% O(2) caused a decrease in the growth rate without affecting viability. Concerted upregulation of the predicted peroxide stress response regulon (PerR) genes was observed in response to the 0.1% O(2) exposure. Several of the candidates also showed increases in protein abundance. Among the remaining small number of transcript changes was the upregulation of the predicted transmembrane tetraheme cytochrome c(3) complex. Other known oxidative stress response candidates remained unchanged during the low-O(2) exposure. To fully understand the results of the 0.1% O(2) exposure, transcriptomics and proteomics data were collected for exposure to air using a similar experimental protocol. In contrast to the 0.1% O(2) exposure, air exposure was detrimental to both the growth rate and viability and caused dramatic changes at both the transcriptome and proteome levels. Interestingly, the transcripts of the predicted PerR regulon genes were downregulated during air exposure. Our results highlight the differences in the cell-wide responses to low and high O(2) levels in D. vulgaris and suggest that while exposure to air is highly detrimental to D. vulgaris, this bacterium can successfully cope with periodic exposure to low O(2) levels in its environment. [Abstract/Link to Full Text]

Bachhawat P, Stock AM
Crystal structures of the receiver domain of the response regulator PhoP from Escherichia coli in the absence and presence of the phosphoryl analog beryllofluoride.
J Bacteriol. 2007 Aug;189(16):5987-95.
The response regulator PhoP is part of the PhoQ/PhoP two-component system involved in responses to depletion of extracellular Mg(2+). Here, we report the crystal structures of the receiver domain of Escherichia coli PhoP determined in the absence and presence of the phosphoryl analog beryllofluoride. In the presence of beryllofluoride, the active receiver domain forms a twofold symmetric dimer similar to that seen in structures of other regulatory domains from the OmpR/PhoB family, providing further evidence that members of this family utilize a common mode of dimerization in the active state. In the absence of activating agents, the PhoP receiver domain crystallizes with a similar structure, consistent with the previous observation that high concentrations can promote an active state of PhoP independent of phosphorylation. [Abstract/Link to Full Text]

Cacciapuoti G, Porcelli M, Moretti MA, Sorrentino F, Concilio L, Zappia V, Liu ZJ, Tempel W, Schubot F, Rose JP, Wang BC, Brereton PS, Jenney FE, Adams MW
The first agmatine/cadaverine aminopropyl transferase: biochemical and structural characterization of an enzyme involved in polyamine biosynthesis in the hyperthermophilic archaeon Pyrococcus furiosus.
J Bacteriol. 2007 Aug;189(16):6057-67.
We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the k(cat)/K(m) value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108 degrees C that increases to 112 degrees C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-A resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation. [Abstract/Link to Full Text]

Daigle DM, Cao L, Fraud S, Wilke MS, Pacey A, Klinoski R, Strynadka NC, Dean CR, Poole K
Protein modulator of multidrug efflux gene expression in Pseudomonas aeruginosa.
J Bacteriol. 2007 Aug;189(15):5441-51.
nalC multidrug-resistant mutants of Pseudomonas aeruginosa show enhanced expression of the mexAB-oprM multidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor of mexAB-oprM expression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [K(D)], 158.0 +/- 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting that mexAB-oprM hyperexpression in nalC mutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression of mexA transcription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in a nalC strain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamed armR (antirepressor for MexR). A representative "noninteracting" mutant MexR protein, MexR(I104F), was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced; K(D), 853.2 +/- 151.1 nM). Consistent with this, MexR(I104F) repressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal alpha-helix of the protein that alone is sufficient for interaction with MexR. [Abstract/Link to Full Text]

Giaquinto L, Curmi PM, Siddiqui KS, Poljak A, DeLong E, DasSarma S, Cavicchioli R
Structure and function of cold shock proteins in archaea.
J Bacteriol. 2007 Aug;189(15):5738-48.
Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despite this, not enough is known about the molecular mechanisms of cold adaptation and no biochemical studies have been performed on stenopsychrophilic archaea (e.g., Methanogenium frigidum). This study examined the structural and functional properties of cold shock proteins (Csps) from archaea, including biochemical analysis of the Csp from M. frigidum. csp genes are present in most bacteria and some eucarya but absent from most archaeal genome sequences, most notably, those of all archaeal thermophiles and hyperthermophiles. In bacteria, Csps are small, nucleic acid binding proteins involved in a variety of cellular processes, such as transcription. In this study, archaeal Csp function was assessed by examining the ability of csp genes from psychrophilic and mesophilic Euryarchaeota and Crenarchaeota to complement a cold-sensitive growth defect in Escherichia coli. In addition, an archaeal gene with a cold shock domain (CSD) fold but little sequence identity to Csps was also examined. Genes encoding Csps or a CSD structural analog from three psychrophilic archaea rescued the E. coli growth defect. The three proteins were predicted to have a higher content of solvent-exposed basic residues than the noncomplementing proteins, and the basic residues were located on the nucleic acid binding surface, similar to their arrangement in E. coli CspA. The M. frigidum Csp was purified and found to be a single-domain protein that folds by a reversible two-state mechanism and to exhibit a low conformational stability typical of cold-adapted proteins. Moreover, M. frigidum Csp was characterized as binding E. coli single-stranded RNA, consistent with its ability to complement function in E. coli. The studies show that some Csp and CSD fold proteins have retained sufficient similarity throughout evolution in the Archaea to be able to function effectively in the Bacteria and that the function of the archaeal proteins relates to cold adaptation. The initial biochemical analysis of M. frigidum Csp has developed a platform for further characterization and demonstrates the potential for expanding molecular studies of proteins from this important archaeal stenopsychrophile. [Abstract/Link to Full Text]

Rodrigues MV, Borges N, Henriques M, Lamosa P, Ventura R, Fernandes C, Empadinhas N, Maycock C, da Costa MS, Santos H
Bifunctional CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, the key enzyme for di-myo-inositol-phosphate synthesis in several (hyper)thermophiles.
J Bacteriol. 2007 Aug;189(15):5405-12.
The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3'-phosphate-1'-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3'-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways. [Abstract/Link to Full Text]

Shao H, James D, Lamont RJ, Demuth DR
Differential interaction of Aggregatibacter (Actinobacillus) actinomycetemcomitans LsrB and RbsB proteins with autoinducer 2.
J Bacteriol. 2007 Aug;189(15):5559-65.
Our previous studies showed that the Aggregatibacter actinomycetemcomitans RbsB protein interacts with cognate and heterologous autoinducer 2 (AI-2) signals and suggested that the rbsDABCK operon encodes a transporter that may internalize AI-2 (D. James et al., Infect. Immun. 74:4021-4029, 2006.). However, A. actinomycetemcomitans also possesses genes related to the lsr operon of Salmonella enterica serovar Typhimurium which function to import AI-2. Here, we show that A. actinomycetemcomitans LsrB protein competitively inhibits the interaction of the Vibrio harveyi AI-2 receptor (LuxP) with AI-2 from either A. actinomycetemcomitans or V. harveyi. Interestingly, LsrB was a more potent inhibitor of LuxP interaction with AI-2 from V. harveyi whereas RbsB competed more effectively with LuxP for A. actinomycetemcomitans AI-2. Inactivation of lsrB in wild-type A. actinomycetemcomitans or in an isogenic RbsB-deficient strain reduced the rate by which intact bacteria depleted A. actinomycetemcomitans AI-2 from solution. Consistent with the results from the LuxP competition experiments, the LsrB-deficient strain depleted AI-2 to a lesser extent than the RbsB-deficient organism. Inactivation of both lsrB and rbsB virtually eliminated the ability of the organism to remove AI-2 from the extracellular environment. These results suggest that A. actinomycetemcomitans possesses two proteins that differentially interact with AI-2 and may function to inactivate or facilitate internalization of AI-2. [Abstract/Link to Full Text]

Alcorlo M, González-Huici V, Hermoso JM, Meijer WJ, Salas M
The phage phi29 membrane protein p16.7, involved in DNA replication, is required for efficient ejection of the viral genome.
J Bacteriol. 2007 Aug;189(15):5542-9.
It is becoming clear that in vivo phage DNA ejection is not a mere passive process. In most cases, both phage and host proteins seem to be involved in pulling at least part of the viral DNA inside the cell. The DNA ejection mechanism of Bacillus subtilis bacteriophage phi29 is a two-step process where the linear DNA penetrates the cell with a right-left polarity. In the first step approximately 65% of the DNA is pushed into the cell. In the second step, the remaining DNA is actively pulled into the cytoplasm. This step requires protein p17, which is encoded by the right-side early operon that is ejected during the first push step. The membrane protein p16.7, also encoded by the right-side early operon, is known to play an important role in membrane-associated phage DNA replication. In this work we show that, in addition, p16.7 is required for efficient execution of the second pull step of DNA ejection. [Abstract/Link to Full Text]

Rabsch W, Ma L, Wiley G, Najar FZ, Kaserer W, Schuerch DW, Klebba JE, Roe BA, Laverde Gomez JA, Schallmey M, Newton SM, Klebba PE
FepA- and TonB-dependent bacteriophage H8: receptor binding and genomic sequence.
J Bacteriol. 2007 Aug;189(15):5658-74.
H8 is derived from a collection of Salmonella enterica serotype Enteritidis bacteriophage. Its morphology and genomic structure closely resemble those of bacteriophage T5 in the family Siphoviridae. H8 infected S. enterica serotypes Enteritidis and Typhimurium and Escherichia coli by initial adsorption to the outer membrane protein FepA. Ferric enterobactin inhibited H8 binding to E. coli FepA (50% inhibition concentration, 98 nM), and other ferric catecholate receptors (Fiu, Cir, and IroN) did not participate in phage adsorption. H8 infection was TonB dependent, but exbB mutations in Salmonella or E. coli did not prevent infection; only exbB tolQ or exbB tolR double mutants were resistant to H8. Experiments with deletion and substitution mutants showed that the receptor-phage interaction first involves residues distributed over the protein's outer surface and then narrows to the same charged (R316) or aromatic (Y260) residues that participate in the binding and transport of ferric enterobactin and colicins B and D. These data rationalize the multifunctionality of FepA: toxic ligands like bacteriocins and phage penetrate the outer membrane by parasitizing residues in FepA that are adapted to the transport of the natural ligand, ferric enterobactin. DNA sequence determinations revealed the complete H8 genome of 104.4 kb. A total of 120 of its 143 predicted open reading frames (ORFS) were homologous to ORFS in T5, at a level of 84% identity and 89% similarity. As in T5, the H8 structural genes clustered on the chromosome according to their function in the phage life cycle. The T5 genome contains a large section of DNA that can be deleted and that is absent in H8: compared to T5, H8 contains a 9,000-bp deletion in the early region of its chromosome, and nine potentially unique gene products. Sequence analyses of the tail proteins of phages in the same family showed that relative to pb5 (Oad) of T5 and Hrs of BF23, the FepA-binding protein (Rbp) of H8 contains unique acidic and aromatic residues. These side chains may promote binding to basic and aromatic residues in FepA that normally function in the adsorption of ferric enterobactin. Furthermore, a predicted H8 tail protein showed extensive identity and similarity to pb2 of T5, suggesting that it also functions in pore formation through the cell envelope. The variable region of this protein contains a potential TonB box, intimating that it participates in the TonB-dependent stage of the phage infection process. [Abstract/Link to Full Text]

Dastidar V, Mao W, Lomovskaya O, Zgurskaya HI
Drug-induced conformational changes in multidrug efflux transporter AcrB from Haemophilus influenzae.
J Bacteriol. 2007 Aug;189(15):5550-8.
In gram-negative bacteria, transporters belonging to the resistance-nodulation-cell division (RND) superfamily of proteins are responsible for intrinsic multidrug resistance. Haemophilus influenzae, a gram-negative pathogen causing respiratory diseases in humans and animals, constitutively produces the multidrug efflux transporter AcrB (AcrB(HI)). Similar to other RND transporters AcrB(HI) associates with AcrA(HI), the periplasmic membrane fusion protein, and the outer membrane channel TolC(HI). Here, we report that AcrAB(HI) confers multidrug resistance when expressed in Escherichia coli and requires for its activity the E. coli TolC (TolC(EC)) protein. To investigate the intracellular dynamics of AcrAB(HI), single cysteine mutations were constructed in AcrB(HI) in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to the hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of AcrAB(HI) and in the presence or absence of AcrA(HI) and TolC(EC). We report that the reactivity of specific cysteines with FM is affected by the presence of some but not all substrates. Our results suggest that substrates induce conformational changes in AcrB(HI). [Abstract/Link to Full Text]

Purdy GE, Fisher CR, Payne SM
IcsA surface presentation in Shigella flexneri requires the periplasmic chaperones DegP, Skp, and SurA.
J Bacteriol. 2007 Aug;189(15):5566-73.
A Shigella flexneri degP mutant, which was defective for plaque formation in Henle cell monolayers, had a reduced amount of IcsA detectable on the bacterial surface with antibody. However, the mutant secreted IcsA to the outer membrane at wild-type levels. This suggests that IcsA adopts an altered conformation in the outer membrane of the degP mutant with reduced exposure on the cell surface. IcsA is, therefore, unlikely to be accessible to actin-nucleating proteins within the eukaryotic cell cytoplasm, which is required for bacterial movement within the host cell and cell-to-cell spread. The degP mutant was somewhat more sensitive to detergents, antibiotics, and the antimicrobial peptide magainin, indicating that the degP phenotype was not limited to IcsA surface presentation. The plaque defect of the degP mutant, which is independent of DegP protease activity, was suppressed by overexpression of the periplasmic chaperone Skp but not by SurA. S. flexneri skp and surA mutants failed to form plaques in Henle cell monolayers and were defective in cell surface presentation and polar localization of IcsA. Therefore, the three periplasmic folding factors DegP, Skp, and SurA were all required for IcsA localization and plaque formation by S. flexneri. [Abstract/Link to Full Text]

White-Ziegler CA, Malhowski AJ, Young S
Human body temperature (37degrees C) increases the expression of iron, carbohydrate, and amino acid utilization genes in Escherichia coli K-12.
J Bacteriol. 2007 Aug;189(15):5429-40.
Using DNA microarrays, we identified 126 genes in Escherichia coli K-12 whose expression is increased at human body temperature (37 degrees C) compared to growth at 23 degrees C. Genes involved in the uptake and utilization of amino acids, carbohydrates, and iron dominated the list, supporting a model in which temperature serves as a host cue to increase expression of bacterial genes needed for growth. Using quantitative real-time PCR, we investigated the thermoregulatory response for representative genes in each of these three categories (hisJ, cysP, srlE, garP, fes, and cirA), along with the fimbrial gene papB. Increased expression at 37 degrees C compared to 23 degrees C was retained in both exponential and stationary phases for all of the genes and in most of the various media tested, supporting the relative importance of this cue in adapting to changing environments. Because iron acquisition is important for both growth and virulence, we analyzed the regulation of the iron utilization genes cirA and fes and found that growth in iron-depleted medium abrogated the thermoregulatory effect, with high-level expression at both temperatures, contrasting with papB thermoregulation, which was not greatly altered by limiting iron levels. A positive role for the environmental regulator H-NS was found for fes, cirA, hisJ, and srlE transcription, whereas it had a primarily negative effect on cysP and garP expression. Together, these studies indicate that temperature is a broadly used cue for regulating gene expression in E. coli and that H-NS regulates iron, carbohydrate, and amino acid utilization gene expression. [Abstract/Link to Full Text]

Glover RT, Kriakov J, Garforth SJ, Baughn AD, Jacobs WR
The two-component regulatory system senX3-regX3 regulates phosphate-dependent gene expression in Mycobacterium smegmatis.
J Bacteriol. 2007 Aug;189(15):5495-503.
Phosphate import is required for the growth of mycobacteria and is regulated by environmental inorganic phosphate (P(i)) concentrations, although the mechanism of this regulation has not been characterized. The expression of genes involved in P(i) acquisition is frequently regulated by two-component regulatory systems (2CRs) consisting of a sensor histidine kinase and a DNA-binding response regulator. In this work, we have identified the senX3-regX3 2CR as a P(i)-dependent regulator of genes involved in phosphate acquisition in Mycobacterium smegmatis. Characterization of senX3 mutants with different PhoA phenotypes suggests a dual role for SenX3 as a phosphatase or a phosphodonor for the response regulator RegX3, depending upon P(i) availability. Expression of PhoA activity required phosphorylation of RegX3, consistent with a role for phosphorylated RegX3 (RegX3 approximately P) as a transcriptional activator of phoA. Furthermore, purified RegX3 approximately P bound to promoter sequences from phoA, senX3, and the high-affinity phosphate transporter component pstS, demonstrating direct transcriptional control of all three genes. DNase I footprinting and primer extension analyses have further defined the DNA-binding region and transcriptional start site within the phoA promoter. A DNA motif consisting of an inverted repeat was identified in each of the promoters bound by RegX3 approximately P. Based upon our findings, we propose a model for P(i)-regulated gene expression mediated by SenX3-RegX3 in mycobacteria. [Abstract/Link to Full Text]

Claggett SB, Grabar TB, Dunn SD, Cain BD
Functional incorporation of chimeric b subunits into F1Fo ATP synthase.
J Bacteriol. 2007 Aug;189(15):5463-71.
F(1)F(o) ATP synthases function by a rotary mechanism. The enzyme's peripheral stalk serves as the stator that holds the F(1) sector and its catalytic sites against the movement of the rotor. In Escherichia coli, the peripheral stalk is a homodimer of identical b subunits, but photosynthetic bacteria have open reading frames for two different b-like subunits thought to form heterodimeric b/b' peripheral stalks. Chimeric b subunit genes have been constructed by substituting sequence from the Thermosynechococcus elongatus b and b' genes in the E. coli uncF gene, encoding the b subunit. The recombinant genes were expressed alone and in combination in the E. coli deletion strain KM2 (Deltab). Although not all of the chimeric subunits were incorporated into F(1)F(o) ATP synthase complexes, plasmids expressing either chimeric b(E39-I86) or b'(E39-I86) were capable of functionally complementing strain KM2 (Deltab). Strains expressing these subunits grew better than cells with smaller chimeric segments, such as those expressing the b'(E39-D53) or b(L54-I86) subunit, indicating intragenic suppression. In general, the chimeric subunits modeled on the T. elongatus b subunit proved to be more stable than the b' subunit in vitro. Coexpression of the b(E39-I86) and b'(E39-I86) subunits in strain KM2 (Deltab) yielded F(1)F(o) complexes containing heterodimeric peripheral stalks composed of both subunits. [Abstract/Link to Full Text]

Ramos AR, Morello JE, Ravindran S, Deng WL, Huang HC, Collmer A
Identification of Pseudomonas syringae pv. syringae 61 type III secretion system Hrp proteins that can travel the type III pathway and contribute to the translocation of effector proteins into plant cells.
J Bacteriol. 2007 Aug;189(15):5773-8.
Pseudomonas syringae translocates effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). T3SS components HrpB, HrpD, HrpF, and HrpP were shown to be pathway substrates and to contribute to elicitation of the plant hypersensitive response and to translocation and secretion of the model effector AvrPto1. [Abstract/Link to Full Text]

Miyamoto Y, Mukai T, Maeda Y, Nakata N, Kai M, Naka T, Yano I, Makino M
Characterization of the fucosylation pathway in the biosynthesis of glycopeptidolipids from Mycobacterium avium complex.
J Bacteriol. 2007 Aug;189(15):5515-22.
The cell envelopes of several species of nontuberculous mycobacteria, including the Mycobacterium avium complex, contain glycopeptidolipids (GPLs) as major glycolipid components. GPLs are highly antigenic surface molecules, and their variant oligosaccharides define each serotype of the M. avium complex. In the oligosaccharide portion of GPLs, the fucose residue is one of the major sugar moieties, but its biosynthesis remains unclear. To elucidate it, we focused on the 5.0-kb chromosomal region of the M. avium complex that includes five genes, two of which showed high levels of similarity to the genes involved in fucose synthesis. For the characterization of this region by deletion and expression analyses, we constructed a recombinant Mycobacterium smegmatis strain that possesses the rtfA gene of the M. avium complex to produce serovar 1 GPL. The results revealed that the 5.0-kb chromosomal region is responsible for the addition of the fucose residue to serovar 1 GPL and that the three genes mdhtA, merA, and gtfD are indispensable for the fucosylation. Functional characterization revealed that the gtfD gene encodes a glycosyltransferase that transfers a fucose residue via 1-->3 linkage to a rhamnose residue of serovar 1 GPL. The other two genes, mdhtA and merA, contributed to the formation of the fucose residue and were predicted to encode the enzymes responsible for the synthesis of fucose from mannose based on their deduced amino acid sequences. These results indicate that the fucosylation pathway in GPL biosynthesis is controlled by a combination of the mdhtA, merA, and gtfD genes. Our findings may contribute to the clarification of the complex glycosylation pathways involved in forming the oligosaccharide portion of GPLs from the M. avium complex, which are structurally distinct. [Abstract/Link to Full Text]

Jakob K, Satorhelyi P, Lange C, Wendisch VF, Silakowski B, Scherer S, Neuhaus K
Gene expression analysis of Corynebacterium glutamicum subjected to long-term lactic acid adaptation.
J Bacteriol. 2007 Aug;189(15):5582-90.
Corynebacteria form an important part of the red smear cheese microbial surface consortium. To gain a better understanding of molecular adaptation due to low pH induced by lactose fermentation, the global gene expression profile of Corynebacterium glutamicum adapted to pH 5.7 with lactic acid under continuous growth in a chemostat was characterized by DNA microarray analysis. Expression of a total of 116 genes was increased and that of 90 genes was decreased compared to pH 7.5 without lactic acid, representing 7% of the genes in the genome. The up-regulated genes encode mainly transcriptional regulators, proteins responsible for export, import, and metabolism, and several proteins of unknown function. As much as 45% of the up-regulated open reading frames code for hypothetical proteins. These results were validated using real-time reverse transcription-PCR. To characterize the functions of 38 up-regulated genes, 36 single-crossover disruption mutants were generated and analyzed for their lactic acid sensitivities. However, only a sigB knockout mutant showed a highly significant negative effect on growth at low pH, suggesting a function in organic-acid adaptation. A sigE mutant already displayed growth retardation at neutral pH but grew better at acidic pH than the sigB mutant. The lack of acid-sensitive phenotypes in 34 out of 36 disrupted genes suggests either a considerable redundancy in acid adaptation response or coincidental effects. Other up-regulated genes included genes for ion transporters and metabolic pathways, including carbohydrate and respiratory metabolism. The enhanced expression of the nrd (ribonucleotide reductase) operon and a DNA ATPase repair protein implies a cellular response to combat acid-induced DNA damage. Surprisingly, multiple iron uptake systems (totaling 15% of the genes induced >or=2-fold) were induced at low pH. This induction was shown to be coincidental and could be attributed to iron-sequestering effects in complex media at low pH. [Abstract/Link to Full Text]

De Silva RS, Kovacikova G, Lin W, Taylor RK, Skorupski K, Kull FJ
Crystal structure of the Vibrio cholerae quorum-sensing regulatory protein HapR.
J Bacteriol. 2007 Aug;189(15):5683-91.
Quorum sensing in Vibrio cholerae involves signaling between two-component sensor protein kinases and the response regulator LuxO to control the expression of the master regulator HapR. HapR, in turn, plays a central role in regulating a number of important processes, such as virulence gene expression and biofilm formation. We have determined the crystal structure of HapR to 2.2-A resolution. Its structure reveals a dimeric, two-domain molecule with an all-helical structure that is strongly conserved with members of the TetR family of transcriptional regulators. The N-terminal DNA-binding domain contains a helix-turn-helix DNA-binding motif and alteration of certain residues in this domain completely abolishes the ability of HapR to bind to DNA, alleviating repression of both virulence gene expression and biofilm formation. The C-terminal dimerization domain contains a unique solvent accessible tunnel connected to an amphipathic cavity, which by analogy with other TetR regulators, may serve as a binding pocket for an as-yet-unidentified ligand. [Abstract/Link to Full Text]

Uehara T, Park JT
An anhydro-N-acetylmuramyl-L-alanine amidase with broad specificity tethered to the outer membrane of Escherichia coli.
J Bacteriol. 2007 Aug;189(15):5634-41.
From its amino acid sequence homology with AmpD, we recognized YbjR, now renamed AmiD, as a possible second 1,6-anhydro-N-acetylmuramic acid (anhMurNAc)-l-alanine amidase in Escherichia coli. We have now confirmed that AmiD is an anhMurNAc-l-Ala amidase and demonstrated that AmpD and AmiD are the only enzymes present in E. coli that are able to cleave the anhMurNAc-l-Ala bond. The activity was present only in the outer membrane fraction obtained from an ampD mutant. In contrast to AmpD, which is specific for the anhMurNAc-l-alanine bond, AmiD also cleaved the bond between MurNAc and l-alanine in both muropeptides and murein sacculi. Unlike the periplasmic murein amidases, AmiD did not participate in cell separation. ampG mutants, which are unable to import GlcNAc-anhMurNAc-peptides into the cytoplasm, released mainly peptides into the medium due to AmiD activity, whereas an ampG amiD double mutant released a large amount of intact GlcNAc-anhMurNAc-peptides into the medium. [Abstract/Link to Full Text]

Kohler PL, Hamilton HL, Cloud-Hansen K, Dillard JP
AtlA functions as a peptidoglycan lytic transglycosylase in the Neisseria gonorrhoeae type IV secretion system.
J Bacteriol. 2007 Aug;189(15):5421-8.
Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis. [Abstract/Link to Full Text]

Guarino E, Salguero I, Jiménez-Sánchez A, Guzmán EC
Double-strand break generation under deoxyribonucleotide starvation in Escherichia coli.
J Bacteriol. 2007 Aug;189(15):5782-6.
Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo "replication fork reversal." This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation. [Abstract/Link to Full Text]

Balasingham SV, Collins RF, Assalkhou R, Homberset H, Frye SA, Derrick JP, Třnjum T
Interactions between the lipoprotein PilP and the secretin PilQ in Neisseria meningitidis.
J Bacteriol. 2007 Aug;189(15):5716-27.
Neisseria meningitidis can be the causative agent of meningitis or septicemia. This bacterium expresses type IV pili, which mediate a variety of functions, including autoagglutination, twitching motility, biofilm formation, adherence, and DNA uptake during transformation. The secretin PilQ supports type IV pilus extrusion and retraction, but it also requires auxiliary proteins for its assembly and localization in the outer membrane. Here we have studied the physical properties of the lipoprotein PilP and examined its interaction with PilQ. We found that PilP was an inner membrane protein required for pilus expression and transformation, since pilP mutants were nonpiliated and noncompetent. These mutant phenotypes were restored by the expression of PilP in trans. The pilP gene is located upstream of pilQ, and analysis of their transcripts indicated that pilP and pilQ were cotranscribed. Furthermore, analysis of the level of PilQ expression in pilP mutants revealed greatly reduced amounts of PilQ only in the deletion mutant, exhibiting a polar effect on pilQ transcription. In vitro experiments using recombinant fragments of PilP and PilQ showed that the N-terminal region of PilP interacted with the middle part of the PilQ polypeptide. A three-dimensional reconstruction of the PilQ-PilP interacting complex was obtained at low resolution by transmission electron microscopy, and PilP was shown to localize around the cap region of the PilQ oligomer. These findings suggest a role for PilP in pilus biogenesis. Although PilQ does not need PilP for its stabilization or membrane localization, the specific interaction between these two proteins suggests that they might have another coordinated activity in pilus extrusion/retraction or related functions. [Abstract/Link to Full Text]

Lu Z, Takeuchi M, Sato T
The LysR-type transcriptional regulator YofA controls cell division through the regulation of expression of ftsW in Bacillus subtilis.
J Bacteriol. 2007 Aug;189(15):5642-51.
We have carried out a functional analysis of LysR family transcriptional regulators in Bacillus subtilis. The cell density of cultures of a yofA insertion mutant declined sharply after the end of exponential growth, as measured by optical density at 600 nm. Complementation in trans and analysis of isopropyl-beta-d-thiogalactopyranoside (IPTG)-dependent growth of an inducible yofA strain confirmed that YofA contributes to the cell density of a culture after the end of exponential growth. Microscopic observation suggested that cell division is inhibited or delayed in the yofA mutant during entry into stationary phase. Analysis of the transcription of cell division genes revealed that the expression of ftsW is inhibited in yofA mutants, and overexpression of yofA, driven by a multiple-copy plasmid, enhances the induction of ftsW expression. These results suggest that YofA is required for the final round of cell division before entry into stationary phase and that YofA positively regulates ftsW expression. The defects caused by mutation of yofA were suppressed in strains carrying P(spac)-ftsW in the presence of IPTG. Furthermore, maximal expression of yofA was observed at the onset of stationary phase, which coincided with the maximal ftsW expression. Our data indicate that YofA is involved in cell division through positive regulation of the expression of ftsW in B. subtilis. [Abstract/Link to Full Text]


Recent Articles in Journal of Clinical Microbiology

Könönen E, Paju S, Pussinen PJ, Hyvönen M, Di Tella P, Suominen-Taipale L, Knuuttila M
Population-based study of salivary carriage of periodontal pathogens in adults.
J Clin Microbiol. 2007 Aug;45(8):2446-51.
Large, general population-based data on carriage rates of periodontal pathogens hardly exist in the current literature. The objectives of the present study were to examine the salivary detection of Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, Campylobacter rectus, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Treponema denticola in a representative sample of the adult population living in southern Finland and to clarify which determinants are associated with the presence of these pathogens in saliva. 16S rRNA-based PCR methods with species-specific primers were employed to determine the presence of the six target bacteria in stimulated saliva samples, which were available from 1,294 subjects aged > or =30 years. The age group, gender, level of education, marital status, smoking history, number of teeth, and number of teeth with deepened pockets were included in the statistical analysis. In general, the carriage of periodontal pathogens was common, since at least one of the examined pathogens was found in 88.2% of the subjects. In descending order, the total detection rates were 56.9%, 38.2%, 35.4%, 31.3%, 20.0%, and 13.9% for T. forsythensis, T. denticola, P. gingivalis, C. rectus, A. actinomycetemcomitans, and P. intermedia, respectively. Age per se was strongly associated with the carriage of P. gingivalis (P = 0.000), and the level of education with that of T. denticola (P = 0.000). There was an association between the number of teeth with deepened pockets and carriage of P. gingivalis (P = 0.000), P. intermedia (P = 0.000), T. denticola (P = 0.000), and A. actinomycetemcomitans (P = 0.004). The data suggest that distinct species have a different carriage profile, depending on variables such as age, educational level, and periodontal status. [Abstract/Link to Full Text]

Sorlozano A, Gutierrez J, Jimenez A, de Dios Luna J, Martínez JL
Contribution of a new mutation in parE to quinolone resistance in extended-spectrum-beta-lactamase-producing Escherichia coli isolates.
J Clin Microbiol. 2007 Aug;45(8):2740-2.
Mutations in the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE were studied in 30 fluoroquinolone-resistant clinical isolates of Escherichia coli producing extended-spectrum beta-lactamases. Ten isolates showed a mutation in parE that was significantly associated with an increase in the MIC for fluoroquinolones. [Abstract/Link to Full Text]

Sábato MF, Shiffman ML, Langley MR, Wilkinson DS, Ferreira-Gonzalez A
Comparison of performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus.
J Clin Microbiol. 2007 Aug;45(8):2529-36.
We evaluated the performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus (HCV) and performed a direct comparison of the systems on the same clinical specimens. Commercial HCV panels (genotype 1b) were used to evaluate linear range, sensitivity, and precision. The Roche COBAS TaqMan HCV test for research use only (RUO) with samples processed on the MagNA Pure LC instrument (Roche RUO-MPLC) and Abbott analyte-specific reagents (ASR) with QIAGEN sample processing (Abbott ASR-Q) showed a sensitivity of 1.0 log(10) IU/ml with a linear dynamic range of 1.0 to 7.0 log(10) IU/ml. The Roche ASR in combination with the High Pure system (Roche ASR-HP) showed a sensitivity of 1.4 log(10) IU/ml with a linear dynamic range of 2.0 to 7.0 log(10) IU/ml. All of the systems showed acceptable reproducibility, the Abbott ASR-Q being the most reproducible of the three systems. Seventy-six clinical specimens (50 with detectable levels of HCV RNA and various titers and genotypes) were tested, and results were compared to those of the COBAS Amplicor HCV Monitor v2.0. Good correlation was obtained for the Roche RUO-MPLC and Abbott ASR-Q (R(2) = 0.84 and R(2) = 0.93, respectively), with better agreement for the Abbott ASR-Q. However, correlation (R(2) = 0.79) and agreement were poor for Roche ASR-HP, with bias relative to concentration and genotype. Roche ASR-HP underestimated HCV RNA for genotypes 3 and 4 as much as 2.19 log(10) IU/ml. Our study demonstrates that Roche RUO-MPLC and Abbott ASR-Q provided acceptable results and agreed sufficiently with the COBAS Amplicor HCV Monitor v2.0. [Abstract/Link to Full Text]

Franzen-Röhl E, Tiveljung-Lindell A, Grillner L, Aurelius E
Increased detection rate in diagnosis of herpes simplex virus type 2 meningitis by real-time PCR using cerebrospinal fluid samples.
J Clin Microbiol. 2007 Aug;45(8):2516-20.
Efficient and sensitive diagnostic methods are needed in the management of virus infections in the central nervous system. There is a demand for an evaluation of the sensitivity of PCR methods for early diagnosis of meningitis due to herpes simplex type 2 (HSV-2) and varicella-zoster virus (VZV). The objective of this study was to evaluate real-time PCR in the detection of HSV-2 and VZV DNA from cerebrospinal fluid (CSF) for etiological diagnoses in clinically well-characterized cases of primary and recurrent aseptic meningitis. Samples from 110 patients, 65 of whom were diagnosed with or were strongly suspected of having HSV-2 meningitis and 45 with acute aseptic meningitis of unknown causes, were analyzed. Results were compared with the outcome of nested PCR for HSV-2 infection. Clinical parameters were analyzed in relation to CSF viral load. With real-time PCR, HSV-2 DNA was found in CSF from 80% (52/65) of patients with clinical HSV-2 meningitis compared to 72% (47/65) found by nested PCR. The sensitivity of real-time HSV-2 PCR was found to be 87% (33/38) in primary and 70% (19/27) in recurrent meningitis. The HSV-2 viral load was significantly higher in primary than in recurrent meningitis and correlated with the degree of inflammation. VZV DNA was detected in 2 of 45 samples (4.4%). Real-time PCR for the diagnosis of HSV-2 meningitis was evaluated in a large, clinically well-characterized sample of material and found to identify more cases than nested PCR in the group of patients with recurrent meningitis. Quantification of DNA enables further research of disease prognosis and treatment. [Abstract/Link to Full Text]

Nakano K, Lapirattanakul J, Nomura R, Nemoto H, Alaluusua S, Grönroos L, Vaara M, Hamada S, Ooshima T, Nakagawa I
Streptococcus mutans clonal variation revealed by multilocus sequence typing.
J Clin Microbiol. 2007 Aug;45(8):2616-25.
Streptococcus mutans is the major pathogen of dental caries, a biofilm-dependent infectious disease, and occasionally causes infective endocarditis. S. mutans strains have been classified into four serotypes (c, e, f, and k). However, little is known about the S. mutans population, including the clonal relationships among strains of S. mutans, in relation to the particular clones that cause systemic diseases. To address this issue, we have developed a multilocus sequence typing (MLST) scheme for S. mutans. Eight housekeeping gene fragments were sequenced from each of 102 S. mutans isolates collected from the four serotypes in Japan and Finland. Between 14 and 23 alleles per locus were identified, allowing us theoretically to distinguish more than 1.2 x 10(10) sequence types. We identified 92 sequence types in these 102 isolates, indicating that S. mutans contains a diverse population. Whereas serotype c strains were widely distributed in the dendrogram, serotype e, f, and k strains were differentiated into clonal complexes. Therefore, we conclude that the ancestral strain of S. mutans was serotype c. No geographic specificity was identified. However, the distribution of the collagen-binding protein gene (cnm) and direct evidence of mother-to-child transmission were clearly evident. In conclusion, the superior discriminatory capacity of this MLST scheme for S. mutans may have important practical implications. [Abstract/Link to Full Text]

Honma S, Chizhikov V, Santos N, Tatsumi M, Timenetsky Mdo C, Linhares AC, Mascarenhas JD, Ushijima H, Armah GE, Gentsch JR, Hoshino Y
Development and validation of DNA microarray for genotyping group A rotavirus VP4 (P[4], P[6], P[8], P[9], and P[14]) and VP7 (G1 to G6, G8 to G10, and G12) genes.
J Clin Microbiol. 2007 Aug;45(8):2641-8.
Previously, we reported the development of a microarray-based method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) (V. Chizhikov et al., J. Clin. Microbiol. 40:2398-2407, 2002). The expanded version of the rotavirus microarray assay presented herein is capable of identifying (i) five clinically relevant human rotavirus VP4 genotypes (P[4], P[6], P[8], P[9], and P[14]) and (ii) five additional human rotavirus VP7 genotypes (G5, G6, G8, G10, and G12) on one chip. Initially, a total of 80 cell culture-adapted human and animal reference rotavirus strains of known P (P[1] to P[12], P[14], P[16], and P[20]) and G (G1-6, G8 to G12, and G14) genotypes isolated in various parts of the world were employed to evaluate the new microarray assay. All rotavirus strains bearing P[4], P[6], P[8], P[9], or P[14] and/or G1 to G6, G8 to G10, or G12 specificity were identified correctly. In addition, cross-reactivity to viruses of genotype G11, G13, or G14 or P[1] to P[3], P[5], P[7], P[10] to P[12], P[16], or P[20] was not observed. Next, we analyzed a total of 128 rotavirus-positive human stool samples collected in three countries (Brazil, Ghana, and the United States) by this assay and validated its usefulness. The results of this study showed that the assay was sensitive and specific and capable of unambiguously discriminating mixed rotavirus infections from nonspecific cross-reactivity; the inability to discriminate mixed infections from nonspecific cross-reactivity is one of the inherent shortcomings of traditional multiplex reverse transcription-PCR genotyping. Moreover, because the hybridization patterns exhibited by rotavirus strains of different genotypes can vary, this method may be ideal for analyzing the genetic polymorphisms of the VP7 or VP4 genes of rotaviruses. [Abstract/Link to Full Text]

Quan PL, Palacios G, Jabado OJ, Conlan S, Hirschberg DL, Pozo F, Jack PJ, Cisterna D, Renwick N, Hui J, Drysdale A, Amos-Ritchie R, Baumeister E, Savy V, Lager KM, Richt JA, Boyle DB, García-Sastre A, Casas I, Perez-Breńa P, Briese T, Lipkin WI
Detection of respiratory viruses and subtype identification of influenza A viruses by GreeneChipResp oligonucleotide microarray.
J Clin Microbiol. 2007 Aug;45(8):2359-64.
Acute respiratory infections are significant causes of morbidity, mortality, and economic burden worldwide. An accurate, early differential diagnosis may alter individual clinical management as well as facilitate the recognition of outbreaks that have implications for public health. Here we report on the establishment and validation of a comprehensive and sensitive microarray system for detection of respiratory viruses and subtyping of influenza viruses in clinical materials. Implementation of a set of influenza virus enrichment primers facilitated subtyping of influenza A viruses through the differential recognition of hemagglutinins 1 through 16 and neuraminidases 1 through 9. Twenty-one different respiratory virus species were accurately characterized, including a recently identified novel genetic clade of rhinovirus. [Abstract/Link to Full Text]

Karpathy SE, Dasch GA, Eremeeva ME
Molecular typing of isolates of Rickettsia rickettsii by use of DNA sequencing of variable intergenic regions.
J Clin Microbiol. 2007 Aug;45(8):2545-53.
Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is found throughout the Americas, where it is associated with different animal reservoirs and tick vectors. No molecular typing system currently exists to allow for the robust differentiation of isolates of R. rickettsii. Analysis of eight completed genome sequences of rickettsial species revealed a high degree of sequence conservation within the coding regions of chromosomes in the genus. Intergenic regions between coding sequences should be under less selective pressure to maintain this conservation and thus should exhibit greater nucleotide polymorphisms. Utilizing these polymorphisms, we developed a molecular typing system that allows for the genetic differentiation of isolates of R. rickettsii. This typing system was applied to a collection of 38 different isolates collected from humans, animals, and tick vectors from different geographic locations. Serotypes 364D, from Dermacentor occidentalis ticks, and Hlp, from Haemaphysalis leporispalustris ticks, appear to be distinct genotypes that may not belong to the species R. rickettsii. We were also able to differentiate 36 historical isolates of R. rickettsii into three different phylogenetic clades containing seven different genotypes. This differentiation correlated well, but not perfectly, with the geographic origin and likely tick vectors associated with the isolates. The few apparent typing discrepancies found suggest that the molecular ecology of R. rickettsii needs more investigation. [Abstract/Link to Full Text]

Gotsch A, Schubert A, Bombis A, Wiedmann M, Zauke M, Schorling S
Nuclease-resistant single-stranded DNA controls for nucleic acid amplification assays.
J Clin Microbiol. 2007 Aug;45(8):2570-4.
Molecular diagnostic tests based on the PCR or alternative nucleic acid amplification technologies are commonly used for pathogen screening at blood drawing centers. Contrived process surveillance using test-specific external and internal controls is critical for the efficient leverage of PCR power. We describe here novel control constructs for use in nucleic acid amplification assays for pathogens with a single-stranded DNA genome, e.g., parvovirus B19. These controls are derived from a deletion mutant of the filamentous phage fd-tet, fKN16, and consist of single-stranded DNA packaged in a protein coat. They are essentially noninfectious to Escherichia coli and highly resistant to nuclease degradation. fKN16 based controls can be readily manufactured and highly purified. Despite their confirmed filamentous morphology, they can be precisely and accurately diluted over a wide range. Stability studies reveal that the novel control constructs are highly resistant to temperature stress, regardless of whether they are tested as concentrated stocks in storage buffer or diluted in buffer or human plasma. Real-time amplification curves derived from recombinant control constructs containing a parvovirus B19 specific sequence fragment match those derived from native virus. In summary, our data demonstrate the feasibility of novel nuclease-resistant single-stranded DNA controls as surrogates for parvovirus B19 and their applicability in routine molecular diagnostics. [Abstract/Link to Full Text]

Lin CY, Chen YC, Lo HJ, Chen KW, Li SY
Assessment of Candida glabrata strain relatedness by pulsed-field gel electrophoresis and multilocus sequence typing.
J Clin Microbiol. 2007 Aug;45(8):2452-9.
In this study, 80 Candida glabrata isolates from intensive care unit and human immunodeficiency virus (HIV)-infected patients were typed by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and mating type class determination. Among the 25 patients with multiple isolates, 19 patients (76%) contained multiple isolates exhibiting identical or highly related PFGE and MLST genotypes, which may indicate the maintenance or microvariation of one C. glabrata strain in each patient. However, isolates from six patients (24%) displayed different sequence types, PFGE genotypes, or mating type classes, which may indicate colonization with more than one clone over time or strain replacement. High correlations among PFGE genotypes, sequence types, and mating types were found (P < 0.01). MLST exhibited less discriminatory power than PFGE with BssHII. The genotypes, sequence types, and mating type classes were independent of anatomic sources, drug susceptibility, and HIV infection status. [Abstract/Link to Full Text]

Friedrichs C, Rodloff AC, Chhatwal GS, Schellenberger W, Eschrich K
Rapid identification of viridans streptococci by mass spectrometric discrimination.
J Clin Microbiol. 2007 Aug;45(8):2392-7.
Viridans streptococci (VS) are responsible for several systemic diseases, such as endocarditis, abscesses, and septicemia. Unfortunately, species identification by conventional methods seems to be more difficult than species identification of other groups of bacteria. The aim of the present study was to evaluate the use of cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the rapid identification of 10 different species of VS. A total of 99 VS clinical isolates, 10 reference strains, and 20 strains from our in-house culture collection were analyzed by MALDI-TOF-MS. To evaluate the mass-spectrometric discrimination results, all strains were identified in parallel by phenotypic and genotypic methods. MALDI-TOF-MS identified 71 isolates as the mitis group, 23 as the anginosus group, and 5 as Streptococcus salivarius. Comparison of the species identification results obtained by the MALDI-TOF-MS analyses and with the phenotypic/genotypic identification systems showed 100% consistency at the species level. Thus, MALDI-TOF-MS seems to be a rapid and reliable method for the identification of species of VS from clinical samples. [Abstract/Link to Full Text]

Gierczy?ski R, Golubov A, Neubauer H, Pham JN, Rakin A
Development of multiple-locus variable-number tandem-repeat analysis for Yersinia enterocolitica subsp. palearctica and its application to bioserogroup 4/O3 subtyping.
J Clin Microbiol. 2007 Aug;45(8):2508-15.
Yersinia enterocolitica bioserogroup 4/O3 is the predominant causative agent of yersiniosis in Europe and North America. Multiple-locus variable-number tandem-repeat analysis (MLVA) was developed to improve the resolution power of classical genotyping methods. MLVA based on six loci was able to distinguish 76 genotypes among 91 Y. enterocolitica isolates of worldwide origin and 41 genotypes among 51 nonepidemiologically linked bioserogroup 4/O3 isolates, proving that it has a high resolution power. However, only a slight correlation of the MLVA genotypes and the geographic distribution of the isolates was observed. Although MLVA was also capable of distinguishing strains of Y. enterocolitica subsp. palearctica O9 and O5,27, there was only a minor correlation between the MLVA genotypes and serogroups. MLVA may be a helpful tool for epidemiological investigations of Y. enterocolitica subsp. palearctica outbreaks. [Abstract/Link to Full Text]

Creuzburg K, Schmidt H
Molecular characterization and distribution of genes encoding members of the type III effector nleA family among pathogenic Escherichia coli strains.
J Clin Microbiol. 2007 Aug;45(8):2498-507.
In this study, we investigated the occurrence of the previously described gene nleA(4795) and variants of nleA, putatively encoding non-locus-of-enterocyte-effacement-encoded type III effector proteins with functions that are unknown. nleA variants were detected in 150 out of 170 enteropathogenic Escherichia coli strains and enterohemorrhagic E. coli strains, two of them being eae negative. Besides the known variants nleA(4795), Z6024, and the espI-like gene, 11 novel nleA variants with different lengths and sequence identities at the deduced amino acid level (between 71% and 96%) have been identified. Whereas most of the serogroups associated with more severe disease were quite homogenous with respect to the presence of a particular nleA variant, other serogroups were not. Moreover, Southern blot hybridization revealed that certain strains carry two copies of nleA in their chromosome, frequently encoding different variants. In most cases, the open reading frame of one of the copies was disrupted, usually by an insertion element. Furthermore, transmission of the type III effector-encoding gene could be shown by transduction of nleA-carrying bacteriophages to a laboratory E. coli strain. [Abstract/Link to Full Text]

Delmas J, Robin F, Schweitzer C, Lesens O, Bonnet R
Evaluation of a new chromogenic medium, ChromID VRE, for detection of vancomycin-resistant Enterococci in stool samples and rectal swabs.
J Clin Microbiol. 2007 Aug;45(8):2731-3.
We compared ChromID VRE medium with Enterococcosel containing vancomycin for the detection of vancomycin-resistant Enterococcus in 1,007 specimens. ChromID VRE in combination with Gram straining provided a higher specificity than Enterococcosel, irrespective of the incubation time and enrichment. [Abstract/Link to Full Text]

Henn JB, Gabriel MW, Kasten RW, Brown RN, Theis JH, Foley JE, Chomel BB
Gray foxes (Urocyon cinereoargenteus) as a potential reservoir of a Bartonella clarridgeiae-like bacterium and domestic dogs as part of a sentinel system for surveillance of zoonotic arthropod-borne pathogens in northern California.
J Clin Microbiol. 2007 Aug;45(8):2411-8.
Two species of Bartonella, a novel Bartonella clarridgeiae-like bacterium and B. vinsonii subsp. berkhoffii, were isolated from rural dogs and gray foxes in northern California. A novel B. clarridgeiae-like species was isolated from 3 (1.7%) of 182 dogs and 22 (42%) of 53 gray foxes, while B. vinsonii subsp. berkhoffii was isolated from 1 dog (0.5%) and 5 gray foxes (9.4%). PCR and DNA sequence analyses of the citrate synthase (gltA) gene and the 16S-23S intergenic spacer region suggested that strains infecting dogs and gray foxes were identical. Fifty-four dogs (29%) and 48 gray foxes (89%) had reciprocal titers of antibodies against Bartonella spp. of > or =64. The high prevalence of bacteremia and seroreactivity to Bartonella spp. in gray foxes suggests that they may act as a reservoir species for the B. clarridgeiae-like species in this region. Domestic dogs were also tested for other arthropod-borne infectious agents. Fifty-one dogs (28%) were positive for Dirofilaria immitis antigen, seventy-four (40%) were seroreactive to Anaplasma phagocytophilum, and five (2.7%) were seropositive for Yersinia pestis. Fourteen dogs (7.6%) were PCR positive for A. phagocytophilum. Polytomous logistic regression models were used to assess the association of Bartonella antibody titer categories with potential risk factors and the presence of other vector-borne agents in domestic dogs. Older dogs were more likely to be seroreactive to Bartonella spp. There was no association between the exposure of dogs to Bartonella and the exposure of dogs to A. phagocytophilum in this study. [Abstract/Link to Full Text]

Houzé S, Munier A, Paoletti X, Kaddouri H, Ringwald P, Le Bras J
Shelf life of predosed plates containing mefloquine, artemisinin, dihydroartemisinin, and artesunate as used for in vitro Plasmodium falciparum susceptibility assessment.
J Clin Microbiol. 2007 Aug;45(8):2734-6.
The shelf lives of preserved antimalarial agent-predosed plates according to the type of wrapping and the temperature of storage were studied by measuring the 50% inhibitory concentrations of drug for Plasmodium falciparum 3D7. The shelf life of mefloquine was 8 weeks at 25 degrees C; and those of artesunate, artemisinin, and dihydroartemisinin were a minimum of 24, 12, and 8 weeks, respectively, at 4 degrees C. [Abstract/Link to Full Text]

Phan TG, Okitsu S, Maneekarn N, Ushijima H, Duan ZJ
Possible misidentification of GSP[6] rotavirus as a novel strain detected in humans for the first time.
J Clin Microbiol. 2007 Jun;45(6):2098; author reply 2099. [Abstract/Link to Full Text]

Duan ZJ, Li DD, Zhang Q, Liu N, Huang CP, Jiang X, Jiang B, Glass R, Steele D, Tang JY, Wang ZS, Fang ZY
Novel human rotavirus of genotype G5P[6] identified in a stool specimen from a Chinese girl with diarrhea.
J Clin Microbiol. 2007 May;45(5):1614-7.
During a rotavirus surveillance conducted in Lulong County, Hebei Province, China, a total of 331 stool specimens collected in 2003 from children under 5 years old with diarrhea were screened. We identified a novel group A human rotavirus of genotype G5P[6]. Phylogenetic analysis confirmed that the VP7 protein of this newly identified strain, LL36755, was closely related to those of the G5 strains. As such, it has 95.4% homology with its counterparts in the porcine G5 strains C134 and CC117 at the amino acid sequence level. On the other hand, the VP4 protein of the LL36755 strain was 94.5% homologous to those of the porcine P[6] strains 134/04-10, 134/04-11, 221/04-7, and 221/04-13. Our findings indicate a dynamic interaction between human and porcine rotaviruses. [Abstract/Link to Full Text]

Sturm PD, Scholle D
Arthritis caused by Corynebacterium striatum: spontaneous infection?
J Clin Microbiol. 2007 Jun;45(6):2097; author reply 2097. [Abstract/Link to Full Text]

Scholle D
A spontaneous joint infection with Corynebacterium striatum.
J Clin Microbiol. 2007 Feb;45(2):656-8.
Corynebacterium striatum is a ubiquitous saprophyte with the potential to cause bacteremia in immunocompromised patients. Until now, spontaneous infection of a natural joint has not been reported. When phenotyping failed, gene sequencing was used to identify the species. The isolate demonstrated high-level resistance to most antibiotics. [Abstract/Link to Full Text]

Curcio D, Fernández F, Jones RN, Ferraro MJ, Reller LB, Schreckenberger PC, Sader HS
Tigecycline disk diffusion breakpoints of Acinetobacter spp.: a clinical point of view.
J Clin Microbiol. 2007 Jun;45(6):2095; author reply 2095-6. [Abstract/Link to Full Text]

Jones RN, Ferraro MJ, Reller LB, Schreckenberger PC, Swenson JM, Sader HS
Multicenter studies of tigecycline disk diffusion susceptibility results for Acinetobacter spp.
J Clin Microbiol. 2007 Jan;45(1):227-30.
Acinetobacter sp. isolates having multidrug resistance (MDR) patterns have become common in many medical centers worldwide, limiting therapeutic options. A five-center study tested 103 contemporary clinical Acinetobacter spp., including MDR strains, by reference broth microdilution and disk diffusion (15-mug disk content) methods against tigecycline. Applying U.S. Food and Drug Administration tigecycline breakpoint criteria for Enterobacteriaceae (susceptibility at < or =2 microg/ml [< or =1 microg/ml by the European Committee on Antimicrobial Susceptibility Testing]; disk diffusion breakpoints at > or =19 mm and < or =14 mm) to Acinetobacter spp. led to an unacceptable error rate (23.3%). However, an adjustment of tigecycline disk diffusion breakpoints (susceptible/resistant) to > or =16/ < or =12 mm reduced intermethod errors to an acceptable level (only 9.7%, all minor). [Abstract/Link to Full Text]

Arendrup MC, Denning DW, Pfaller MA, Diekema DJ, Rex JH
Does one voriconazole breakpoint suit all Candida species?
J Clin Microbiol. 2007 Jun;45(6):2093; author reply 2094. [Abstract/Link to Full Text]

Pfaller MA, Diekema DJ, Rex JH, Espinel-Ingroff A, Johnson EM, Andes D, Chaturvedi V, Ghannoum MA, Odds FC, Rinaldi MG, Sheehan DJ, Troke P, Walsh TJ, Warnock DW
Correlation of MIC with outcome for Candida species tested against voriconazole: analysis and proposal for interpretive breakpoints.
J Clin Microbiol. 2006 Mar;44(3):819-26.
Developing interpretive breakpoints for any given organism-drug combination requires integration of the MIC distribution, pharmacokinetic and pharmacodynamic parameters, and the relationship between the in vitro activity and outcome from both in vivo and clinical studies. Using data generated by standardized broth microdilution and disk diffusion test methods, the Antifungal Susceptibility Subcommittee of the Clinical and Laboratory Standards Institute has now proposed interpretive breakpoints for voriconazole and Candida species. The MIC distribution for voriconazole was determined using a collection of 8,702 clinical isolates. The overall MIC90 was 0.25 microg/ml and 99% of the isolates were inhibited at < or = 1 microg/ml of voriconazole. Similar results were obtained for 1,681 Candida isolates (16 species) from the phase III clinical trials. Analysis of the available data for 249 patients from six phase III voriconazole clinical trials demonstrated a statistically significant correlation (P = 0.021) between MIC and investigator end-of-treatment assessment of outcome. Consistent with parallel pharmacodynamic analyses, these data support the following MIC breakpoints for voriconazole and Candida species: susceptible (S), < or = 1 microg/ml; susceptible dose dependent (SDD), 2 microg/ml; and resistant (R), > or = 4 microg/ml. The corresponding disk test breakpoints are as follows: S, > or = 17 mm; SDD, 14 to 16 mm; and R, < or = 13 mm. [Abstract/Link to Full Text]

Madisch I, Heim A
Extent of circulation of incorrectly labeled adenovirus 50 and 51 prototype preparations.
J Clin Microbiol. 2007 Jun;45(6):2092; discussion 2092. [Abstract/Link to Full Text]

de Jong JC, Niesters HG, van Doornum GJ
Circulation of incorrectly labeled adenovirus 50 and 51 prototype preparations.
J Clin Microbiol. 2006 Sep;44(9):3468. [Abstract/Link to Full Text]

Iorio NL, Ferreira RB, Schuenck RP, Malvar KL, Brilhante AP, Nunes AP, Bastos CC, Dos Santos KR
Simplified and reliable scheme for species-level identification of Staphylococcus clinical isolates.
J Clin Microbiol. 2007 Aug;45(8):2564-9.
Reliable and rapid identification of staphylococcal strains continues to be a problem faced by many microbiology laboratories. This study evaluates a simplified method that uses a flowchart to assist in the identification of 12 clinical species of Staphylococcus, including eight subspecies. A total of 198 isolates and 11 control strains were identified by the reference method, which employed 22 tests. The results were compared with those obtained by two other methods: an automated system (MicroScan WalkAway) and a simplified method composed of nine tests. The simplified scheme showed an accuracy of 98.5%, while the automated method showed an accuracy of 79.3% (P < 0.001), in identifying staphylococcal species. Atypical phenotypic profiles were detected by both the reference (55.6%) and the simplified (19.7%) methods. The simplified method proposed here was shown to be reliable, with the advantage of being more practical and economic than the reference method. [Abstract/Link to Full Text]

Cruz AT, Goytia VK, Starke JR
Mycobacterium simiae complex infection in an immunocompetent child.
J Clin Microbiol. 2007 Aug;45(8):2745-6.
Nontuberculous mycobacteria are ubiquitous in the environment but rarely infect immunocompetent patients. We describe a pediatric case of Mycobacterium simiae complex lymphadenitis in an immunocompetent child and review the natural history, clinical manifestations, diagnosis, and current management of the disease. [Abstract/Link to Full Text]

van Hal SJ, Stark D, Lockwood B, Marriott D, Harkness J
Methicillin-resistant Staphylococcus aureus (MRSA) detection: comparison of two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) with three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA) for use with infection-control swabs.
J Clin Microbiol. 2007 Aug;45(8):2486-90.
Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing problem. Rapid detection of MRSA-colonized patients has the potential to limit spread of the organism. We evaluated the sensitivities and specificities of MRSA detection by two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) and three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA), using 205 (101 nasal, 52 groin, and 52 axillary samples) samples from consecutive known MRSA-infected and/or -colonized patients. All detection methods had higher MRSA detection rates for nasal swabs than for axillary and groin swabs. Detection of MRSA by IDI-MRSA was the most sensitive method, independent of the site (94% for nasal samples, 80% for nonnasal samples, and 90% overall). The sensitivities of the GenoType MRSA Direct assay and the MRSA ID, MRSASelect, and CHROMagar MRSA agars with nasal swabs were 70%, 72%, 68%, and 75%, respectively. All detection methods had high specificities (95 to 99%), independent of the swab site. Extended incubation for a further 24 h with selective MRSA agars increased the detection of MRSA, with a corresponding decline in specificity secondary to a significant increase in false-positive results. There was a noticeable difference in test performance of the GenoType MRSA Direct assay in detection of MRSA (28/38 samples [74%]) compared with detection of nonmultiresistant MRSA (17/31 samples [55%]) (susceptible to two or more non-beta-lactam antibiotics). This was not observed with selective MRSA agar plates or IDI-MRSA. Although it is more expensive, in addition to rapid turnaround times of 2 to 4 h, IDI-MRSA offers greater detection of MRSA colonization, independent of the swab site, than do conventional selective agars and GenoType MRSA Direct. [Abstract/Link to Full Text]

Schlievert PM, Case LC, Strandberg KL, Tripp TJ, Lin YC, Peterson ML
Vaginal Staphylococcus aureus superantigen profile shift from 1980 and 1981 to 2003, 2004, and 2005.
J Clin Microbiol. 2007 Aug;45(8):2704-7.
We determined vaginal Staphylococcus aureus superantigens. Staphylococci were quantified from tampons/diaphragms in 2003 to 2005, with counts compared to those determined in 1980 and 1981. In 2003 to 2005, more women were colonized than in 1980 and 1981 (23 versus 12%). Enterotoxins G and I and enterotoxin-like superantigens M and N declined, but enterotoxin-like superantigens K, L, and Q increased. [Abstract/Link to Full Text]

Sharma D, Sethi S, Mehta SD, Sharma M
In-house growth-promoting transport system for Neisseria gonorrhoeae.
J Clin Microbiol. 2007 Aug;45(8):2743-4.
Eno powder (GlaxoSmithKline), an antacid preparation readily available over the counter, was used instead of a CO(2) generator for the growth of 15 strains of Neisseria gonorrhoeae obtained from men with urethritis. Due to its easy accessibility and low cost, Eno powder can be useful in developing countries for transporting clinical specimens from resource-poor peripheral labs to reference laboratories. [Abstract/Link to Full Text]

Holtfreter S, Grumann D, Schmudde M, Nguyen HT, Eichler P, Strommenger B, Kopron K, Kolata J, Giedrys-Kalemba S, Steinmetz I, Witte W, Bröker BM
Clonal distribution of superantigen genes in clinical Staphylococcus aureus isolates.
J Clin Microbiol. 2007 Aug;45(8):2669-80.
Staphylococcus aureus is both a successful human commensal and a major pathogen. The elucidation of the molecular determinants of virulence, in particular assessment of the contributions of the genetic background versus those of mobile genetic elements (MGEs), has proved difficult in this variable species. To address this, we simultaneously determined the genetic backgrounds (spa typing) and the distributions of all 19 known superantigens and the exfoliative toxins A and D (multiplex PCR) as markers for MGEs. Methicillin- sensitive S. aureus strains from Pomerania, 107 nasal and 88 blood culture isolates, were investigated. All superantigen-encoding MGEs were linked more or less tightly to the genetic background. Thus, each S. aureus clonal complex was characterized by a typical repertoire of superantigen and exfoliative toxin genes. However, within each S. aureus clonal complex and even within the same spa type, virulence gene profiles varied remarkably. Therefore, virulence genes of nasal and blood culture isolates were separately compared in each clonal complex. The results indicated a role in infection for the MGE harboring the exfoliative toxin D gene. In contrast, there was no association of superantigen genes with bloodstream invasion. In summary, we show here that the simultaneous assessment of virulence gene profiles and the genetic background increases the discriminatory power of genetic investigations into the mechanisms of S. aureus pathogenesis. [Abstract/Link to Full Text]

Schwetz I, Hinrichs G, Reisinger EC, Krejs GJ, Olschewski H, Krause R
Delayed processing of blood samples influences time to positivity of blood cultures and results of Gram stain-acridine orange leukocyte Cytospin test.
J Clin Microbiol. 2007 Aug;45(8):2691-4.
We investigated in vitro whether storage of blood samples influences the time to positivity used for the calculation of the differential time to positivity (DTP) and the results of the Gram stain-acridine orange leukocyte Cytospin (AOLC) test. A 24-hour storage of blood samples at room temperature may lead to false-negative DTP and false-positive Gram stain-AOLC test results, whereas storage at 4 degrees C does not. [Abstract/Link to Full Text]

Evander M, Eriksson I, Pettersson L, Juto P, Ahlm C, Olsson GE, Bucht G, Allard A
Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome.
J Clin Microbiol. 2007 Aug;45(8):2491-7.
Puumala virus (PUUV) is the endemic hantavirus in northern Sweden and causes nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome. There is a need for fast and reliable diagnostics to differentiate the disease from other infections. By aligning virus RNA sequences isolated from 11 different bank voles and one human patient, we designed a real-time reverse transcriptase (RT) PCR method for detection of PUUV RNA. The real-time RT-PCR assay showed linearity from 20 to 2 x 10(6) virus copies with a correlation coefficient above 0.98 to 0.99 for all experiments. The detection threshold for PUUV cDNA was two copies per reaction. A two-step qualitative RT-PCR to detect PUUV RNA showed 100% concordance with the real-time RT-PCR assay. PUUV RNA viremia was detected in 33 of 34 PUUV immunoglobulin M (IgM)-positive patients with typical clinical NE disease from the region of endemicity. One PUUV IgM-negative sample had PUUV RNA, and 4 days later, the patient was IgM positive. Of samples with indeterminate IgM, 43% were PUUV RNA positive. The kinetics of antibody titers and PUUV viremia were studied, and five of six NE patients displayed a decrease in PUUV viremia a few days after disease outbreak coupled with an increase in PUUV IgM and IgG. In one patient with continuously high PUUV RNA levels but low IgM and no IgG response, the infection was lethal. These findings demonstrated that real-time RT-PCR is a useful method for diagnosis of PUUV viremia and for detecting PUUV RNA at early time points, before the appearance of IgM antibodies. [Abstract/Link to Full Text]

Chernesky M, Jang D, Portillo E, Chong S, Smieja M, Luinstra K, Petrich A, Macritchie C, Ewert R, Hayhoe B, Sarabia A, Thompson F
Abilities of APTIMA, AMPLICOR, and ProbeTec assays to detect Chlamydia trachomatis and Neisseria gonorrhoeae in PreservCyt ThinPrep Liquid-based Pap samples.
J Clin Microbiol. 2007 Aug;45(8):2355-8.
Infections with Chlamydia trachomatis and Neisseria gonorrhoeae are often asymptomatic. Liquid-based Pap (L-Pap) screening may provide samples for testing by commercial assays. Women attending a health clinic or a street youth clinic had a PreservCyt ThinPrep sample and a cervical swab (CS) collected. The L-Pap sample was tested for cytopathology; then 1 ml was transferred to an L-Pap specimen transfer tube for testing by the Gen-Probe APTIMA assays (APTIMA Combo 2 [AC2], APTIMA C. trachomatis [ACT], and APTIMA N. gonorrhoeae [AGC]). The residual L-Pap sample was tested for C. trachomatis and N. gonorrhoeae using Roche AMPLICOR (AMP) and Becton Dickinson ProbeTec (PT). The CS was tested by AC2. A patient was considered infected if two specimens were positive or if a single specimen was positive in two tests. The prevalence of infection was 10% (29/290) for C. trachomatis and 2.4% (7/290) for N. gonorrhoeae. Most of the positive patients had specimens that were reactive in all assays (20/29 for C. trachomatis; 6/7 for N. gonorrhoeae). Four patients had double infections. The sensitivities and specificities of the various tests for the specimens tested were as follows. For C. trachomatis on L-Pap, sensitivity and specificity were 100 and 98.1%, respectively, for ACT, 93.1 and 98.8% for AC2, 86.2 and 91.2% for AMP, and 72.4 and 92.7% for PT. For N. gonorrhoeae on L-Pap, sensitivity and specificity were 100% for both AGC and AC2, 85.7 and 100% for AMP, and 85.7 and 100% for PT. For AC2 with CSs, sensitivity and specificity were 93.1 and 98.5%, respectively, for C. trachomatis, and both were 100% for N. gonorrhoeae. There were significant differences in sensitivity and specificity (P < 0.001). The APTIMA assays were more sensitive and specific than AMP or PT for detecting women's C. trachomatis and/or N. gonorrhoeae infections by testing ThinPrep samples. [Abstract/Link to Full Text]

Thibault VC, Grayon M, Boschiroli ML, Hubbans C, Overduin P, Stevenson K, Gutierrez MC, Supply P, Biet F
New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing.
J Clin Microbiol. 2007 Aug;45(8):2404-10.
Mycobacterium avium subsp. paratuberculosis, the etiological agent of paratuberculosis, affects a wide range of domestic ruminants and has been suggested to be involved in Crohn's disease in humans. Most available methods for identifying and differentiating strains of this difficult species are technically demanding and have limited discriminatory power. Here, we report the identification of novel PCR-based typing markers consisting of variable-number tandem repeats (VNTRs) of genetic elements called mycobacterial interspersed repetitive units (MIRUs). Eight markers were applied to 183 M. avium subsp. paratuberculosis isolates from bovine, caprine, ovine, cervine, leporine, and human origins from 10 different countries and to 82 human isolates of the closely related species M. avium from France. Among the M. avium subsp. paratuberculosis isolates, 21 patterns were found by MIRU-VNTR typing, with a discriminatory index of 0.751. The predominant R01 IS900 restriction fragment length polymorphism type, comprising 131 isolates, was divided into 15 MIRU-VNTR types. Among the 82 M. avium isolates, the eight MIRU-VNTR loci distinguished 30 types, none of which was shared by M. avium subsp. paratuberculosis isolates, resulting in a discriminatory index of 0.889. Our results suggest that MIRU-VNTR typing is a fast typing method that, in combination with other methods, might prove to be optimal for PCR-based molecular epidemiological studies of M. avium/M. avium subsp. paratuberculosis pathogens. In addition, presumably identical M. avium subsp. paratuberculosis 316F vaccine strains originating from the Weybridge laboratory and from different commercial batches from Mérial actually differed by one or both typing methods. These results indicate a substantial degree of genetic drift among different vaccine preparations, which has important implications for prophylactic approaches. [Abstract/Link to Full Text]

Ticehurst JR, Hamzeh FM, Thomas DL
Factors affecting serum concentrations of hepatitis C virus (HCV) RNA in HCV genotype 1-infected patients with chronic hepatitis.
J Clin Microbiol. 2007 Aug;45(8):2426-33.
The serum concentration of hepatitis C virus (HCV) RNA is usually stable (4 to 8 log(10) IU/ml) in untreated patients with chronic hepatitis C. While this baseline HCV RNA concentration ([HCV RNA](BL)) is predictive of a sustained virologic response to treatment, its determinants are only partially identified. We therefore analyzed the baseline characteristics of 2,472 HCV genotype 1-infected patients to identify correlations with gender, age, race, weight, body mass index (BMI), HCV acquisition mode, HCV subtype, alanine aminotransferase concentration, or histopathologic changes in the liver. After separation of the data according to four [HCV RNA](BL) groups (< or =5.0, >5.0 to 5.6, >5.6 to 5.9, and >5.9 log(10) IU/ml), we determined that increasing [HCV RNA](BL) correlated (P < 0.05) with increasing proportions of patients who were male, >40 years of age, or heavier (a weight of >85 kg or a BMI of >27 kg/m(2)). Histologic activity index (HAI) data were available for 1,304 of these patients: increasing [HCV RNA](BL) correlated with higher fibrosis and necrosis-inflammation scores. As a continuous variable, [HCV RNA](BL) correlated with age, gender, weight (continuous or < or =85 versus >85 kg), BMI (continuous or < or =27 versus >27 kg/m(2)), subtype, fibrosis score, and necrosis-inflammation score; however, multiple-regression analysis yielded P values of <0.1 only for age, gender, BMI (< or =27 versus >27 kg/m(2)), and fibrosis score. While our findings are suggestive of a role for these factors in maintenance of the pretreatment state of HCV infection, the multiple-regression model accounted for only < or =4.6% of the [HCV RNA](BL) differences between individuals (R(2) = 0.046 for 1,304 patients with HAI scores; 0.043 for all 2,472 patients). [Abstract/Link to Full Text]

Nasri D, Bouslama L, Omar S, Saoudin H, Bourlet T, Aouni M, Pozzetto B, Pillet S
Typing of human enterovirus by partial sequencing of VP2.
J Clin Microbiol. 2007 Aug;45(8):2370-9.
The sequencing of the VP1 hypervariable region of the human enterovirus (HEV) genome has become the reference test for typing field isolates. This study describes a new strategy for typing HEV at the serotype level that uses a reverse transcription-PCR assay targeting the central part of the VP2 capsid protein. Two pairs of primers were used to amplify a fragment of 584 bp (with reference to the PV-1 sequence) or a part of it (368 bp) for typing. For a few strains not amplified by the first PCR, seminested primers enhanced the sensitivity (which was found to be approximately 10(-1) and 10(-4) 50% tissue culture infective dose per reaction tube for the first and seminested assay, respectively). The typing method was then applied to 116 clinical and environmental strains of HEV. Sixty-one typeable isolates were correctly identified at the serotype level by comparison to seroneutralization. Forty-eight of 55 "untypeable" strains (87.3%) exhibited the same serotype using VP1 and VP2 sequencing methods. For six strains (four identified as EV-71, one as E-9, and one as E-30 by the VP2 method), no amplification was obtained by the VP1 method. The last strain, typed as CV-B4 by VP1 and CV-B3 by VP2 and monovalent antiserum, could exhibit recombination within the capsid region. Although the VP2 method was tested on only 36 of the 68 HEV serotypes, it appears to be a promising strategy for typing HEV strains isolated on a routine basis. The good sensitivity of the seminested technique could avoid cell culture and allow HEV typing directly from PCR products. [Abstract/Link to Full Text]


Recent Articles in Journal of Immune Based Therapies and Vaccines

Newell MK, Melamede R, Villalobos-Menuey E, Swartzendruber D, Trauger R, Camley RE, Crisp W
The effects of chemotherapeutics on cellular metabolism and consequent immune recognition.
J Immune Based Ther Vaccines. 2004 Feb 2;2(1):3.
Awidely held view is that oncolytic agents induce death of tumor cells directly. In this report we review and discuss the apoptosis-inducing effects of chemotherapeutics, the effects of chemotherapeutics on metabolic function, and the consequent effects of metabolic function on immune recognition. Finally, we propose that effective chemotherapeutic and/or apoptosis-inducing agents, at concentrations that can be achieved physiologically, do not kill tumor cells directly. Rather, we suggest that effective oncolytic agents sensitize immunologically altered tumor cells to immune recognition and immune-directed cell death. [Abstract/Link to Full Text]

Malley R, Stack AM, Husson RN, Thompson CM, Fleisher GR, Saladino RA
Development of a model of focal pneumococcal pneumonia in young rats.
J Immune Based Ther Vaccines. 2004 Jan 23;2(1):2.
BACKGROUND: A recently licensed pneumococcal conjugate vaccine has been shown to be highly effective in the prevention of bacteremia in immunized children but the degree of protection against pneumonia has been difficult to determine. METHODS: We sought to develop a model of Streptococcus pneumoniae pneumonia in Sprague-Dawley rats. We challenged three-week old Sprague-Dawley pups via intrapulmonary injection of S. pneumoniae serotypes 3 and 6B. Outcomes included bacteremia, mortality as well histologic sections of the lungs. RESULTS: Pneumonia was reliably produced in animals receiving either 10 or 100 cfu of type 3 pneumococci, with 30% and 50% mortality respectively. Similarly, with type 6B, the likelihood of pneumonia increased with the inoculum, as did the mortality rate. Prophylactic administration of a preparation of high-titered anticapsular antibody prevented the development of type 3 pneumonia and death. CONCLUSION: We propose that this model may be useful for the evaluation of vaccines for the prevention of pneumococcal pneumonia. [Abstract/Link to Full Text]

Mohamadzadeh M, Luftig R
Dendritic cells: In the forefront of immunopathogenesis and vaccine development - A review.
J Immune Based Ther Vaccines. 2004 Jan 13;2(1):1.
Dendritic cellls (DCs) comprise an essential component of the immune system. These cells, as antigen presenting cells (APCs) to naďve T cells, are crucial in the initiation of antigen specific immune responses. In the past years, several DC subsets have been identified in different organs which exert different effects in order to elicit adaptive immune responses. Thus, identification of such DC subsets has led to a better understanding of their distribution and function in the body. In this review, several key properties of the immunobiology, immunopathogenesis and vaccine strategies using DCs will be discussed. [Abstract/Link to Full Text]

Agrawal L, Haq W, Hanson CV, Rao DN
Generating neutralizing antibodies, Th1 response and MHC non restricted immunogenicity of HIV-I env and gag peptides in liposomes and ISCOMs with in-built adjuvanticity.
J Immune Based Ther Vaccines. 2003 Nov 25;1(1):5.
For enhancing immunogenicity and develop vaccine strategies using peptide based constructs against HIV-1, a chimeric peptide containing V3 loop and transmembrane sequence of gp41 with two glycine motifs as spacer was constructed. The V3-gp41, gp41 peptide and p17 and p24 peptides separately or in a cocktail were entrapped with or without MA729 as an immunoadjuvant in liposomes or ISCOMs. The immunogenicity, antigen induced T-cell proliferation and cytokine profiles of various formulations were studied in four different inbred strains of mice of H-2d, H-2b, H-2k and H-2q haplotypes, keeping alum as a control adjuvant. Both liposomes and ISCOM preparations elicited high titer and long lasting antibody response (60 days and above). When compared to the alum formulation, the liposomes co-entrapped with MA729 produced high antibody levels, comparable with that induced by ISCOMs. Peptide in alum, liposomes and ISCOMs enhanced both antigen specific IgG2a and IgG2b isotypes and high T-cell stimulation index. Peptide formulations also induced antibodies with high affinity and in vitro neutralizated the formation of HIV-1 syncytia. T-cell supernatants contained high levels of IFN-gamma and IL-2. Thus formulation in these adjuvants induced a predominant Th1 like response with MA729 as a versatile novel delivery vehicle for stimulating the appropriate arm of the immune response that can selectively modulate MHC class I or MHC class II response. The above peptide can be of wide vaccination interest as a means to improve immune responses to several other HIV-1 antigens and may serve as candidates for vaccine development. [Abstract/Link to Full Text]

Ritvo P, Irvine J, Klar N, Wilson K, Brown L, Bremner KE, Rinfret A, Remis R, Krahn MD
A Canadian national survey of attitudes and knowledge regarding preventive vaccines.
J Immune Based Ther Vaccines. 2003 Nov 5;1(1):3.
BACKGROUND: Vaccines have virtually eliminated many diseases, but public concerns about their safety could undermine future public health initiatives. OBJECTIVE: To determine Canadians' attitudes and knowledge about vaccines, particularly in view of increasing public concern about bioterrorism and the possible need for emergency immunizations after weaponized anthrax incidents and the events of September 11, 2001. METHOD: A 20-question survey based on well-researched dimensions of vaccine responsiveness was telephone-administered to a random sample of N = 1330 adult Canadians in January, 2002. RESULTS: 1057 (79.5%) completed the survey. Respondents perceived vaccines to be highly effective and demonstrated considerable support for further vaccine research. However, results also indicate a lack of knowledge about vaccines and uncertainty regarding the safety. CONCLUSIONS: Support for vaccines is broad but shallow. While Canadians hold generally positive attitudes about vaccines, support could be undermined by widely publicized adverse events. Better public education is required to maintain support for future public health initiatives. [Abstract/Link to Full Text]

Moss RB
Welcome to the Journal of Immune Based Therapies and Vaccines (JIBTV).
J Immune Based Ther Vaccines. 2003 Nov 6;1(1):4. [Abstract/Link to Full Text]

Faure K, Fujimoto J, Shimabukuro DW, Ajayi T, Shime N, Moriyama K, Spack EG, Wiener-Kronish JP, Sawa T
Effects of monoclonal anti-PcrV antibody on Pseudomonas aeruginosa-induced acute lung injury in a rat model.
J Immune Based Ther Vaccines. 2003 Aug 13;1(1):2.
BACKGROUND: The effects of the murine monoclonal anti-PcrV antibody Mab166 on acute lung injury induced by Pseudomonas aeruginosa were analyzed in a rat model. METHODS: Lung injury was induced by the instillation of P. aeruginosa strain PA103 directly into the left lungs of anesthetized rats. One hour after the bacterial instillation, rabbit polyclonal anti-PcrV IgG, murine monoclonal anti-PcrV IgG Mab166 or Mab166 Fab-fragments were administered intratracheally directly into the lungs. The degree of alveolar epithelial injury, amount of lung edema, decrease in oxygenation and extent of lung inflammation by histology were evaluated as independent parameters of acute lung injury. RESULTS: These parameters improved in rats that had received intratracheal instillation of either rabbit polyclonal anti-PcrV IgG, murine monoclonal anti-PcrV IgG Mab166 or Mab166 Fab-fragments in comparison with the control group. CONCLUSION: Mab166 and its Fab fragments have potential as adjuvant therapy for acute lung injury due to P. aeruginosa pneumonia. [Abstract/Link to Full Text]

Andersen ML, Ruhwald M, Thorn M, Pedersen AE, Mathiassen S, Buus S, Claesson MH
Tumor-associated antigens identified by mRNA expression profiling as tumor rejection epitopes.
J Immune Based Ther Vaccines. 2003 Jan 29;1(1):1.
Thirteen H-2b-binding peptides derived from six potentially overexpressed proteins in p53-/- thymoma (SM7) cells were studied for immunogenecity and vaccine-induced prevention of tumor growth in mice inoculated with SM7 tumor cells. Six of the peptides generated specific CTL responses after immunization, but only two of these peptides (RAD23-31 and RAD24-31) were capable of generating a weak vaccination-induced protection against adoptive tumor growth. SM7 inoculated mice treated with a blocking antibody against the inhibitory T cell signal transducing molecule CTLA4 appeared to delay tumor take, suggesting that SM7 thymoma cells are recognized by the adaptive immune system of the host. However, prophylactic vaccination with RAD23-31 and RAD24-31 peptides combined with anti-CTLA4 Ab treatment and did not improve tumor resistance. Our data would indicate that vaccination with immunogenic peptides derived from potentially overexpressed tumor proteins, as identified by mRNA expression profiling of p53-/- thymoma cells, at best results in a weak tumor protection thus questioning this way of detection of new tumor rejection epitopes. [Abstract/Link to Full Text]


Recent Articles in Journal of Virology

Chang PC, Li M
Kaposi's sarcoma-associated herpesvirus K-cyclin interacts with Cdk9 and stimulates Cdk9-mediated phosphorylation of p53 tumor suppressor.
J Virol. 2008 Jan;82(1):278-90.
K-cyclin, encoded by Kaposi's sarcoma-associated herpesvirus, has previously been demonstrated to activate cyclin-dependent kinase 6 (Cdk6) to induce the phosphorylation of various cell cycle regulators. In this study, we identified Cdk9 as a new K-cyclin-associated Cdk and showed that K-cyclin interacted with Cdk9 through its basic domain. We hypothesized that K-cyclin served as a regulatory subunit for the activity of Cdk9. Recent reports show that Cdk9 phosphorylates tumor suppressor p53, and we found that the K-cyclin/Cdk9 interaction greatly enhanced the kinase activity of Cdk9 toward p53. The phosphorylation site(s) of K-cyclin/Cdk9 kinase complexes was mapped in the transactivation domain of p53. We showed that the ectopic expression of K-cyclin led to a sustained increase of p53 phosphorylation on Ser(33) in vivo, and the phosphorylation could be inhibited by a dominant negative Cdk9 mutant, dn-Cdk9. Using p53-positive U2OS and p53-null SaOS2 cells, we demonstrated that K-cyclin-induced growth arrest was associated with the presence of p53. In addition, K-cyclin-induced p53-dependent growth arrest was rescued by the dn-Cdk9- or Cdk9-specific short hairpin RNA in SaOS2 cells. Together, our findings for the first time demonstrated the interaction of K-cyclin and Cdk9 and revealed a new molecular link between K-cyclin and p53. [Abstract/Link to Full Text]

Depla E, Van der Aa A, Livingston BD, Crimi C, Allosery K, De Brabandere V, Krakover J, Murthy S, Huang M, Power S, Babé L, Dahlberg C, McKinney D, Sette A, Southwood S, Philip R, Newman MJ, Meheus L
Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections.
J Virol. 2008 Jan;82(1):435-50.
Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2(bxd) (BALB/c x C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice. [Abstract/Link to Full Text]

Chou S, Marousek GI
Accelerated evolution of maribavir resistance in a cytomegalovirus exonuclease domain II mutant.
J Virol. 2008 Jan;82(1):246-53.
A human cytomegalovirus (CMV) UL54 pol exonuclease domain II mutation, D413A, found in a clinical specimen, conferred ganciclovir (GCV) and cidofovir resistance but not foscarnet resistance when incorporated into laboratory strain T2294. After several passages without drug, mutation was observed in five of eight plaques of T2294, and its plating efficiency under foscarnet was increased approximately 30-fold over that of a control strain. When T2294 was serially passed under maribavir (MBV), phenotypic changes and viral UL97 mutations were detected by passage 5, much earlier than previously reported for other CMV strains. By passage 15, mutations included two cases of H411Y, one each of H411L and H411N, three of T409M, five of V353A, and one of L397R. Five instances of codon 409 or 411 mutations evolved into double mutations including V353A. Marker transfer experiments showed H411N/Y/L to confer 9- to 70-fold-increased MBV resistance and combinations of H411L/Y and V353A to confer >150-fold-increased MBV resistance, but no GCV resistance. These findings are consistent with defective exonuclease activity of the pol D413A mutant T2294, leading to the accelerated evolution of UL97 mutations under MBV. This recapitulated the known resistance mutations V353A, L397R, and T409M; suggested their relative frequency; and identified new ones at codon 411. These UL97 mutations predict an MBV binding region overlapping the kinase ATP binding site and located upstream of known GCV resistance mutations. The existence of viable pol D413A mutants may facilitate the selection of additional drug resistance mutations in vivo and the study of these mutations in vitro. [Abstract/Link to Full Text]

Cooper D, Wright KJ, Calderon PC, Guo M, Nasar F, Johnson JE, Coleman JW, Lee M, Kotash C, Yurgelonis I, Natuk RJ, Hendry RM, Udem SA, Clarke DK
Attenuation of recombinant vesicular stomatitis virus-human immunodeficiency virus type 1 vaccine vectors by gene translocations and g gene truncation reduces neurovirulence and enhances immunogenicity in mice.
J Virol. 2008 Jan;82(1):207-19.
Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made. [Abstract/Link to Full Text]

Halasz P, Holloway G, Turner SJ, Coulson BS
Rotavirus replication in intestinal cells differentially regulates integrin expression by a phosphatidylinositol 3-kinase-dependent pathway, resulting in increased cell adhesion and virus yield.
J Virol. 2008 Jan;82(1):148-60.
Changes in the interactions between intestinal cells and their surrounding environment during virus infection have not been well documented. The growth and survival of intestinal epithelial cells, the main targets of rotavirus infection, are largely dependent on the interaction of cell surface integrins with the extracellular matrix. In this study, we detected alterations in cellular integrin expression following rotavirus infection, identified the signaling components required, and analyzed the subsequent effects on cell binding to the matrix component collagen. After rotavirus infection of intestinal cells, expression of alpha2beta1 and beta2 integrins was up-regulated, whereas that of alphaVbeta3, alphaVbeta5, and alpha5beta1 integrins, if present, was down-regulated. This differential regulation of integrins was reflected at the transcriptional level. It was unrelated to the use of integrins as rotavirus receptors, as both integrin-using and integrin-independent viruses induced integrin regulation. Using pharmacological agents that inhibit kinase activity, integrin regulation was shown to be dependent on phosphatidylinositol 3-kinase (PI3K) but independent of the activities of the mitogen-activated protein kinases p38 and ERK1/2, and cyclooxygenase-2. Replication-dependent activation of the PI3K/Akt pathway was observed following infection of intestinal and nonintestinal cell lines. Rotavirus activation of PI3K was important for regulation of alpha2beta1 expression. Blockade of integrin regulation by PI3K inhibition led to decreased adherence of infected intestinal cells to collagen and a concomitant decrease in virus titer. These findings indicate that rotavirus-induced PI3K activation causes regulation of integrin expression in intestinal cells, leading to prolonged adherence of infected cells to collagen and increased virus production. [Abstract/Link to Full Text]

Lakhashe SK, Thakar MR, Bharucha KE, Paranjape RS
Quantitation of HLA proteins incorporated by human immunodeficiency virus type 1 and assessment of neutralizing activity of anti-HLA antibodies.
J Virol. 2008 Jan;82(1):428-34.
Human anti-human leukocyte antigen (HLA) antibodies were assessed for neutralizing activity against human immunodeficiency virus type 1 (HIV-1) carrying HLA alleles with matching specificity. Multiparous women carrying anti-HLA antibodies were identified. Plasma samples from those women were confirmed as having antibodies that specifically bound to HLA proteins expressed on the peripheral blood mononuclear cells (PBMCs) of their husbands. A primary HIV-1 isolate was cultured in the husband's PBMCs so that the virus carried matching HLA alleles. To determine the HIV-1-neutralizing activity of anti-HLA antibodies, the infectivity of the virus for GHOST cells (which express green fluorescent protein after HIV infection) was investigated in the presence of a plasma sample positive for the respective anti-HLA antibody. A neutralization assay was also performed using purified immunoglobulin G (IgG) from two plasma samples, and two plasma samples were investigated in the presence of complement. The prerequisite for anti-HLA antibody-mediated neutralization is incorporation of HLA proteins by HIV-1. Therefore, the extent of incorporation of HLA proteins by the primary HIV-1 isolate was estimated. The ratios of HLA class I protein to HIV-1 capsid (p24) protein cultured in the PBMCs of two healthy individuals were 0.017 and 0.054. These ratios suggested that the HIV-1 strain used in the assay incorporated more HLA proteins than gp160 trimers. Anti-HLA antibody-positive plasma was found to contain antibodies that specifically reacted to HIV-1 carrying cognate HLA alleles. However, incubation of HIV-1 with anti-HLA antibody- positive plasma or purified IgG did not show a reduction in viral infectivity. HIV-1-neutralizing activity was also not detected in the presence of complement. This study shows that HIV-1 primary isolates cultured in PBMCs contain significant amounts of HLA proteins. However, the binding of antibodies to those HLA proteins does not mediate a reduction in viral infectivity. [Abstract/Link to Full Text]

Tran K, Mahr JA, Choi J, Teodoro JG, Green MR, Spector DH
Accumulation of substrates of the anaphase-promoting complex (APC) during human cytomegalovirus infection is associated with the phosphorylation of Cdh1 and the dissociation and relocalization of APC subunits.
J Virol. 2008 Jan;82(1):529-37.
Cell cycle dysregulation upon human cytomegalovirus (HCMV) infection of human fibroblasts is associated with the inactivation of the anaphase-promoting complex (APC), a multisubunit E3 ubiquitin ligase, and accumulation of its substrates. Here, we have further elucidated the mechanism(s) by which HCMV-induced inactivation of the APC occurs. Our results show that Cdh1 accumulates in a phosphorylated form that may prevent its association with and activation of the APC. The accumulation of Cdh1, but not its phosphorylation, appears to be cyclin-dependent kinase dependent. The lack of an association of exogenously added Cdh1 with the APC from infected cells indicates that the core APC also may be impaired. This is further supported by an examination of the localization and composition of the APC. Coimmunoprecipitation studies show that both Cdh1 and the subunit APC1 become dissociated from the complex. In addition, immunofluorescence analysis demonstrates that as the infection progresses, several subunits redistribute to the cytoplasm, while APC1 remains nuclear. Dissociation of the core complex itself would account for not only the observed inactivity but also its inability to bind to Cdh1. Taken together, these results illustrate that HCMV has adopted multiple mechanisms to inactivate the APC, which underscores its importance for a productive infection. [Abstract/Link to Full Text]

Leen AM, Christin A, Khalil M, Weiss H, Gee AP, Brenner MK, Heslop HE, Rooney CM, Bollard CM
Identification of hexon-specific CD4 and CD8 T-cell epitopes for vaccine and immunotherapy.
J Virol. 2008 Jan;82(1):546-54.
Adenoviral infections in the immunocompromised host are associated with significant morbidity and mortality. Although the adoptive transfer of adenovirus-specific T cells may prevent and treat such infections, the T-cell immune response to the multiplicity of adenovirus serotypes and subspecies that infect humans has not been well characterized, impeding the development of such approaches. We have, therefore, analyzed the specificities of T-cell responses to the viral capsid hexon antigen, since this structure is highly conserved in human pathogens. We screened 25 human cytotoxic T-cell lines with adenovirus specificity to extensively characterize their responses to adenoviral hexon and to identify a panel of novel CD4(+) and CD8(+) T-cell epitopes. Using a peptide library spanning the entire sequence of the hexon protein, we confirmed the responsiveness of these cytotoxic T-cell lines to seven peptides described previously and also identified 33 new CD4- or CD8-restricted hexon epitopes. Importantly, the majority of these epitopes were shared among different adenovirus subspecies, suggesting that T cells with such specificities could recognize and be protective against multiple serotypes, simplifying the task of effective adoptive transfer or vaccine-based immunotherapy for treating infection by this virus. [Abstract/Link to Full Text]

Subramanian A, Hegde S, Porayette P, Yon M, Hankey P, Paulson RF
Friend virus utilizes the BMP4-dependent stress erythropoiesis pathway to induce erythroleukemia.
J Virol. 2008 Jan;82(1):382-93.
More than 50 years of genetic analysis has identified a number of host genes that are required for the expansion of infected cells during the progression of Friend-virus-induced erythroleukemia. In this report, we show that Friend virus induces the bone morphogenetic protein 4 (BMP4)-dependent stress erythropoiesis pathway in the spleen, which rapidly amplifies target cells, propagating their infection and resulting in acute splenomegaly. This mechanism mimics the response to acute anemia, in which BMP4 expressed in the spleen drives the expansion of a specialized population of stress erythroid progenitors. Previously we demonstrated that these progenitors, termed stress BFU-E, are targets for Friend virus in the spleen (A. Subramanian, H. E. Teal, P. H. Correll, and R. F. Paulson, J. Virol. 79:14586-14594, 2005). Here, we extend those findings by showing that Friend virus infects two distinct populations of bone marrow cells. One population, when infected, differentiates into mature erythrocytes in an Epo-independent manner, while a second population migrates to the spleen after infection, where it induces BMP4 expression and acts as a reservoir of virus. The activation of the stress erythropoiesis pathway in the spleen by Friend virus results in the rapid expansion of stress BFU-E, providing abundant target cells for viral infection. These observations suggest a novel mechanism by which a virus induces a stress response pathway that amplifies target cells for the virus, leading to acute expansion of infected cells. [Abstract/Link to Full Text]

Kapasi AJ, Spector DH
Inhibition of the cyclin-dependent kinases at the beginning of human cytomegalovirus infection specifically alters the levels and localization of the RNA polymerase II carboxyl-terminal domain kinases cdk9 and cdk7 at the viral transcriptosome.
J Virol. 2008 Jan;82(1):394-407.
We previously reported that defined components of the host transcription machinery are recruited to human cytomegalovirus immediate-early (IE) transcription sites, including cdk9 and cdk7 (S. Tamrakar, A. J. Kapasi, and D. H. Spector, J. Virol. 79:15477-15493, 2005). In this report, we further document the complexity of this site, referred to as the transcriptosome, through identification of additional resident proteins, including viral UL69 and cellular cyclin T1, Brd4, histone deacetylase 1 (HDAC1), and HDAC2. To examine the role of cyclin-dependent kinases (cdks) in the establishment of this site, we used roscovitine, a specific inhibitor of cdk1, cdk2, cdk7, and cdk9, that alters processing of viral IE transcripts and inhibits expression of viral early genes. In the presence of roscovitine, IE2, cyclin T1, Brd4, HDAC1, and HDAC2 accumulate at the transcriptosome. However, accumulation of cdk9 and cdk7 was specifically inhibited. Roscovitine treatment also resulted in decreased levels of cdk9 and cdk7 RNA. There was a corresponding reduction in cdk9 protein but only a modest decrease in cdk7 protein. However, overexpression of cdk9 does not compensate for the effects of roscovitine on cdk9 localization or viral gene expression. Delaying the addition of roscovitine until 8 h postinfection prevented all of the observed effects of the cdk inhibitor. These data suggest that IE2 and multiple cellular factors needed for viral RNA synthesis accumulate within the first 8 h at the viral transcriptosome and that functional cdk activity is required for the specific recruitment of cdk7 and cdk9 during this time interval. [Abstract/Link to Full Text]

Tavalai N, Papior P, Rechter S, Stamminger T
Nuclear domain 10 components promyelocytic leukemia protein and hDaxx independently contribute to an intrinsic antiviral defense against human cytomegalovirus infection.
J Virol. 2008 Jan;82(1):126-37.
Infection with DNA viruses commonly results in the association of viral genomes with a cellular subnuclear structure known as nuclear domain 10 (ND10). Recent studies demonstrated that individual ND10 components, like hDaxx or promyelocytic leukemia protein (PML), mediate an intrinsic immune response against human cytomegalovirus (HCMV) infection, strengthening the assumption that ND10 components are part of a cellular antiviral defense mechanism. In order to further define the role of hDaxx and PML for HCMV replication, we generated either primary human fibroblasts with a stable, individual knockdown of PML or hDaxx (PML-kd and hDaxx-kd, respectively) or cells exhibiting a double knockdown. Comparative analysis of HCMV replication in PML-kd or hDaxx-kd cells revealed that immediate-early (IE) gene expression increased to a similar extent, regardless of which ND10 constituent was depleted. Since a loss of PML, the defining component of ND10, results in a dispersal of the entire nuclear substructure, the increased replication efficacy of HCMV in PML-kd cells could be a consequence of the dissociation of the repressor protein hDaxx from its optimal subnuclear localization. However, experiments using three different recombinant HCMVs revealed a differential growth complementation in PML-kd versus hDaxx-kd cells, strongly arguing for an independent involvement in suppressing HCMV replication. Furthermore, infection experiments using double-knockdown cells devoid of both PML and hDaxx illustrated an additional enhancement in the replication efficacy of HCMV compared to the single-knockdown cells. Taken together, our data indicate that both proteins, PML and hDaxx, mediate an intrinsic immune response against HCMV infection by contributing independently to the silencing of HCMV IE gene expression. [Abstract/Link to Full Text]

Buchkovich NJ, Maguire TG, Yu Y, Paton AW, Paton JC, Alwine JC
Human cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone BiP/GRP78, which is required for virion assembly.
J Virol. 2008 Jan;82(1):31-9.
The endoplasmic reticulum (ER) chaperone BiP/GRP78 regulates ER function and the unfolded protein response (UPR). Human cytomegalovirus infection of human fibroblasts induces the UPR but modifies it to benefit viral replication. BiP/GRP78 protein levels are tightly regulated during infection, rising after 36 h postinfection (hpi), peaking at 60 hpi, and decreasing thereafter. To determine the effects of this regulation on viral replication, BiP/GRP78 was depleted using the SubAB subtilase cytotoxin, which rapidly and specifically cleaves BiP/GRP78. Toxin treatment of infected cells for 12-h periods beginning at 36, 48, 60, and 84 hpi caused complete loss of BiP but had little effect on viral protein synthesis. However, progeny virion formation was significantly inhibited, suggesting that BiP/GRP78 is important for virion formation. Electron microscopic analysis showed that infected cells were resistant to the toxin and showed none of the cytotoxic effects seen in uninfected cells. However, all viral activity in the cytoplasm ceased, with nucleocapsids remaining in the nucleus or concentrated in the cytoplasmic space just outside of the outer nuclear membrane. These data suggest that one effect of the controlled expression of BiP/GRP78 in infected cells is to aid in cytoplasmic virion assembly and egress. [Abstract/Link to Full Text]

Scott CA, Rossiter JP, Andrew RD, Jackson AC
Structural abnormalities in neurons are sufficient to explain the clinical disease and fatal outcome of experimental rabies in yellow fluorescent protein-expressing transgenic mice.
J Virol. 2008 Jan;82(1):513-21.
Under natural conditions and in some experimental models, rabies virus infection of the central nervous system causes relatively mild histopathological changes, without prominent evidence of neuronal death despite its lethality. In this study, the effects of rabies virus infection on the structure of neurons were investigated with experimentally infected transgenic mice expressing yellow fluorescent protein (YFP) in neuronal subpopulations. Six-week-old mice were inoculated in the hind-limb footpad with the CVS strain of fixed virus or were mock infected with vehicle (phosphate-buffered saline). Brain regions were subsequently examined by light, epifluorescent, and electron microscopy. In moribund CVS-infected mice, histopathological changes were minimal in paraffin-embedded tissue sections, although mild inflammatory changes were present. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase-3 immunostaining showed only a few apoptotic cells in the cerebral cortex and hippocampus. Silver staining demonstrated the preservation of cytoskeletal integrity in the cerebral cortex. However, fluorescence microscopy revealed marked beading and fragmentation of the dendrites and axons of layer V pyramidal neurons in the cerebral cortex, cerebellar mossy fibers, and axons in brainstem tracts. At an earlier time point, when mice displayed hind-limb paralysis, beading was observed in a few axons in the cerebellar commissure. Toluidine blue-stained resin-embedded sections from moribund YFP-expressing animals revealed vacuoles within the perikarya and proximal dendrites of pyramidal neurons in the cerebral cortex and hippocampus. These vacuoles corresponded with swollen mitochondria under electron microscopy. Vacuolation was also observed ultrastructurally in axons and in presynaptic nerve endings. We conclude that the observed structural changes are sufficient to explain the severe clinical disease with a fatal outcome in this experimental model of rabies. [Abstract/Link to Full Text]

DeGottardi MQ, Lew SK, Piatak M, Jia B, Feng Y, Lee SJ, Brenchley JM, Douek DC, Kodama T, Lifson JD, Evans DT
Comparison of plasma viremia and antibody responses in macaques inoculated with envelope variants of single-cycle simian immunodeficiency virus differing in infectivity and cellular tropism.
J Virol. 2008 Jan;82(1):321-34.
Molecular differences in the envelope glycoproteins of human immunodeficiency virus type 1 and simian immunodeficiency virus (SIV) determine virus infectivity and cellular tropism. To examine how these properties contribute to productive infection in vivo, rhesus macaques were inoculated with strains of single-cycle SIV (scSIV) engineered to express three different envelope glycoproteins with full-length (TM(open)) or truncated (TM(stop)) cytoplasmic tails. The 239 envelope uses CCR5 for infection of memory CD4(+) T cells, the 316 envelope also uses CCR5 but has enhanced infectivity for primary macrophages, and the 155T3 envelope uses CXCR4 for infection of both naive and memory CD4(+) T cells. Separate groups of six rhesus macaques were inoculated intravenously with mixtures of TM(open) and TM(stop) scSIV(mac)239, scSIV(mac)316, and scSIV(mac)155T3. A multiplex real-time PCR assay specific for unique sequence tags engineered into each virus was then used to measure viral loads for each strain independently. Viral loads in plasma peaked on day 4 for each strain and were resolved below the threshold of detection within 4 to 10 weeks. Truncation of the envelope cytoplasmic tail significantly increased the peak of viremia for all three envelope variants and the titer of SIV-specific antibody responses. Although peak viremias were similar for both R5- and X4-tropic viruses, clearance of scSIV(mac)155T3 TM(stop) was significantly delayed relative to the other strains, possibly reflecting the infection of a CXCR4(+) cell population that is less susceptible to the cytopathic effects of virus infection. These studies reveal differences in the peaks and durations of a single round of productive infection that reflect envelope-specific differences in infectivity, chemokine receptor specificity, and cellular tropism. [Abstract/Link to Full Text]

Alam SM, Scearce RM, Parks RJ, Plonk K, Plonk SG, Sutherland LL, Gorny MK, Zolla-Pazner S, Vanleeuwen S, Moody MA, Xia SM, Montefiori DC, Tomaras GD, Weinhold KJ, Karim SA, Hicks CB, Liao HX, Robinson J, Shaw GM, Haynes BF
Human immunodeficiency virus type 1 gp41 antibodies that mask membrane proximal region epitopes: antibody binding kinetics, induction, and potential for regulation in acute infection.
J Virol. 2008 Jan;82(1):115-25.
Two human monoclonal antibodies (MAbs) (2F5 and 4E10) against the human immunodeficiency virus type 1 (HIV-1) envelope g41 cluster II membrane proximal external region (MPER) broadly neutralize HIV-1 primary isolates. However, these antibody specificities are rare, are not induced by Env immunization or HIV-1 infection, and are polyspecific and also react with lipids such as cardiolipin or phosphatidylserine. To probe MPER anti-gp41 antibodies that are produced in HIV-1 infection, we have made two novel murine MAbs, 5A9 and 13H11, against HIV-1 gp41 envelope that partially cross-blocked 2F5 MAb binding to Env but did not neutralize HIV-1 primary isolates or bind host lipids. Competitive inhibition assays using labeled 13H11 MAb and HIV-1-positive patient plasma samples demonstrated that cluster II 13H11-blocking plasma antibodies were made in 83% of chronically HIV-1 infected patients and were acquired between 5 to 10 weeks after acute HIV-1 infection. Both the mouse 13H11 MAb and the three prototypic cluster II human MAbs (98-6, 126-6, and 167-D) blocked 2F5 binding to gp41 epitopes to variable degrees; the combination of 98-6 and 13H11 completely blocked 2F5 binding. These data provide support for the hypothesis that in some patients, B cells make nonneutralizing cluster II antibodies that may mask or otherwise down-modulate B-cell responses to immunogenic regions of gp41 that could be recognized by B cells capable of producing antibodies like 2F5. [Abstract/Link to Full Text]

Vestergaard G, Aramayo R, Basta T, Häring M, Peng X, Brügger K, Chen L, Rachel R, Boisset N, Garrett RA, Prangishvili D
Structure of the acidianus filamentous virus 3 and comparative genomics of related archaeal lipothrixviruses.
J Virol. 2008 Jan;82(1):371-81.
Four novel filamentous viruses with double-stranded DNA genomes, namely, Acidianus filamentous virus 3 (AFV3), AFV6, AFV7, and AFV8, have been characterized from the hyperthermophilic archaeal genus Acidianus, and they are assigned to the Betalipothrixvirus genus of the family Lipothrixviridae. The structures of the approximately 2-mum-long virions are similar, and one of them, AFV3, was studied in detail. It consists of a cylindrical envelope containing globular subunits arranged in a helical formation that is unique for any known double-stranded DNA virus. The envelope is 3.1 nm thick and encases an inner core with two parallel rows of protein subunits arranged like a zipper. Each end of the virion is tapered and carries three short filaments. Two major structural proteins were identified as being common to all betalipothrixviruses. The viral genomes were sequenced and analyzed, and they reveal a high level of conservation in both gene content and gene order over large regions, with this similarity extending partly to the earlier described betalipothrixvirus Sulfolobus islandicus filamentous virus. A few predicted gene products of each virus, in addition to the structural proteins, could be assigned specific functions, including a putative helicase involved in Holliday junction branch migration, a nuclease, a protein phosphatase, transcriptional regulators, and glycosyltransferases. The AFV7 genome appears to have undergone intergenomic recombination with a large section of an AFV2-like viral genome, apparently resulting in phenotypic changes, as revealed by the presence of AFV2-like termini in the AFV7 virions. Shared features of the genomes include (i) large inverted terminal repeats exhibiting conserved, regularly spaced direct repeats; (ii) a highly conserved operon encoding the two major structural proteins; (iii) multiple overlapping open reading frames, which may be indicative of gene recoding; (iv) putative 12-bp genetic elements; and (v) partial gene sequences corresponding closely to spacer sequences of chromosomal repeat clusters. [Abstract/Link to Full Text]

Alvarez R, Reading J, King DF, Hayes M, Easterbrook P, Farzaneh F, Ressler S, Yang F, Rowley D, Vyakarnam A
WFDC1/ps20 is a novel innate immunomodulatory signature protein of human immunodeficiency virus (HIV)-permissive CD4+ CD45RO+ memory T cells that promotes infection by upregulating CD54 integrin expression and is elevated in HIV type 1 infection.
J Virol. 2008 Jan;82(1):471-86.
Understanding why human immunodeficiency virus (HIV) preferentially infects some CD4(+) CD45RO(+) memory T cells has implications for antiviral immunity and pathogenesis. We report that differential expression of a novel secreted factor, ps20, previously implicated in tissue remodeling, may underlie why some CD4 T cells are preferentially targeted. We show that (i) there is a significant positive correlation between endogenous ps20 mRNA in diverse CD4 T-cell populations and in vitro infection, (ii) a ps20(+) permissive cell can be made less permissive by antibody blockade- or small-interference RNA-mediated knockdown of endogenous ps20, and (iii) conversely, a ps20(low) cell can be more permissive by adding ps20 exogenously or engineering stable ps20 expression by retroviral transduction. ps20 expression is normally detectable in CD4 T cells after in vitro activation and interleukin-2 expansion, and such oligoclonal populations comprise ps20(positive) and ps20(low/negative) isogenic clones at an early differentiation stage (CD45RO(+)/CD25(+)/CD28(+)/CD57(-)). This pattern is altered in chronic HIV infection, where ex vivo CD4(+) CD45RO(+) T cells express elevated ps20. ps20 promoted HIV entry via fusion and augmented CD54 integrin expression; both of these effects were reversed by anti-ps20 antibody. We therefore propose ps20 to be a novel signature of HIV-permissive CD4 T cells that promotes infection in an autocrine and paracrine manner and that HIV has coopted a fundamental role of ps20 in promoting cell adhesion for its benefit. Disrupting the ps20 pathway may therefore provide a novel anti-HIV strategy. [Abstract/Link to Full Text]

Loo YM, Fornek J, Crochet N, Bajwa G, Perwitasari O, Martinez-Sobrido L, Akira S, Gill MA, García-Sastre A, Katze MG, Gale M
Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity.
J Virol. 2008 Jan;82(1):335-45.
Alpha/beta interferon immune defenses are essential for resistance to viruses and can be triggered through the actions of the cytoplasmic helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Signaling by each is initiated by the recognition of viral products such as RNA and occurs through downstream interaction with the IPS-1 adaptor protein. We directly compared the innate immune signaling requirements of representative viruses of the Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae for RIG-I, MDA5, and interferon promoter-stimulating factor 1 (IPS-1). In cultured fibroblasts, IPS-1 was essential for innate immune signaling of downstream interferon regulatory factor 3 activation and interferon-stimulated gene expression, but the requirements for RIG-I and MDA5 were variable. Each was individually dispensable for signaling triggered by reovirus and dengue virus, whereas RIG-I was essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus. Functional genomics analyses identified cellular genes triggered during influenza A virus infection whose expression was strictly dependent on RIG-I and which are involved in processes of innate or adaptive immunity, apoptosis, cytokine signaling, and inflammation associated with the host response to contemporary and pandemic strains of influenza virus. These results define IPS-1-dependent signaling as an essential feature of host immunity to RNA virus infection. Our observations further demonstrate differential and redundant roles for RIG-I and MDA5 in pathogen recognition and innate immune signaling that may reflect unique and shared biologic properties of RNA viruses whose differential triggering and control of gene expression may impact pathogenesis and infection. [Abstract/Link to Full Text]

Jung A, Kato H, Kumagai Y, Kumar H, Kawai T, Takeuchi O, Akira S
Lymphocytoid choriomeningitis virus activates plasmacytoid dendritic cells and induces a cytotoxic T-cell response via MyD88.
J Virol. 2008 Jan;82(1):196-206.
Toll-like receptors (TLRs) and retinoic acid-inducible gene I-like helicases (RLHs) are two major machineries recognizing RNA virus infection of innate immune cells. Intracellular signaling for TLRs and RLHs is mediated by their cytoplasmic adaptors, i.e., MyD88 or TRIF and IPS-1, respectively. In the present study, we investigated the contributions of TLRs and RLHs to the cytotoxic T-lymphocyte (CTL) response by using lymphocytoid choriomeningitis virus (LCMV) as a model virus. The generation of virus-specific cytotoxic T lymphocytes was critically dependent on MyD88 but not on IPS-1. Type I interferons (IFNs) are known to be important for the development of the CTL response to LCMV infection. Serum levels of type I IFNs and proinflammatory cytokines were mainly dependent on the presence of MyD88, although IPS-1(-/-) mice showed a decrease in IFN-alpha levels but not in IFN-beta and proinflammatory cytokine levels. Analysis of Ifna6(+/GFP) reporter mice revealed that plasmacytoid dendritic cells (DCs) are the major source of IFN-alpha in LCMV infection. MyD88(-/-) mice were highly susceptible to LCMV infection in vivo. These results suggest that recognition of LCMV by plasmacytoid DCs via TLRs is responsible for the production of type I IFNs in vivo. Furthermore, the activation of a MyD88-dependent innate mechanism induces a CTL response, which eventually leads to virus elimination. [Abstract/Link to Full Text]

Cao J, McNevin J, McSweyn M, Liu Y, Mullins JI, McElrath MJ
Novel cytotoxic T-lymphocyte escape mutation by a three-amino-acid insertion in the human immunodeficiency virus type 1 p6Pol and p6Gag late domain associated with drug resistance.
J Virol. 2008 Jan;82(1):495-502.
Cytolytic T lymphocytes (CTL) play a major role in controlling human immunodeficiency virus type 1 (HIV-1) infection. To evade immune pressure, HIV-1 is selected at targeted CTL epitopes, which may consequentially alter viral replication fitness. In our longitudinal investigations of the interplay between T-cell immunity and viral evolution following acute HIV-1 infection, we observed in a treatment-naďve patient the emergence of highly avid, gamma interferon-secreting, CD8(+) CTL recognizing an HLA-Cw*0102-restricted epitope, NSPTRREL (NL8). This epitope lies in the p6(Pol) protein, located in the transframe region of the Gag-Pol polyprotein. Over the course of infection, an unusual viral escape mutation arose within the p6(Pol) epitope through insertion of a 3-amino-acid repeat, NSPT(SPT)RREL, with a concomitant insertion in the p6(Gag) late domain, PTAPP(APP). Interestingly, this p6(Pol) insertion mutation is often selected in viruses with the emergence of antiretroviral drug resistance, while the p6(Gag) late-domain PTAPP motif binds Tsg101 to permit viral budding. These results are the first to demonstrate viral evasion of immune pressure by amino acid insertions. Moreover, this escape mutation represents a novel mechanism whereby HIV-1 can alter its sequence within both the Gag and Pol proteins with potential functional consequences for viral replication and budding. [Abstract/Link to Full Text]

Mulupuri P, Zimmerman JJ, Hermann J, Johnson CR, Cano JP, Yu W, Dee SA, Murtaugh MP
Antigen-specific B-cell responses to porcine reproductive and respiratory syndrome virus infection.
J Virol. 2008 Jan;82(1):358-70.
Porcine reproductive and respiratory syndrome virus (PRRSV) causes an acute, viremic infection of 4 to 6 weeks, followed by a persistent infection lasting for several months. We characterized antibody and B-cell responses to viral proteins in acute and persistent infection to better understand the immunological basis of the prolonged infection. The humoral immune response to PRRSV was robust overall and varied among individual viral proteins, with the important exception of a delayed and relatively weak response to envelope glycoprotein 5 (GP5). Memory B cells were in secondary lymphoid organs, not in bone marrow or Peyer's patches, in contrast to the case for many mammalian species. Potent anti-PRRSV memory responses were elicited to recall antigen in vitro, even though a second infection did not increase the B-cell response in vivo, suggesting that productive reinfection does not occur in vivo. Antibody titers to several viral proteins decline over time, even though abundant antigen is known to be present in lymphoid tissues, possibly indicating ineffective antigen presentation. The appearance of antibodies to GP5 is delayed relative to the resolution of viremia, suggesting that anti-GP5 antibodies are not crucial for resolving viremia. Lastly, viral infection had no immunosuppressive effect on the humoral response to a second, unrelated antigen. Taking these data together, the active effector and memory B-cell responses to PRRSV are robust, and over time the humoral immune response to PRRSV is effective. However, the delayed response against GP5 early in infection may contribute to the prolonged acute infection and the establishment of persistence. [Abstract/Link to Full Text]

Klingen Y, Conzelmann KK, Finke S
Double-labeled rabies virus: live tracking of enveloped virus transport.
J Virol. 2008 Jan;82(1):237-45.
Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo. [Abstract/Link to Full Text]

Ireland DD, Stohlman SA, Hinton DR, Atkinson R, Bergmann CC
Type I interferons are essential in controlling neurotropic coronavirus infection irrespective of functional CD8 T cells.
J Virol. 2008 Jan;82(1):300-10.
Neurotropic coronavirus infection induces expression of both beta interferon (IFN-beta) RNA and protein in the infected rodent central nervous system (CNS). However, the relative contributions of type I IFN (IFN-I) to direct, cell-type-specific virus control or CD8 T-cell-mediated effectors in the CNS are unclear. IFN-I receptor-deficient (IFNAR(-/-)) mice infected with a sublethal and demyelinating neurotropic virus variant and those infected with a nonpathogenic neurotropic virus variant both succumbed to infection within 9 days. Compared to wild-type (wt) mice, replication was prominently increased in all glial cell types and spread to neurons, demonstrating expanded cell tropism. Furthermore, increased pathogenesis was associated with significantly enhanced accumulation of neutrophils, tumor necrosis factor alpha, interleukin-6, chemokine (C-C motif) ligand 2, and IFN-gamma within the CNS. The absence of IFN-I signaling did not impair induction or recruitment of virus-specific CD8 T cells, the primary adaptive mediators of virus clearance in wt mice. Despite similar IFN-gamma-mediated major histocompatibility complex class II upregulation on microglia in infected IFNAR(-/-) mice, class I expression was reduced compared to that on microglia in wt mice, suggesting a synergistic role of IFN-I and IFN-gamma in optimizing class I antigen presentation. These data demonstrate a critical direct antiviral role of IFN-I in controlling virus dissemination within the CNS, even in the presence of potent cellular immune responses. By limiting early viral replication and tropism, IFN-I controls the balance of viral replication and immune control in favor of CD8 T-cell-mediated protective functions. [Abstract/Link to Full Text]

Warke RV, Martin KJ, Giaya K, Shaw SK, Rothman AL, Bosch I
TRAIL is a novel antiviral protein against dengue virus.
J Virol. 2008 Jan;82(1):555-64.
Dengue fever is an important tropical illness for which there is currently no virus-specific treatment. To shed light on mechanisms involved in the cellular response to dengue virus (DV), we assessed gene expression changes, using Affymetrix GeneChips (HG-U133A), of infected primary human cells and identified changes common to all cells. The common response genes included a set of 23 genes significantly induced upon DV infection of human umbilical vein endothelial cells (HUVECs), dendritic cells (DCs), monocytes, and B cells (analysis of variance, P < 0.05). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one of the common response genes, was identified as a key link between type I and type II interferon response genes. We found that DV induces TRAIL expression in immune cells and HUVECs at the mRNA and protein levels. The induction of TRAIL expression by DV was found to be dependent on an intact type I interferon signaling pathway. A significant increase in DV RNA accumulation was observed in anti-TRAIL antibody-treated monocytes, B cells, and HUVECs, and, conversely, a decrease in DV RNA was seen in recombinant TRAIL-treated monocytes. Furthermore, recombinant TRAIL inhibited DV titers in DV-infected DCs by an apoptosis-independent mechanism. These data suggest that TRAIL plays an important role in the antiviral response to DV infection and is a candidate for antiviral interventions against DV. [Abstract/Link to Full Text]

Yang K, Poon AP, Roizman B, Baines JD
Temperature-sensitive mutations in the putative herpes simplex virus type 1 terminase subunits pUL15 and pUL33 preclude viral DNA cleavage/packaging and interaction with pUL28 at the nonpermissive temperature.
J Virol. 2008 Jan;82(1):487-94.
Terminases comprise essential components of molecular motors required to package viral DNA into capsids in a variety of DNA virus systems. Previous studies indicated that the herpes simplex virus type 1 U(L)15 protein (pU(L)15) interacts with the pU(L)28 moiety of a pU(L)28-pU(L)33 complex to form the likely viral terminase. In the current study, a novel temperature-sensitive mutant virus was shown to contain a mutation in U(L)33 codon 61 predicted to change threonine to proline. At the nonpermissive temperature, this virus, designated ts8-22, replicated viral DNA and produced capsids that became enveloped at the inner nuclear membrane but failed to form plaques or to cleave or package viral DNA. Incubation at the nonpermissive temperature also precluded coimmunoprecipitation of U(L)33 protein with its normal interaction partners encoded by U(L)28 and U(L)15 in ts8-22-infected cells and with pU(L)28 in transient-expression assays. Moreover, a temperature-sensitive mutation in U(L)15 precluded coimmunoprecipitation of pU(L)15 with the U(L)28 and U(L)33 proteins at the nonpermissive temperature. We conclude that interactions between putative terminase components are tightly linked to successful viral DNA cleavage and packaging. [Abstract/Link to Full Text]

Yaiw KC, Bingham J, Crameri G, Mungall B, Hyatt A, Yu M, Eaton B, Shamala D, Wang LF, Thong Wong K
Tioman virus, a paramyxovirus of bat origin, causes mild disease in pigs and has a predilection for lymphoid tissues.
J Virol. 2008 Jan;82(1):565-8.
Disease manifestation, pathology, and tissue tropism following infection with Tioman virus (TioPV), a newly isolated, bat-derived paramyxovirus, was investigated in subcutaneously (n = 12) and oronasally (n = 4) inoculated pigs. Pigs were either asymptomatic or developed pyrexia, but all of the animals produced neutralizing antibodies. The virus (viral antigen and/or genome) was detected in lymphocytes of the thymus, tonsils, spleen, lymph nodes and Peyer's patches (ileum), tonsillar epithelium, and thymic epithelioreticular cells. Virus was isolated from oral swabs but not from urine. Our findings suggest that the pig could act as an intermediate or amplifying host for TioPV and that oral secretion is a possible means of viral transmission. [Abstract/Link to Full Text]

Li S, Gowans EJ, Chougnet C, Plebanski M, Dittmer U
Natural regulatory T cells and persistent viral infection.
J Virol. 2008 Jan;82(1):21-30. [Abstract/Link to Full Text]

Smith I
Misleading messengers? Interpreting baculovirus transcriptional array profiles.
J Virol. 2007 Jul;81(14):7819-20; author reply 7820-1. [Abstract/Link to Full Text]

Jiang SS, Chang IS, Huang LW, Chen PC, Wen CC, Liu SC, Chien LC, Lin CY, Hsiung CA, Juang JL
Temporal transcription program of recombinant Autographa californica multiple nucleopolyhedrosis virus.
J Virol. 2006 Sep;80(18):8989-99.
Baculoviruses, a family of large, rod-shaped viruses that mainly infect lepidopteran insects, have been widely used to transduce various cells for exogenous gene expression. Nonetheless, how a virus controls its transcription program in cells is poorly understood. With a custom-made baculovirus DNA microarray, we investigated the recombinant Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) gene expression program in lepidopteran Sf21 cells over the time course of infection. Our analysis of transcription kinetics in the cells uncovered sequential viral gene expression patterns possibly regulated by different mechanisms during different phases of infection. To gain further insight into the regulatory network, we investigated the transcription program of a mutant virus deficient in an early transactivator (pe38) and uncovered several pe38-dependent and pe38-independent genes. This study of baculovirus dynamic transcription programs in different virus genetic backgrounds provides new molecular insights into how gene expression in viruses is regulated. [Abstract/Link to Full Text]

Pignatelli S, Dal Monte P, Landini MP, Severi B, Nassiri R, Gilloteaux J, Papadimitriou JM, Shellam GR, Mertens T, Buser C, Michel D, Walther P
Cytomegalovirus primary envelopment at large nuclear membrane infoldings: what's new?
J Virol. 2007 Jul;81(13):7320-1; author reply 7321-2. [Abstract/Link to Full Text]

Buser C, Walther P, Mertens T, Michel D
Cytomegalovirus primary envelopment occurs at large infoldings of the inner nuclear membrane.
J Virol. 2007 Mar;81(6):3042-8.
We have investigated the morphogenesis of human and murine cytomegalovirus by transmission electron microscopy after high-pressure freezing, freeze substitution, and plastic embedding. We observed large tubular infoldings of the inner nuclear membrane that were free of lamina and active in primary envelopment and subsequent transport of capsids to the nuclear periphery. Semiquantitative determinations of the enlarged inner nuclear membrane area and the location of the primary envelopment of nucleocapsids demonstrated that this structure represents a virus-induced specialized membrane domain at which the particles are preferentially enveloped. This is a previously undescribed structural element relevant in cytomegalovirus morphogenesis. [Abstract/Link to Full Text]

Manrique A, Rusert P, Joos B, Fischer M, Kuster H, Leemann C, Niederöst B, Weber R, Stiegler G, Katinger H, Günthard HF, Trkola A
In vivo and in vitro escape from neutralizing antibodies 2G12, 2F5, and 4E10.
J Virol. 2007 Aug;81(16):8793-808.
Recently, passive immunization of human immunodeficiency virus (HIV)-infected individuals with monoclonal antibodies (MAbs) 2G12, 2F5, and 4E10 provided evidence of the in vivo activity of 2G12 but raised concerns about the function of the two membrane-proximal external region (MPER)-specific MAbs (A. Trkola, H. Kuster, P. Rusert, B. Joos, M. Fischer, C. Leemann, A. Manrique, M. Huber, M. Rehr, A. Oxenius, R. Weber, G. Stiegler, B. Vcelar, H. Katinger, L. Aceto, and H. F. Gunthard, Nat. Med. 11:615-622, 2005). In the light of MPER-targeting vaccines under development, we performed an in-depth analysis of the emergence of mutations conferring resistance to these three MAbs to further elucidate their activity. Clonal analysis of the MPER of plasma virus samples derived during antibody treatment confirmed that no changes in this region had occurred in vivo. Sequence analysis of the 2G12 epitope relevant N-glycosylation sites of viruses derived from 13 patients during the trial supported the phenotypic evaluation, demonstrating that mutations in these sites are associated with resistance. In vitro selection experiments with isolates of four of these individuals corroborated the in vivo finding that virus strains rapidly escape 2G12 pressure. Notably, in vitro resistance mutations differed, in most cases, from those found in vivo. Importantly, in vitro selection with 2F5 and 4E10 demonstrated that resistance to these MAbs can be difficult to achieve and can lead to selection of variants with impaired infectivity. This remarkable vulnerability of the virus to interference within the MPER calls for a further evaluation of the safety and efficacy of MPER-targeting therapeutic and vaccination strategies. [Abstract/Link to Full Text]


Recent Articles in Medical Immunology

No recent articles are currently available.

Recent Articles in Microbiology and Immunology

Asakura H, Panutdaporn N, Kawamoto K, Igimi S, Yamamoto S, Makino S
Proteomic characterization of enterohemorrhagic escherichia coli O157:H7 in the oxidation-induced viable but non-culturable state.
Microbiol Immunol. 2007;51(9):875-81.
Enterohemorrhagic Escherichia coli (EHEC) O157 strain F2, a food isolate of an outbreak, is resistant to oxidative stress, but has increased stress-sensitivity after passage through mice. The stress-sensitive variant of F2 (designated MP37) has decreased culturability, but retains membrane integrity under stress conditions, indicating that the cells enter a viable but non-culturable (VBNC) state. Proteomic analyses revealed that MP37 in the VBNC state had decreased levels of some oxidation-responsive factors (AhpCF, AceF), but it markedly increased levels of outer membrane protein W (OmpW). Because F2 expressed higher levels of some ribosome-associated proteins (RaiA, S6, Bcp) than MP37, the effect of animal passage on the induction of the VBNC state in the EHEC O157 cells might be due to ribosomal activity. [Abstract/Link to Full Text]

Ikewaki N, Fujii N, Onaka T, Ikewaki S, Inoko H
Immunological actions of Sophy beta-glucan (beta-1,3-1,6 glucan), currently available commercially as a health food supplement.
Microbiol Immunol. 2007;51(9):861-73.
We examined the immunological actions of Sophy beta-glucan(Ikewaki N., et al. United States Patent 6956120 and Japan Patent 2004-329077), a type of beta-1,3-1,6 glucan produced by the black yeast Aureobasidium pullulans (A. pullulans) strain AFO-202, currently available commercially as a health food supplement, using different human in vitro experimental systems. Sophy beta-glucan significantly (P<0.01) stimulated the (3)H-thymidine incorporation rates (marker of DNA synthesis) in human peripheral blood mononuclear cells (PBMCs) obtained from normal adult donors, in vitro. Enzyme-linked immunoassays (EIAs) revealed that Sophy beta-glucan stimulated the production of interleukin-8 (IL-8) or soluble Fas (sFas), but not that of IL-1beta, IL-2, IL-6, IL-12 (p70+40), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) or soluble Fas ligand (sFasL), in either cultured PBMCs or cells of the human monocyte-like cell line, U937. The induction by Sophy beta-glucan of DNA synthesis in PBMCs was completely blocked by the addition of monoclonal antibodies (mAbs) to CD11a, CD54, human leukocyte antigen-class II (HLA-class II), Toll-like receptor-2 (TLR-2), and Toll-like receptor-4 (TLR-4). In these blocking experiments using the mAbs, the main differences in the results between PBMCs and U937 cells were that the mAbs against TLR-2 and TLR-4 did not block the Sophy beta-glucan-induced production of IL-8 in the U937 cells. Furthermore, a mAb to the beta-glucan receptor, Dectin-1, significantly (P<0.05) blocked the Sophy beta-glucan induced DNA synthesis in the PBMCs, and Sophy beta-glucan-induced production of IL-8 in the U937 cells. The Sophy beta-glucan-induced production of IL-8 in the U937 cells was significantly (P<0.01) blocked by the conventional protein kinase C (PKC) inhibitor Go6976, the novel PKC inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89, and the protein tyrosine kinase (PTK) inhibitor herbimycin A. Among these, the blocking effect of the novel PKC (PKC delta isoenzyme) inhibitor Rottlerin was the most pronounced. Studies employing reverse transcriptase-polymerase chain reaction (RT-PCR) showed that Sophy beta-glucan stimulated the expression of IL-8 mRNA in the U937 cells, and that this induction was inhibited by Rottlerin. Sophy beta-glucan also blocked the stimulator cell induction of DNA synthesis and IFN-gamma production in the responder cells in a one-way mixed lymphocyte reaction (MLR) using allogenic PBMCs. Interestingly, immunoglobulin G (IgG), but not IgM to Sophy beta-glucan was detected in the sera derived from normal adult donors and from the umbilical cord blood of neonates. Taken together, these findings strongly suggest that the Sophy beta-glucan may have unique immune regulatory or enhancing properties that could be exploited by the health food, medical and pharmaceutical industries. [Abstract/Link to Full Text]

Nameda S, Miura NN, Adachi Y, Ohno N
Antibiotics protect against septic shock in mice administered beta-glucan and indomethacin.
Microbiol Immunol. 2007;51(9):851-9.
We have developed an animal model of sepsis in mice by repeatedly administering beta-glucan, a biological response modifier, and indomethacin (IND), a nonsteroidal anti-inflammatory drug. The combination of these drugs induced bacteremia by translocation of the enterobacterial flora, resulting in increasing the number of activated leukocytes, and inducing hyper cytokinemia. In the present study, we examined the effect of antibiotics on beta-glucan and IND-induced septic shock. Treatment with antibiotics inhibited microbial translocation, inhibited contraction of the colon, reduced lipopolysaccharides (LPS)-elicited production of TNF-alpha and IL-6, and finally prolonged survival. However, the efficacy of antibiotics treatment was limited in mice administered IND orally. These findings strongly suggested that the antibiotics controlled the gut-associated action of IND and reduced various symptoms accompanying sepsis. [Abstract/Link to Full Text]

Ekström JO, Tolf C, Edman KA, Lindberg AM
Physicochemical properties of the Ljungan virus prototype virion in different environments: inactivated by heat but resistant to acidic pH, detergents and non-physiological environments such as Virkon-containing solutions.
Microbiol Immunol. 2007;51(9):841-50.
It is of great importance to know how a virus particle is affected by environmental conditions. Physicochemical properties of the virion will affect the virus viability in different environments, viral transmission between hosts, and will also be important for safe handling of the virus. The physicochemical properties of the Ljungan virus (LV) prototype, 87-012, adapted to grow in cell culture were evaluated using both LV in crude cell extracts and purified virions. Replication of LV was completely inhibited by heat. Titers of LV were unaffected by acidic pH, reduced but not completely abolished by alkaline pH, and unaffected by exposure to the detergents Triton X-100 and SDS. Surprisingly, viable LV was still detected after incubation in the acidic, oxidising and detergent-containing environment produced by the commonly used disinfectant Virkon. In conclusion, LV is resilient to extreme pH, detergents and also to oxidising environments, but is sensitive to heat treatment. [Abstract/Link to Full Text]

Yamagata J, Ahmed K, Khawplod P, Mannen K, Xuyen DK, Loi HH, Dung NV, Nishizono A
Molecular epidemiology of rabies in Vietnam.
Microbiol Immunol. 2007;51(9):833-40.
The present study was done to determine the molecular epidemiology of rabies virus (RV) in Vietnam. The nucleoprotein (N) and glycoprotein (G) genes of RVs were amplified from the brains of ten rabid dogs of Ho Chi Minh City, Vietnam. The nucleotide sequences of these genes were compared with those of other Asian strains to find the possible relationship among them. Phylogenetic analysis revealed that the Asian N gene segregated into three main branches, namely South-East Asia 1 (SEA 1), South-East Asia 2 (SEA 2) and Indian subcontinent (ISC) genotypes. The SEA 1 genotype comprised RVs from Malaysia, Vietnam and Thailand. The SEA 2 genotype contained strains from the Philippines, and the ISC genotype comprised strains from Sri Lanka and India. Phylogenetically G genes of RVs from Vietnam and Thailand were clustered together. Our study suggests that Vietnamese and Thai RVs are closely related and might have originated from a common ancestor. [Abstract/Link to Full Text]

Fukutome A, Watashi K, Kawakami N, Ishikawa H
Mathematical modeling of severe acute respiratory syndrome nosocomial transmission in Japan: the dynamics of incident cases and prevalent cases.
Microbiol Immunol. 2007;51(9):823-32.
An outbreak of Severe Acute Respiratory Syndrome (SARS) occurred in Hong Kong in late February 2003, resulting in 8,096 cumulative cases with 774 deaths. The outbreak was amplified by nosocomial transmission in many hospitals. Using mathematical modeling, we simulated the number of new incident and prevalent cases of SARS after one infected person was admitted to a hospital (index case). The simulation was tested stochastically using the SEIR model based on previously reported Gamma distributions. We estimated the duration time until 10 beds in negative pressure rooms in Chiyoda-ku, one of the 23 wards in Tokyo, were fully occupied with SARS-infected patients. We determined the impact of an increasing number of days on the number of prevalent cases until the index case was isolated. The prevalent cases increase exponentially along with the increase of the non-isolation period of the index case, and all the beds were fully occupied if the index case was not isolated until more than 6 days. However even 2 days non-isolation period of the index case could fill up all the beds when 16% of secondary infections are transmitted outside the hospital. There is a possibility that an epidemic will occur with the isolation of the index case even at early days if the infection is transmitted outside the hospital. The simulation results revealed that it was important to recognize and isolate SARS patients as early as possible and also to prevent the transmission spreading outside the hospital to control an epidemic. [Abstract/Link to Full Text]

Saitoh M, Kimura H, Kozawa K, Nishio O, Shoji A
Detection and phylogenetic analysis of norovirus in Corbicula fluminea in a freshwater river in Japan.
Microbiol Immunol. 2007;51(9):815-22.
To study the molecular epidemiology of noroviruses (NoVs) in bivalves residing in freshwater rivers, we detected, quantified and phylogenetically analyzed the NoV genome in purified concentrates obtained from the gills and digestive diverticula of Corbicula fluminea in a freshwater river in Gunma Prefecture, Japan. We detected the NoV genome in 35 of the 58 C. fluminea samples. Based on our phylogenetic analysis, the NoV genome detected in the samples was classified into 4 genotypes (GI/1, GI/2, GI/3 and GI/4) in genogroup I and 5 genotypes (GII/3, GII/4, GII/5, GII/8 and GII/12) in genogroup II. The phylogenetic tree showed wide genetic diversity among the genogroups. In addition, more than 10(4) copies of the NoV genome were detected in 2 of 35 samples. These results suggest that the freshwater bivalve C. fluminea is a reservoir for NoVs, similar to seawater bivalves such as oysters. [Abstract/Link to Full Text]

Sumi A, Kamo K, Ohtomo N, Kobayashi N
Study of the effect of vaccination on periodic structures of measles epidemics in Japan.
Microbiol Immunol. 2007;51(9):805-14.
The purpose of this paper is to investigate the effect of vaccination on periodic structures of measles epidemics in Japan. We carried out spectral analysis for time series data of measles notifications collected in Japan. It was confirmed that the interepidemic period, which corresponds to the interval between major epidemics of measles, increases as the vaccination ratio increases. This result was supported by a theory based on a mathematical model for epidemics of infectious diseases. It was suggested that the interepidemic period is useful to estimate the effect of vaccination on measles incidences quantitatively. [Abstract/Link to Full Text]

Izgü F, Altinbay D, Türeli AE
In vitro susceptibilities of Candida spp. to Panomycocin, a novel exo-beta-1,3-glucanase isolated from Pichia anomala NCYC 434.
Microbiol Immunol. 2007;51(9):797-803.
Panomycocin, the killer toxin of Pichia anomala NCYC 434 (K5), is a 49 kDa monomeric glycoprotein with exo-beta-1,3-glucanase activity (patent pending). In this study we evaluated the in vitro activity of panomycocin against a panel of 109 human isolates of seven different pathogenic Candida spp. using microdilution and time-kill methods. Panomycocin was most active against C. tropicalis, C. pseudotropicalis and C. glabrata with MIC(90) values of 1 microg/ml. It displayed significant activity against C. albicans and C. parapsilosis with MIC(90) values of 4 and 2 microg/ml, respectively. For C. krusei, the MIC(90) value was 8 microg/ml. Panomycocin was fungicidal against all the tested Candida spp. The MFC values were only one or 2 dilutions higher than the MICs with the exception of C. krusei isolates with MFCs greater than or equal to 4xMIC. Results of this study indicated that panomycocin could be considered as a natural antifungal agent against Candida infections and has significant potential for further investigation. [Abstract/Link to Full Text]

Martins A, Cunha Mde L
Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: epidemiological and molecular aspects.
Microbiol Immunol. 2007;51(9):787-95.
Infections caused by the genus Staphylococcus are of great importance for human health. Staphylococcus species are divided into coagulase-positive staphylococci, represented by S. aureus, a pathogen that can cause infections of the skin and other organs in immunocompetent patients, and coagulase-negative staphylococci (CNS) which comprise different species normally involved in infectious processes in immunocompromised patients or patients using catheters. Oxacillin has been one of the main drugs used for the treatment of staphylococcal infections; however, a large number of S. aureus and CNS isolates of nosocomial origin are resistant to this drug. Methicillin resistance is encoded by the mecA gene which is inserted in the SCC mec cassette. This cassette is a mobile genetic element consisting of five different types and several subtypes. Oxacillin-resistant strains are detected by phenotypic and genotypic methods. Epidemiologically, methicillin-resistant S. aureus strains can be divided into five large pandemic clones, called Brazilian, Hungarian, Iberian, New York/Japan and Pediatric. The objective of the present review was to discuss aspects of resistance, epidemiology, genetics and detection of oxacillin resistance in Staphylococcus spp., since these microorganisms are increasingly more frequent in Brazil. [Abstract/Link to Full Text]

Nintasen R, Utrarachkij F, Siripanichgon K, Bhumiratana A, Suzuki Y, Suthienkul O
Enhancement of Legionella pneumophila culture isolation from microenvironments by macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction.
Microbiol Immunol. 2007;51(8):777-85.
The combination of a Legionella pneumophila culture isolation technique and macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction (PCR) is pivotal for effective routine use in an environmental water system laboratory. Detection of Legionella organisms in 169 environmental samples was performed by using modified buffered charcoal yeast extract (MBCYE) agar for conventional culture. Nested PCR specific for L. pneumophila was performed using boiled genomic DNA extracts from filtered and Chelex 100-treated water samples, or by using silica-gel membrane spin column-eluted DNA from concentrated pond, canal and river samples. Overall, the nested PCR was twelvefold more sensitive than the culture method. The target amplicons (471 basepairs) of all 4 biochemically characterized L. pneumophila isolates were sequenced. They had homology at the DNA and protein levels to 3' proximity of the mip-coding gene of L. pneumophila deposited in genome databases. EcoRI- or KpnI-digested PCR fragments with expected sizes were also confirmed in all 52 PCR-positive samples that were isolated from cooling towers and condenser drains. Viable but nonculturable L. pneumophila might have been present in 48 PCR-positive samples. This study demonstrates that detection of the genetically stable mip gene by nested PCR with a modified process of water sample preparation can be rapidly and effectively used to enhance isolation of the L. pneumophila taxon from microenvironments. [Abstract/Link to Full Text]

Soejima T, Iida K, Qin T, Taniai H, Seki M, Takade A, Yoshida S
Photoactivated ethidium monoazide directly cleaves bacterial DNA and is applied to PCR for discrimination of live and dead bacteria.
Microbiol Immunol. 2007;51(8):763-75.
Ethidium monoazide (EMA) is a DNA intercalating agent and a eukaryotic topoisomerase II poison. We found that EMA treatment and subsequent visible light irradiation (photoactivation or photolysis) shows a bactericidal effect, hence the mechanism was analyzed. When bacterial cells were treated with more than 10 microg/ml of EMA for 1 hr plus photoactivation for 20 min, cleavage of bacterial DNA was confirmed by agarose gel electrophoresis and electron microscopic studies. The cleavage of chromosomal DNA was seen when it was treated in vitro with EMA and photolysis, which showed that the cleavage directly took place without the assistance of DNA gyrase/topoisomerase IV and the DNA repair enzymes of bacteria. It was also verified, by using negatively supercoiled pBR322 DNA, that medium/high concentrations of EMA (1 to 100 microg/ml) led to breaks of double-stranded DNA and that low concentrations of EMA (10 to 100 ng/ml) generated a single-stranded break. EMA is known to easily penetrate dead but not live bacteria. After treatment of 10 microg/ml of EMA for 30 min and photoactivation for 5 min, EMA cleaved the DNA of dead but not live Klebsiella oxytoca. When the cleaved DNA was used for templates in PCR targeting 16S rDNA, PCR product from the dead bacteria was completely suppressed. We demonstrated that EMA and photolysis directly cleaved bacterial DNA and are effective tools for discriminating live from dead bacteria by PCR. [Abstract/Link to Full Text]

Morishita Y, Uenaka A, Kaya S, Sato S, Aji T, Nakayama E
HLA-DRB1*0410-restricted recognition of XAGE-1b37-48 peptide by CD4 T cells.
Microbiol Immunol. 2007;51(8):755-62.
XAGE-1b belongs to cancer/testis (CT) antigens, and has been shown to be expressed frequently in lung cancers and to elicit an antibody response in patients with XAGE-1b-expressing tumors. In this study, we investigated an XAGE-1b peptide recognized by CD4 T cells. CD4 T cells were purified from PBMC of a healthy donor and stimulated with pooled 25-mer peptides overlapped with 15 amino acids spanning the entire XAGE-1b protein. The generation of XAGE-1b-specific CD4 T cells was shown by IFNgamma secretion assay. A CD4 T cell clone OHD1 was obtained by limiting dilution. OHD1 recognized two overlapping peptides, XAGE1-b(33-49) and XAGE-1b(37-52), by ELISPOT assay. A peptide XAGE-1b(38-46) which was included in both XAGE-1b(33-49) and XAGE-1b(37-52) was predicted to be a DRB1*0410-restricted 9-mer peptide by a computer-based program. We identified the 12-mer peptide XAGE-1b(37-48) as a new XAGE-1b epitope restricted to HLA-DRB1*0410. [Abstract/Link to Full Text]

Okamoto M, Benno Y, Leung KP, Maeda N
Metascardovia criceti Gen. Nov., Sp. Nov., from hamster dental plaque.
Microbiol Immunol. 2007;51(8):747-54.
A novel microorganism, Metascardovia criceti gen. nov., sp. nov., was isolated from dental plaque of golden hamsters fed with a high-carbohydrate diet. The three isolated strains, OMB104, OMB105, and OMB107, were Gram-positive, facultative anaerobic rods that lacked catalase activity. Analyses of the partial 16S rRNA and heat-shock protein 60 (HSP60) gene sequences of these isolates indicated that they belonged to the family Bifidobacteriaceae. However, in contrast to Bifidobacterium, one of the genera under this family, these isolates grew under aerobic conditions, and the DNA G + C contents were lower (53 mol%) than those of Bifidobacterium. On the basis of phylogenetic analyses using phenotypic characterization, and partial 16S rRNA and HSP60 gene sequences data, we propose a novel taxa, Metascardovia criceti for OMB105(T) (type strain=JCM 13493(T)=DSM 17774(T)) for this newly described isolate. [Abstract/Link to Full Text]

Kinjo T, Miyagi K, Nakamura K, Higa F, Gang X, Miyazato A, Kaku M, Fujita J, Kawakami K
Adjuvant effect of CpG-oligodeoxynucleotide in anti-fungal chemotherapy against fatal infection with Cryptococcus neoformans in mice.
Microbiol Immunol. 2007;51(8):741-6.
Cryptococcal meningoencephalitis is a life-threatening infectious disease in immunocompromised patients. Unmethylated CpG-oligodeoxynucleotides (CpG-ODN) protects hosts in a mouse model. In the present study, we tested the adjuvant effect of CpG-ODN in anti-fungal chemotherapy. Administration of either fluconazole (FLCZ) or CpG-ODN was effective in extending survival, accelerating clearance of fungi and preventing disseminated infection. Combination of both agents provided more beneficial effect than either agent alone. Cytokine balance in the infected lungs was biased to Th1-type response by CpGODN, while FLCZ did not further promote. These results suggest that CpG-ODN is a promising adjuvant in chemotherapy against this infection. [Abstract/Link to Full Text]

Mohapatra BR, Broersma K, Nordin R, Mazumder A
Evaluation of repetitive extragenic palindromic-PCR for discrimination of fecal Escherichia coli from humans, and different domestic- and wild-animals.
Microbiol Immunol. 2007;51(8):733-40.
The objective of this study was to investigate the potential of repetitive extragenic palindromic anchored polymerase chain reaction (rep-PCR) in differentiating fecal Escherichia coli isolates of human, domestic- and wild-animal origin that might be used as a molecular tool to identify the possible source(s) of fecal pollution of source water. A total of 625 fecal E. coli isolates of human, 3 domestic- (cow, dog and horse) and 7 wild-animal (black bear, coyote, elk, marmot, mule deer, raccoon and wolf) species were characterized by rep-PCR DNA fingerprinting technique coupled with BOX A1R primer and discriminant analysis. Discriminant analysis of rep-PCR DNA fingerprints of fecal E. coli isolates from 11 host sources revealed an average rate of correct classification of 79.89%, and 84.6%, 83.8%, 83.3%, 82.5%, 81.6%, 80.8%, 79.8%, 79.3%, 77.4%, 73.2% and 63.6% of elk, human, marmot, mule deer, cow, coyote, raccoon, horse, dog, wolf and black bear fecal E. coli isolates were assigned to the correct host source. These results suggest that rep-PCR DNA fingerprinting procedures can be used as a source tracking tool for detection of human- as well as animal-derived fecal contamination of water. [Abstract/Link to Full Text]

Tamura H, Yamada A, Kato H
Identification and characterization of a dextranase gene of Streptococcus criceti.
Microbiol Immunol. 2007;51(8):721-32.
The dextranase gene, dex, was identified in Streptococcus criceti strain E49 by degenerate PCR and sequenced completely by the gene-walking method. A sequence of 3,960 nucleotides was determined. The dex gene encodes a 1,200-amino acid protein, which has a calculated molecular mass of 128,129.91 and pI of 4.15 and is predicted to be a cell-surface protein. The deduced amino acid sequence of dex showed homology to S. downei dextranase (63.9% identity). Phylogenetic analysis revealed the similarity of the deduced amino acid sequence of dextranases in S. criceti, S. sobrinus, and S. downei. A recombinant form of the protein with six histidine residues tagged in the C-terminus was partially purified and showed dextranase activity on blue-dextran sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDSPAGE) followed by renaturation. We also detected dextranase activity in S. criceti cell extracts and culture supernatant by renatured BD-SDS-PAGE, whereas no dextranase activity of the cells was observed on blue-dextran brain heart infusion (BD-BHI) agar plates. Furthermore, PCR-based mutations of dextranase indicated that a deletion mutant of the C-terminal region could hydrolyze blue dextrans and that the D453E mutation, W793L mutation, and double mutations (W793L and deletion of the C-terminal region) resulted in a loss of dextranase activity. These findings suggest that Asp-453 and Trp-793 residues of S. criceti dextranase are critical to the enzyme's activity. [Abstract/Link to Full Text]

Oliveira MA, Carvalho LP, Gomes Mde S, Bacellar O, Barros TF, Carvalho EM
Microbiological and immunological features of oral candidiasis.
Microbiol Immunol. 2007;51(8):713-9.
Candida albicans(C. albicans) is the major infectious agent of oral candidiasis, and both innate immunity and cell-mediated immune response participate in the control of the fungal infections. The aim of this study was to correlate the clinical forms of oral candidiasis with the number of colony forming units (CFU) of C. albicans in saliva and to characterize T cell response in patients with oral candidiasis. Participants included 75 subjects: 36 with lesions of candidiasis and 39 without lesions of oral candidiasis. A 2-ml sample of saliva was collected from all subjects for microbiological analysis. Cytokine levels were determined by ELISA in supernatants of peripheral blood mononuclear cells of 25 patients with oral candidiasis, after in vitro stimulation with C. albicans antigens. In 48% of patients, no association was observed with denture use. C. albicans was detected in the saliva of 91.7% of patients with oral candidiasis, and there was an association between the number of CFU and the presence of oral lesions. A type Th1 immune response was observed in supernatants of peripheral blood mononuclear cells stimulated with C. albicans antigens. In contrast, IL-5 and IL-10 levels were very low or undetectable. Together, this study shows an association between clinical forms of oral candidiasis and the number of colonies of C. albicans in saliva, and that a systemic immune response characterized by the production of TNF-alpha and IFN-gamma is observed in patients with oral candidiasis. [Abstract/Link to Full Text]

Nozawa T, Tanikawa T, Hasegawa H, Takahashi C, Ando Y, Matsushita M, Nakagawa Y, Matsuyama T
Rhamnolipid-dependent spreading growth of Pseudomonas aeruginosa on a high-agar medium: marked enhancement under CO2-rich anaerobic conditions.
Microbiol Immunol. 2007;51(8):703-12.
Anaerobiosis of Pseudomonas aeruginosa in infected organs is now gaining attention as a unique physiological feature. After anaerobic cultivation of P. aeruginosa wild type strain PAO1 T, we noticed an unexpectedly expanding colony on a 1.5% agar medium. The basic factors involved in this spreading growth were investigated by growing the PAO1 T strain and its isogenic mutants on a Davis high-agar minimal synthetic medium under various experimental conditions. The most promotive environment for this spreading growth was an O(2)-depleted 8% CO(2) condition. From mutational analysis of this spreading growth, flagella and type IV pili were shown to be ancillary factors for this bacterial activity. On the other hand, a rhamnolipid-deficient rhlA mutant TR failed to exhibit spreading growth on a high-agar medium. Complementation of the gene defect of the mutant TR with a plasmid carrying the rhlAB operon resulted in the restoration of the spreading growth. In addition, an external supply of rhamnolipid or other surfactants (surfactin from Bacillus subtilis or artificial product Tween 80) also restored the spreading growth of the mutant TR. Such activity of surfactants on bacterial spreading on a hard-agar medium was unique to P. aeruginosa under CO(2)-rich anaerobic conditions. [Abstract/Link to Full Text]

Yano A, Komatsu T, Ishibashi M, Udaka K
Potent CTL induction by a whole cell pertussis vaccine in anti-tumor peptide immunotherapy.
Microbiol Immunol. 2007;51(7):685-99.
Promising yet limited clinical responses have been reported for peptide based immunotherapy against tumors. In order to induce more potent cytolytic CD8 T cell responses, we investigated the use of Bordetella pertussis vaccine as an adjuvant for peptide immunization. A whole cell (Wc) vaccine has been known to induce a Th1 biased immune response while an acellular (Ac) vaccine tends to induce that of the Th2 type. Natural infection by B. pertussis helps to maintain a robust Th1 memory in the host population. To examine the adjuvant activity of the pertussis vaccine, we immunized mice with an ovalbumin peptide as a model tumor antigen, and monitored the development of anti-tumor activities. The addition of either the Ac or the Wc vaccine helped expand the specific CD8 T cells. However, there was a marked difference in the induced cytolytic activity where the Wc vaccine was superior to the Ac. The Wc vaccine was also more effective in inducing in vivo tumor rejection. The adjuvant activity was not only effective against ovalbumin, but was also evident when an endogenous tumor antigen, Wilms' tumor 1 gene product, was targeted. These results indicate that, although the Wc vaccine does not share the same antigen specificity with tumor cells, it can aid in the development of highly cytolytic CD8 T cells as an adjuvant at the site of peptide immunization. [Abstract/Link to Full Text]

Kaida A, Kubo H, Goto K, Shiomi M, Kohdera U, Iritani N
Co-infection of human metapneumovirus with adenovirus or respiratory syncytial virus among children in Japan.
Microbiol Immunol. 2007;51(7):679-83.
Human metapneumovirus (hMPV) is one of the etiological agents of acute respiratory tract infections. From June 2005 to May 2006, we collected 185 clinical specimens from children in Osaka City, Japan, and detected 41 hMPV RNA. Of the 41 specimens, four (9.8%) also contained other viruses (3 with adenovirus [AdV] and 1 with respiratory syncytial virus [RSV]). The clinical symptoms of patients coinfected with AdV were indistinct from those of patients mono-infected with hMPV. The symptoms of the one patient co-infected with RSV were clinically severe. Further research is needed to clarify the effect of hMPV on other respiratory viruses or vice versa. [Abstract/Link to Full Text]

Asakura H, Morita-Ishihara T, Yamamoto S, Igimi S
Genetic characterization of thermal tolerance in Enterobacter sakazakii.
Microbiol Immunol. 2007;51(7):671-7.
Enterobacter sakazakii is an opportunistic pathogen that causes meningitis and necrotizing enterocolitis in neonates. Here we characterized the thermal tolerance of E. sakazakii isolates obtained from powdered infant formula and other food products in Japan. Isolates were categorized into three classes according to their thermal tolerance, and differential gene expression analysis showed that the heat-resistant clones expressed a higher level of infB (which encodes a translation initiation factor), than did the heat-sensitive isolates. Gene expression and DNA polymorphism analyses suggested that this gene target might be useful to unequivocally detect and identify heat-resistant clones, permitting epidemiological surveillance for this pathogen. [Abstract/Link to Full Text]

Uryu M, Karino A, Kamihara Y, Horiuchi M
Characterization of prion susceptibility in Neuro2a mouse neuroblastoma cell subclones.
Microbiol Immunol. 2007;51(7):661-9.
In this study, we established Neuro2a (N2a) neuroblastoma subclones and characterized their susceptibility to prion infection. The N2a cells were treated with brain homogenates from mice infected with mouse prion strain Chandler. Of 31 N2a subclones, 19 were susceptible to prion as those cells became positive for abnormal isoform of prion protein (PrP(Sc)) for up to 9 serial passages, and the remaining 12 subclones were classified as unsusceptible. The susceptible N2a subclones expressed cellular prion protein (PrP(C)) at levels similar to the parental N2a cells. In contrast, there was a variation in PrP(C) expression in unsusceptible N2a subclones. For example, subclone N2a-1 expressed PrP(C) at the same level as the parental N2a cells and prion-susceptible subclones, whereas subclone N2a-24 expressed much lower levels of PrP mRNA and PrP(C) than the parental N2a cells. There was no difference in the binding of PrP(Sc) to prion-susceptible and unsusceptible N2a subclones regardless of their PrP(C) expression level, suggesting that the binding of PrP(Sc) to cells is not a major determinant for prion susceptibility. Stable expression of PrP(C) did not confer susceptibility to prion in unsusceptible subclones. Furthermore, the existence of prion-unsusceptible N2a subclones that expressed PrP(C) at levels similar to prion-susceptible subclones, indicated that a host factor(s) other than PrP(C) and/or specific cellular microenvironments are required for the propagation of prion in N2a cells. The prion-susceptible and -unsusceptible N2a subclones established in this study should be useful for identifying the host factor(s) involved in the prion propagation. [Abstract/Link to Full Text]

Iwabuchi N, Takahashi N, Xiao JZ, Miyaji K, Iwatsuki K
In vitro Th1 cytokine-independent Th2 suppressive effects of bifidobacteria.
Microbiol Immunol. 2007;51(7):649-60.
A comparison between 17 strains of lactic acid bacteria and 15 strains of bifidobacteria indicated that bifidobacteria induced significantly lower levels of interleukin-12 (IL-12) in murine splenic cells. The present study aims to evaluate the effect and mechanism of Bifidobacterium longum BB536, a probiotic strain, in suppressing antigen-induced Th2 immune response in vitro. BB536 suppressed immunoglobulin (Ig) E and IL-4 production by ovalbumin-sensitized splenic cells, but induction of Th1-inducing cytokine production, such as IL-12 and gamma interferon (IFN-gamma) tended to be lower compared with lactic acid bacteria. Neutralization with antibodies to IL-12, IFN-gamma, IL-10 and transforming growth factor beta indicated negative involvement of Th1-inducing cytokines and regulatory cytokines in the suppression of Th2 immune response by BB536, especially when treated at higher doses of BB536 (>10 microg cells/ml). Furthermore, BB536 induced the maturation of immature bone marrow-derived dendritic cells (BM-DCs), and suppressed antigen-induced IL-4 production mediated by BM-DCs. These results suggested that BB536 suppressed Th2 immune responses, partially independent of Th1-inducing cytokines and independent of regulatory cytokines, mediated by antigen-presenting cells such as dendritic cells. [Abstract/Link to Full Text]

Iida K, Amako K, Takade A, Ueda Y, Yoshida S
Electron microscopic examination of the dormant spore and the sporulation of Paenibacillus motobuensis strain MC10.
Microbiol Immunol. 2007;51(7):643-8.
We previously reported a new species Paenibacillus motobuensis. The type strain MC10 was stained gram-negative, but had a gram-positive cell wall structure and its spore had a characteristic star shape. The spore and sporulation process of P. motobuensis strain MC10 were examined by electron microscopy using the technique of freeze-substitution in thin sectioning. The structure of the dormant spore was basically the same as that of the other Bacillus spp. The core of the spore was enveloped with two main spore components, the cortex and the spore coat. In thin section, the spore showed a star-shaped image, which was derived from the structure of the spore coat, which is composed of three layers, namely the inner, middle and outer spore coat. The middle coat was an electron-dense thick layer and had a characteristic ridge. By scanning electron microscopic observation, the ridges were seen running parallel to the long axis of the oval-shaped spore. The process of sporulation was essentially the same as that of the other Bacillus spp. The forespore was engulfed by the mother cell membrane, then the spore coat and the cortex were accumulated in the space between the mother cell membrane and forespore membrane. The mother cell membrane seemed to participate in the synthesis of the spore coat. MC10 strain showed almost identical heat resistance to that of B. subtilis. [Abstract/Link to Full Text]

Furuhata K, Goto K, Kato Y, Saitou K, Sugiyama J, Hara M, Yoshida S, Fukuyama M
Characteristics of a pink-pigmented bacterium isolated from biofilm in a cooling tower in Tokyo, Japan.
Microbiol Immunol. 2007;51(6):637-41.
Strain K-20, a Gram-negative, non-motile, non-spore-forming and strictly aerobic rod, which produces a pale pink pigment, was isolated from biofilm in a cooling tower in Tokyo, Japan. The taxonomic feature of the strain was studied using phenotypic tests and phylogenetic analysis. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain was related to Roseomonas gilardii subsp. rosea, Roseomonas gilardii subsp. gilardii, Roseomonas cervicalis and Roseomonas mucosa at 94.3-94.6 sequence similarities. Growth occurred at 25-40 C and pH 5.0-10.0, optimal at 35 C and pH 7.0. Growth did not occur in the presence of >or=2% NaCl. The API 20NE identification system gave a positive result for urease, L-arabinose, potassium gluconate, adipic acid, malic acid and trisodium citrate (API code number 0201465). The predominant fatty acids of strain K-20 were C18:1Delta11 (50.8%) and C16:1 (17.2%). Cells contained ubiquinone 10 (Q-10) as the major quinone and the G+C content was 72.0 mol%. Based on phenotypic, chemotaxonomic and phylogenetic data, it was assumed that strain K-20 (=JCM 14634) is a novel species of the genus Roseomonas. [Abstract/Link to Full Text]

Mekha N, Sugita T, Ikeda R, Nishikawa A, Poonwan N
Real-time PCR assay to detect DNA in sera for the diagnosis of deep-seated trichosporonosis.
Microbiol Immunol. 2007;51(6):633-5.
Deep-seated trichosporonosis due to Trichosporon asahii is life-threatening and has high mortality. A real-time PCR assay to detect T. asahii DNA in sera for diagnosis of this fungal infection was developed. The assay showed a higher sensitivity than polysaccharide antigen detection method. Our new real-time PCR assay may be used for diagnosing deep-seated trichosporonosis due to T. asahii. [Abstract/Link to Full Text]

Takano T, Saito K, Teng LJ, Yamamoto T
Spread of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) in hospitals in Taipei, Taiwan in 2005, and comparison of its drug resistance with previous hospital-acquired MRSA.
Microbiol Immunol. 2007;51(6):627-32.
Panton-Valentine leucocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (PVL+ MRSA) is an emerging pathogen in the community worldwide. The incidence of PVL+ MRSA in Taipei, Taiwan was 23.3% for hospital MRSA. PVL+ MRSA was isolated from both outpatients and inpatients. Some PVL+ (mecA+) strains (36.8%) showed low MIC values (<or=2 microg/ml) to oxacillin. A major PVL+ MRSA resistance pattern was oxacillin and clindamycin resistance (81%). There was no multidrug resistance over three drugs, in contrast to patient PVL- MRSA with resistance to five drugs as a major resistance pattern. The majority of PVL+ MRSA belonged to multilocus sequence (ST) type 59, while PVL+ MRSA belonged to ST239, ST59 and ST5. The data suggests that although PVL+ CA-MRSA is isolated at a high incidence from hospitals in Taipei, the drug resistance is mostly selected in the community and less prominent compared with previous PVL- hospital-acquired MRSA. [Abstract/Link to Full Text]

Arakawa M, Yamashiro T, Uechi G, Tadano M, Nishizono A
Construction of human Fab (gamma1/kappa) library and identification of human monoclonal Fab possessing neutralizing potency against Japanese encephalitis virus.
Microbiol Immunol. 2007;51(6):617-25.
A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from Japanese encephalitis virus hyper-immune volunteers on pComb3H phagemid vector. The size of the constructed Fab library was 3.3x10(8) Escherichia coli transformants. The library was panned 3 times on the purified Japanese encephalitis virus (JEV) virion, and phage clones displaying JEV antigen-specific Fab were enriched. The enriched phage pool was then screened for clones producing Fab molecule with JEV neutralizing activity by the focus reduction-neutralizing test. Among 188 randomly selected clones, 9 Fab preparations revealed neutralizing activities against JEV strain Nakayama. An E. coli transformed with TJE12B02 clone, which produced human monoclonal Fab with the highest neutralizing activity was cultured in a large scale, and the Fab molecule was purified using affinity chromatography. The purified FabTJE12B02 showed the 50% focus reduction endpoint at the concentration of 50.2 microg/ml (ca. 1,000 nM) when JEV strain Nakayama was used. The FabTJE12B02 recognized E protein of JEV strain Nakayama, and the dissociation equilibrium constant (Kd) of the FabTJE12B02 against purified JEV antigen was calculated as 1.21x10(-8) M. Sequence analysis demonstrated that TJE12B02 used a VH sequence homologous to the VH3 family showing 88.8% homology to germline VH3-23, and used a Vkappa sequence homologous to the VkappaII subgroup showing 92.8% homology to germline A17. [Abstract/Link to Full Text]

Abe M, Okada K, Hayashida K, Matsuo F, Shiosaki K, Miyazaki C, Ueda K, Kino Y
Duration of neutralizing antibody titer after Japanese encephalitis vaccination.
Microbiol Immunol. 2007;51(6):609-16.
In paired serum samples collected from 17 children, we measured neutralizing antibody (NTAb) titers after the second series of routine Japanese encephalitis (JE) vaccination in Japan to estimate the duration of NTAb titer when children did not receive the third series of routine vaccination by applying a random coefficient model. We also measured NTAb titers in adult serum samples to confirm the duration of NTAb titer estimated in the analysis of pediatric serum samples. In the absence of the third series of routine vaccination, 18% (3/17), 47% (8/17), 82% (14/17) and 100% (17/17) of children were estimated to become NTAb negative at 5, 10, 15, and 20 years after the second series of routine vaccination, respectively. Of 38 adults, 39.5% (15/38) became NTAb negative; the percentage was somewhat lower than that of antibody-negative children. The results suggested that JE vaccination schedule should be reevaluated in the future. [Abstract/Link to Full Text]


Recent Articles in International Microbiology

Termens M
DOI: The "Big Brother" in the dissemination of scientific documentation.
Int Microbiol. 2006 Jun;9(2):139-42. [Abstract/Link to Full Text]

Testa J
The Thomson Scientific journal selection process.
Int Microbiol. 2006 Jun;9(2):135-8. [Abstract/Link to Full Text]

de Lyra Mdo C, Lopez-Baena FJ, Madinabeitia N, Vinardell JM, Espuny Mdel R, Cubo MT, Belloguin RA, Ruiz-Sainz JE, Ollero FJ
Inactivation of the Sinorhizobium fredii HH103 rhcJ gene abolishes nodulation outer proteins (Nops) secretion and decreases the symbiotic capacity with soybean.
Int Microbiol. 2006 Jun;9(2):125-33.
It has been postulated that nodulation outer proteins (Nops) avoid effective nodulation of Sinorhizobium fredii USDA257 to nodulate with American soybeans. S. fredii HH103 naturally nodulates with both Asiatic (non-commercial) and American (commercial) soybeans. Inactivation of the S. fredii HH103 gene rhcJ, which belongs to the tts (type III secretion) cluster, abolished Nop secretion and decreased its symbiotic capacity with the two varieties of soybeans. S. fredii strains HH103 and USDA257, that only nodulates with Asian soybeans, showed different SDS-PAGE Nop profiles, indicating that these strains secrete different sets of Nops. In coinoculation experiments, the presence of strain USDA257 provoked a clear reduction of the nodulation ability of strain HH103 with the American soybean cultivar Williams. These results suggest that S. fredii Nops can act as either detrimental or beneficial symbiotic factors in a strain-cultivar-dependent manner. Differences in the flavonoid-mediated expression of rhcJ with respect to nodA were also detected. In addition, one of the Nops secreted by strain HH103 was identified as NopA. [Abstract/Link to Full Text]

Sánchez O, Ferrera I, Vigués N, Garcia de Oteyza T, Grimalt JO, Mas J
Presence of opportunistic oil-degrading microorganisms operating at the initial steps of oil extraction and handling.
Int Microbiol. 2006 Jun;9(2):119-24.
Hydrocarbon-degrading microorganisms from natural environments have been isolated and identified using culture-dependent or molecular techniques. However, there has been little research into the occurrence of microorganisms incorporated into crude oil in the initial steps of extraction and handling, which can reduce the quality of stored petroleum. In the present study, a packed-column reactor filled with autoclaved perlite soaked with crude oil was subjected to a continuous flow of sterile medium in order to determine the presence of potential hydrocarbon degraders. Microorganisms developed on the surface of the perlite within a period of 73 days. DNA was extracted from the biofilm and then PCR-amplified using 16S rRNA bacterial and archaeal primers and 18S rRNA eukaryotic primers. No amplification was obtained using archaeal primers. However, denaturing gradient gel electrophoresis (DGGE) revealed the presence of unique bands indicating bacterial and eukaryotic amplification. Excision of these bands, sequencing, and subsequent BLAST search showed that they corresponded to Bacillus sp. and Aspergillus versicolor. The fungus was later isolated from intact perlite in agar plates. A bacterial clone library was used to confirm the presence in the biofilm of a unique hydrocarbon-degrading bacterium closely related to Bacillus sp. Analysis of the petroleum components by gas chromatography showed that there n-alkanes, aromatic hydrocarbons, and carbazoles were degraded. [Abstract/Link to Full Text]

Lisboa MP, Bonatto D, Bizani D, Henriques JA, Brandelli A
Characterization of a bacteriocin-like substance produced by Bacillus amyloliquefaciens isolated from the Brazilian Atlantic forest.
Int Microbiol. 2006 Jun;9(2):111-8.
A Bacillus strain producing a bacteriocin-like substance was characterized by biochemical profiling and 16S rDNA sequencing. Phylogenetic analysis indicated that the strain has high sequence similarity with Bacillus amyloliquefaciens. The antimicrobial substance was inhibitory to pathogenic and food-spoilage bacteria, such as Listeria monocytogenes, Bacillus cereus, Serratia marcescens, and Pasteurella haemolytica. It was stable over a wide temperature range, but lost activity when the temperature reached 121 degrees C/15 min. Maximum activity was observed at acidic and neutral pH values, but not at alkaline pH. The antimicrobial substance was sensitive to the proteolytic action of trypsin, papain, proteinase K, and pronase E. Except for iturins, other antimicrobial peptides have not been described for B. amyloliquefaciens. The identification of a bacteriocin-like inhibitory substance active against L. monocytogenes addresses an important aspect of food protection. [Abstract/Link to Full Text]

Blanco M, Blanco JE, Dahbi G, Alonso MP, Mora A, Coira MA, Madrid C, Juárez A, Bernárdez MI, González EA, Blanco J
Identification of two new intimin types in atypical enteropathogenic Escherichia coli.
Int Microbiol. 2006 Jun;9(2):103-10.
Stool specimens of patients with diarrhea or other gastrointestinal alterations who were admitted to Xeral-Calde Hospital (Lugo, Spain) were analyzed for the prevalence of typical and atypical enteropathogenic Escherichia coli (EPEC). Atypical EPEC strains (eae+ bfp-) were detected in 105 (5.2%) of 2015 patients, whereas typical EPEC strains (eae+ bfp+) were identified in only five (0.2%) patients. Atypical EPEC strains were (after Salmonella) the second most frequently recovered enteropathogenic bacteria. In this study, 110 EPEC strains were characterized. The strains belonged to 43 O serogroups and 69 O:H serotypes, including 44 new serotypes not previously reported among human EPEC. However, 29% were of one of three serogroups (O26, O51, and O145) and 33% belonged to eight serotypes (O10:H-, O26:H11, O26:H-, O51:H49, O123:H19, O128:H2, O145:H28, and O145:H-). Only 14 (13%) could be assigned to classical EPEC serotypes. Fifteen intimin types, namely, alpha1 (6 strains), alpha2 (4 strains), beta1 (34 strains), xiR/b2 (6 strains), gamma1 (13 strains), gamma2/q (16 strains), delta/k (5 strains), epsilon1 (9 strains), nuR/e2 (5 strains), zeta (6 strains), iota1 (1 strain), muR/iota2 (1 strain), nuB (1 strain), xiB (1 strain), and o (2 strains), were detected among the 110 EPEC strains, but none of the strains was positive for intimin types mu1, mu2, lambda, or muB. In addition, in atypical EPEC strains of serotypes O10:H-, O84:H-, and O129:H-, two new intimin genes (eae-nuB and eae-o) were identified. These genes showed less than 95% nucleotide sequence identity with existing intimin types. Phylogenetic analysis revealed six groups of closely related intimin genes: (i) alpha1, alpha2, zeta, nuB, and o; (ii) iota1 and muR/iota2; (iii) beta1, xiR/beta2B, delta/beta2O, and kappa; (iv) epsilon1, xiB, eta1,eta2, and nuR/epsilon2; (v) gamma1, muB, gamma2, and theta; and (vi) lambda. These results indicate that atypical EPEC strains belonging to large number of serotypes and with different intimin types might be frequently isolated from human clinical stool samples in Spain. [Abstract/Link to Full Text]

Berlanga M, Montero MT, Fernández-Borrell J, Guerrero R
Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats.
Int Microbiol. 2006 Jun;9(2):95-102.
Microbial mat ecosystems are characterized by both seasonal and diel fluctuations in several physicochemical variables, so that resident microorganisms must frequently adapt to the changing conditions of their environment. It has been pointed out that, under stress conditions, bacterial cells with higher contents of poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA content. In the present study, PHA-producing strains from Ebro Delta microbial mats were selected using the Nile red dying technique and the relative accumulation of PHA was monitored during further laboratory cultivation. The number of heterotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/g microbial mat. Of these, 100 randomly chosen colonies were replicated on mineral salt agar limited in nitrogen, and Nile red was added to the medium to detect PHA. Orange fluorescence, produced upon binding of the dye to polymer granules in the cell, was detected in approximately 10% of the replicated heterotrophic isolates. The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans were compared with those of several of the environmental isolates spectrofluorometry. PHA accumulation, measured as relative fluorescence intensity, resulted in a steady-state concentration after 48 h of incubation in all strains assayed. At 72 h, the maximum fluorescence intensity of each strain incubated with glucose and fructose was usually similar. MAT-28 strain accumulated more PHA than the other isolates. The results show that data obtained from environmental isolates can highly improve studies based on modeling-simulation programs, and that microbial mats constitute an excellent source for the isolation of PHA-producing strains with industrial applications. [Abstract/Link to Full Text]

Alonso PL
Malaria: deploying a candidate vaccine (RTS,S/AS02A) for an old scourge of humankind.
Int Microbiol. 2006 Jun;9(2):83-93.
Malaria is an infectious disease caused by the protist Plasmodium spp. and it currently kills more than one million people annually. The burden of malaria is concentrated in sub-Saharan Africa, India, and Southeast Asia. The parasite's resistance to commonly used anti-malarial drugs has worsened the situation in the poorest countries. The World Health Organization (WHO) estimates that more than 100 countries suffer from endemic malaria episodes. In addition to numerous control measures and treatments, several vaccines are at different research stages and trials. We have assayed RTS,S/AS02A, a pre-erythrocytic candidate vaccine that has shown promising protection levels in phase IIb trials in Mozambique. The vaccine is directed against the sporozoite form of the parasite, which is injected by the mosquito Anopheles spp. The vaccine induces a strong antibody response and stimulates Th1 cells-a subset of helper T cells that participates in cell-mediated immunity. Recent interest by international funding agencies has provided new inputs into initiatives and programs to fight malaria, which, under normal welfare and adequate social development conditions, is a curable disease. [Abstract/Link to Full Text]

Navarro F
Acquisition and horizontal diffusion of beta-lactam resistance among clinically relevant microorganisms.
Int Microbiol. 2006 Jun;9(2):79-81. [Abstract/Link to Full Text]

Capanna E
Grassi versus Ross: who solved the riddle of malaria?
Int Microbiol. 2006 Mar;9(1):69-74. [Abstract/Link to Full Text]

Mishra PK, Tewari JP, Clear RM, Turkington TK
Genetic diversity and recombination within populations of Fusarium pseudograminearum from western Canada.
Int Microbiol. 2006 Mar;9(1):65-8.
Genetic diversity within populations of Fusarium pseudograminearum isolated from wheat grains from the Canadian provinces of Alberta and Saskatchewan was investigated. Three restriction enzymes (EcoRI, HaeIII, and PstI) were used to carry out restriction analysis of the nuclear ribosomal DNA (nrDNA) intergenic spacer region (IGS region) and eight primers were used to generate inter-simple sequence-repeat (ISSR) molecular markers. Our study indicated substantially high genetic diversity within these two populations, but low genetic differentiation and frequent gene flow among populations. The IGS data showed no genetic distinction between the two Alberta populations and only minor genetic differentiation between the Saskatchewan and Alberta populations. Analysis of molecular variance indicated that most genetic variability resulted from differences among isolates within populations. Multilocus linkage disequilibrium analysis suggested a panmictic population genetic structure and the occurrence of significant recombination in F. pseudograminearum. Regular gene flow and random mating between isolates from different populations could result in novel genotypes with both improved pathological and biological traits. [Abstract/Link to Full Text]

Lapeńa MA, Vicente-Soler J, Soto T, Madrid M, Núńez A, García E, Cansado J, Gacto M
Light-induced rhythmic changes in thermotolerance in stationary-phase cells of Candida utilis.
Int Microbiol. 2006 Mar;9(1):61-4.
In synchronized light-dark cycles, stationary-phase cultures of the budding yeast Candida utilis were able to survive heat treatment at 50 degees C with an apparent circadian-like rhythm related to the onset of light. However, in continuous darkness this pattern did not run freely and was markedly dampened. We discuss these findings in terms of the potential circadian control of heat tolerance, which has been described in the fission yeast Schizosaccharomyces pombe. Our results suggest that the resistance pattern observed in C. utilis is most likely an adaptive response to the light-induced generation of reactive oxygen species rather than the occurrence of a truly endogenous circadian rhythm. [Abstract/Link to Full Text]

Blanco M, Lazo L, Blanco JE, Dahbi G, Mora A, López C, González EA, Blanco J
Serotypes, virulence genes, and PFGE patterns of enteropathogenic Escherichia coli isolated from Cuban pigs with diarrhea.
Int Microbiol. 2006 Mar;9(1):53-60.
Thirty-six enteropathogenic Escherichia coli strains isolated from Cuban pigs with diarrhea were serotyped and screened by PCR for the presence of virulence genes. The 36 isolates belonged to 11 O serogroups and 14 O:H serotypes, with 53% of the isolates belonging to only two serotypes: O141:H- (13 isolates) and O157:H19 (6 isolates). Genes coding for STb, STa, VT2e, and LT toxins were identified in 69, 61, 53, and 6% of the isolates, respectively. The most prevalent fimbrial adhesin was F18, detected in 22 (61%) isolates. The gene encoding F6 (P987) colonization factor was identified in three (8%) isolates. None of the 36 isolates assayed contained genes encoding F4 (K88), F5 (K99), or F41. The seropathotype O141:H-:STa/STb/VT2e/F18 (13 isolates) was the most frequently detected, followed by O157:H19:VT2e/F18 (5 isolates). A genetic diversity study, carried out by pulsed-field gel electrophoresis (PFGE) of 24 representative isolates, revealed 21 distinct restriction patterns clustered in 18 groups (I-XVIII). Isolates of the same serotype were placed together in a dendrogram, but isolates of serotype O157:H19 showed a high degree of polymorphism. The results of this study demonstrate the presence in Cuba of different clusters among one of the most prevalent serotypes isolated from pigs with diarrhea. Further experiments are needed to determine whether some of these clusters have appeared recently; if so, their evolution, as well as their possible association with pathogenicity in farms should be studied. [Abstract/Link to Full Text]

Ivanova EP, Alexeeva YV, Pham DK, Wright JP, Nicolau DV
ATP level variations in heterotrophic bacteria during attachment on hydrophilic and hydrophobic surfaces.
Int Microbiol. 2006 Mar;9(1):37-46.
A survey of the extracellular ATP levels of 86 heterotrophic bacteria showed that gram-negative bacteria of the genera Sulfitobacter, Staleya, and Marinobacter secreted elevated amounts of extracellular ATP, ranging from 6.0 to 9.8 pM ATP/colony forming unit (cfu), and that gram-positive bacteria of the genera Kocuria and Planococcus secreted up to 4.1 pM ATP/cfu. Variations in the levels of extracellular and intracellular ATP-dependent luminescence were monitored in living cells of Sulfitobacter mediterraneus ATCC 700856T and Planococcus maritimus F 90 during 48 h of attachment on hydrophobic (poly[tert-butyl methacrylate], PtBMA) and hydrophilic (mica) surfaces. The bacteria responded to different polymeric surfaces by producing either intracellular or extracellular ATP. The level of intracellular ATP in S. mediterraneus ATCC 700856T attached to either surface was as high as 50-55 pM ATP/cfu, while in P. maritimus F 90 it was 120 and 250 pM ATP/cfu on PtBMA and mica, respectively. S. mediterraneus ATCC 700856T generated about 20 and 50 pM of extracellular ATP/cfu on PtBMA and mica, respectively, while the amount generated by P. maritimus F 90 was about the same for both surfaces, 6 pM ATP/cfu. The levels of extracellular ATP generated by S. mediterraneus during attachment on PtBMA and mica were two to five times higher than those detected during the initial screening. High-resolution atomic force microscopy imaging revealed a potentially interesting correlation between the porous cell-surface of certain alpha- and gamma-proteobacteria and their ability to secrete high amounts of ATP. [Abstract/Link to Full Text]

Becker F, Rhiel E
Immuno-electron microscopic quantification of the fucoxanthin chlorophyll a/c binding polypeptides Fcp2, Fcp4, and Fcp6 of Cyclotella cryptica grown under low- and high-light intensities.
Int Microbiol. 2006 Mar;9(1):29-36.
The diatom Cyclotella cryptica was grown under low- and high-intensity white light of 50 and 500 micromol photons m-2 s-1, respectively. Western immunoblotting showed that the diatom adapted its light-harvesting apparatus, giving rise to different amounts of distinct fucoxanthin chlorophyll a/c binding polypeptides (Fcp). The amount of Fcp2 was approximately two-fold higher under low-light than under high-light conditions, whereas the amount of Fcp6 increased four- to five-fold under high-light conditions. For Fcp4, no significant differences were detected in response to either light regime. Cells of Cyclotella grown under high- and low-light intensity were subjected to immunoelectron microscopy. Quantification of the gold label, expressed as gold particles per microm2, confirmed the results obtained by Western immunoblotting. Exposure to low light resulted in the detection of approximately six times more Fcp2-bound gold particles per microm2 in thylakoid membranes, whereas in cells grown under high light the number of Fcp6-bound gold particles increased ten-fold. For Fcp4, similar amounts of gold particles per microm2 were counted under the two light regimes. These immunocytochemical results confirmed molecular data derived from phylogenetic analyses of the sequences of genes encoding fucoxanthin chlorophyll a/c binding polypeptides (fcp genes) and from measurements of steady-state fcp mRNA concentrations. The results show that Fcp2 and Fcp6 accumulate under low- and high-light intensity, respectively, whereas Fcp4 seems to be constitutively synthesized. [Abstract/Link to Full Text]

Lasa I
Towards the identification of the common features of bacterial biofilm development.
Int Microbiol. 2006 Mar;9(1):21-8.
Microorganisms can live and proliferate as individual cells swimming freely in the environment, or they can grow as highly organized, multicellular communities encased in a self-produced polymeric matrix in close association with surfaces and interfaces. This microbial lifestyle is referred to as biofilms. The intense search over the last few years for factors involved in biofilm development has revealed that distantly related bacterial species recurrently make use of the same elements to produce biofilms. These common elements include a group of proteins containing GGDEF/EAL domains, surface proteins homologous to Bap of Staphylococcus aureus, and some types of exopolysaccharides, such as cellulose and the poly-beta-1,6-N-acetylglucosamine. This review summarizes current knowledge about these three common elements and their role in biofilm development. [Abstract/Link to Full Text]

Liras P, Martín JF
Gene clusters for beta-lactam antibiotics and control of their expression: why have clusters evolved, and from where did they originate?
Int Microbiol. 2006 Mar;9(1):9-19.
While beta-lactam compounds were discovered in filamentous fungi, actinomycetes and gram-negative bacteria are also known to produce different types of beta-lactams. All beta-lactam compounds contain a four-membered beta-lactam ring. The structure of their second ring allows these compounds to be classified into penicillins, cephalosporins, clavams, carbapenens or monobactams. Most beta-lactams inhibits bacterial cell wall biosynthesis but others behave as beta-lactamase inhibitors (e.g., clavulanic acid) and even as antifungal agents (e.g., some clavams). Due to the nature of the second ring in beta-lactam molecules, the precursors and biosynthetic pathways of clavams, carbapenems and monobactams differ from those of penicillins and cephalosporins. These last two groups, including cephamycins and cephabacins, are formed from three precursor amino acids that are linked into the alpha-aminoadipyl-L-cysteinyl-D-valine tripeptide. The first two steps of their biosynthetic pathways are common. The intermediates of these pathways, the characteristics of the enzymes involved, the lack of introns in the genes and bioinformatic analysis suggest that all of them should have evolved from an ancestral gene cluster of bacterial origin, which was surely transferred horizontally in the soil from producer to non-producer microorganisms. The receptor strains acquired fragments of the original bacterial cluster and occasionally inserted new genes into the clusters, which once modified, acquired new functions and gave rise to the final compounds that we know. When the order of genes in the Streptomyces genome is analyzed, the antibiotic gene clusters are highlighted as gene islands in the genome. Nonetheless, the assemblage of the ancestral beta-lactam gene cluster remains a matter of speculation. [Abstract/Link to Full Text]

Maloy S, Schaechter M
The era of microbiology: a golden phoenix.
Int Microbiol. 2006 Mar;9(1):1-7.
The discoveries over the last decade have demonstrated that microbiology is a central scientific discipline with practical applications in agriculture, medicine, bioremediation, biotechnology, engineering, and other fields. It is clear that the roles of microbes in nature are so diverse that the process of mining this genetic variation for new applications will continue long into the future. Moreover, the rapid rate of microbial evolution ensures that there will be no permanent solution to agricultural, medical, or environmental problems caused by microbes. These problems will demand a continual stream of creative new approaches that evolve along with the microbes. Thus, the excitement of this field will continue long into the future. However, these opportunities and imperatives demand a deep understanding of basic microbial physiology, genetics, and ecology. Major challenges that lay ahead are to impart the broad training needed to entice and enable the next generation of microbiologists, and to educate the public and government representatives about the continued and critical importance of this field for health and the economy. [Abstract/Link to Full Text]

Di Conza J, Ayala JA, Porto A, Mollerach M, Gutkind G
Molecular characterization of InJR06, a class 1 integron located in a conjugative plasmid of Salmonella enterica ser. Typhimurium.
Int Microbiol. 2005 Dec;8(4):287-90.
The presence of class 1, 2, and 3 integrons was investigated in four pediatric isolates of Salmonella enterica ser. Typhimurium (S. Typhimurium). A class 1 integron was detected in one S. Typhimurium strain, the only one that also showed resistance to various aminoglycoside antibiotics. This integron, called InJR06, and the aminoglycoside resistance determinants were located in pS06, a large (> or = 55 kb) conjugative plasmid. A single mobile cassette (encoding the aminoglycoside adenylyltransferase ANT(3'')-Ia) was detected in the variable region of InJR06, while the architecture of the attI1 and attC sites was conserved. [Abstract/Link to Full Text]

Camacho EM, Serna A, Casadesús J
Regulation of conjugal transfer by Lrp and Dam methylation in plasmid R100.
Int Microbiol. 2005 Dec;8(4):279-85.
Conjugal transfer of the F-like plasmid R100 occurs at higher frequencies in the absence of DNA adenine methylation. Lower levels of R100-encoded FinP RNA were found in a Dam host, suggesting that Dam methylation regulates FinP RNA synthesis. Lack of the leucine-responsive regulatory protein (Lrp) causes a decrease in R100 plasmid transfer, indicating that Lrp is an activator of R100-mediated conjugation. Hence, host-encoded regulators previously described for the Salmonella virulence plasmid (pSLT) seem to play analogous roles in R100. Repression of conjugal transfer in rich medium is an additional trait shared by R100 and pSLT. DNA sequence comparisons in regulatory loci support the view that R100 and pSLT are closely related. [Abstract/Link to Full Text]

Elena SF, Whittam TS, Winkworth CL, Riley MA, Lenski RE
Genomic divergence of Escherichia coli strains: evidence for horizontal transfer and variation in mutation rates.
Int Microbiol. 2005 Dec;8(4):271-8.
This report describes the sequencing in the Escherichia coli B genome of 36 randomly chosen regions that are present in most or all of the fully sequenced E. coli genomes. The phylogenetic relationships among E. coli strains were examined, and evidence for the horizontal gene transfer and variation in mutation rates was determined. The overall phylogenetic tree indicated that E. coli B and K-12 are the most closely related strains, with E. coli O157:H7 being more distantly related, Shigella flexneri 2a even more, and E. coli CFT073 the most distant strain. Within the B, K-12, and O157:H7 clusters, several regions supported alternative topologies. While horizontal transfer may explain these phylogenetic incongruities, faster evolution at synonymous sites along the O157:H7 lineage was also identified. Further interpretation of these results is confounded by an association among genes showing more rapid evolution and results supporting horizontal transfer. Using genes supporting the B and K-12 clusters, an estimate of the genomic mutation rate from a long-term experiment with E. coli B, and an estimate of 200 generations per year, it was estimated that B and K-12 diverged several hundred thousand years ago, while O157:H7 split off from their common ancestor about 1.5-2 million years ago. [Abstract/Link to Full Text]

Martínez-Murcia AJ, Soler L, Saavedra MJ, Chacón MR, Guarro J, Stackebrandt E, Figueras MJ
Phenotypic, genotypic, and phylogenetic discrepancies to differentiate Aeromonas salmonicida from Aeromonas bestiarum.
Int Microbiol. 2005 Dec;8(4):259-69.
The taxonomy of the "Aeromonas hydrophila" complex (comprising the species A. hydrophila, A. bestiarum, A. salmonicida, and A. popoffii) has been controversial, particularly the relationship between the two relevant fish pathogens A. salmonicida and A. bestiarum. In fact, none of the biochemical tests evaluated in the present study were able to separate these two species. One hundred and sixteen strains belonging to the four species of this complex were identified by 16S rDNA restriction fragment length polymorphism (RFLP). Sequencing of the 16S rDNA and cluster analysis of the 16S-23S intergenic spacer region (ISR)-RFLP in selected strains of A. salmonicida and A. bestiarum indicated that the two species may share extremely conserved ribosomal operons and demonstrated that, due to an extremely high degree of sequence conservation, 16S rDNA cannot be used to differentiate these two closely related species. Moreover, DNA-DNA hybridization similarity between the type strains of A. salmonicida subsp. salmonicida and A. bestiarum was 75.6 %, suggesting that they may represent a single taxon. However, a clear phylogenetic divergence between A. salmonicida and A. bestiarum was ascertained from an analysis based on gyrB and rpoD gene sequences, which provided evidence of a lack of congruence of the results obtained from 16S rDNA, 16S-23S ISR-RFLP, DNA-DNA pairing, and biochemical profiles. [Abstract/Link to Full Text]

Iddar A, Valverde F, Assobhei O, Serrano A, Soukri A
Widespread occurrence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria.
Int Microbiol. 2005 Dec;8(4):251-8.
The non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDHN, NADP+-specific, EC 1.2.1.9) is present in green eukaryotes and some Streptococcus strains. The present report describes the results of activity and immunoblot analyses, which were used to generate the first survey of bacterial GAPDHN distribution in a number of Bacillus, Streptococcus and Clostridium strains. Putative gapN genes were identified after PCR amplification of partial 700-bp sequences using degenerate primers constructed from highly conserved protein regions. Alignment of the amino acid sequences of these fragments with those of known sequences from other eukaryotic and prokaryotic GAPDHNs, demonstrated the presence of conserved residues involved in catalytic activity that are not conserved in aldehyde dehydrogenases, a protein family closely linked to GAPDHNs. The results confirm that the basic structural features of the members of the GAPDHN family have been conserved throughout evolution and that no identity exists with phosphorylating GAPDHs. Furthermore, phylogenetic trees generated from multiple sequence alignments suggested a close relationship between plant and bacterial GAPDHN families. [Abstract/Link to Full Text]

Nikel PI, Pettinari MJ, Méndez BS, Galvagno MA
Statistical optimization of a culture medium for biomass and poly(3-hydroxybutyrate) production by a recombinant Escherichia coli strain using agroindustrial byproducts.
Int Microbiol. 2005 Dec;8(4):243-50.
A statistically based Plackett-Burman screening design identified milk whey and corn steep liquor concentrations as well as ionic strength (based on phosphate buffer concentration) as the three main independent components of the culture medium that significantly (p < 0.05) influenced biomass and poly(3-hydroxybutyrate) (PHB) production in recombinant cells of Escherichia coli. This strain carries a plasmid encoding phb genes from a natural isolate of Azotobacter sp. Response surface methodology, using a central composite rotatable design, demonstrated that the optimal concentrations of the three components, defined as those yielding maximal biomass and PHB production in shaken flasks, were 37.96 g deproteinated milk whey powder/l, 29.39 g corn steep liquor/l, and 23.76 g phosphates/l (r2 = 0.957). The model was validated by culturing the recombinant cells in medium containing these optimal concentrations, which yielded 9.41 g biomass/l and 6.12 g PHB/l in the culture broth. Similar amounts of PHB were obtained following batch fermentations in a bioreactor. These results show that PHB can be produced efficiently by culturing the recombinant strain in medium containing cheap carbon and nitrogen sources. [Abstract/Link to Full Text]

Ramírez-Moreno S, Martínez-Alonso M, Méndez-Alvarez S, Gaju N
Seasonal microbial ribotype shifts in the sulfurous karstic lakes Cisó and Vilar, in northeastern Spain.
Int Microbiol. 2005 Dec;8(4):235-42.
Spatio-temporal changes in two sulfurous lakes from the karstic area of Banyoles (Girona, Spain), holomictic lake Cisó and meromictic lake Vilar, were studied over one year. Samples were collected at different depths from the two lakes on the same days, during each of the four seasons, and several physico-chemical variables (temperature, light, pH, conductivity, sulfide, oxygen concentration, pigment concentrations, etc.) were measured. To fingerprint bacterial populations from each sample, DNA was extracted, bacterial 16S rRNA genes were amplified by PCR, and restriction fragment length polymorphism (RFLP) analyses of the total bacterial 16S rDNAs were performed. Each 16S rDNA pool was independently digested with three restriction endonucleases (AluI, HinfI, and RsaI) and separated electrophoretically. More restriction fragments were obtained from the Lake Vilar samples than from the Lake Cisó samples. Moreover, intrasample and intersample differences were observed in each lake. RFLP patterns were compared by scoring similarities using the Jaccard coefficient and then building a multidimensional scaling (MDS) map from the resulting similarities matrix. In both lakes, results indicated that seasonality was mostly responsible for the observed fluctuations in the RFLP patterns, while the effect of stratification was less pronounced. [Abstract/Link to Full Text]

Sarró MI, García AM, Moreno DA
Biofilm formation in spent nuclear fuel pools and bioremediation of radioactive water.
Int Microbiol. 2005 Sep;8(3):223-30.
Microbiological studies of spent nuclear fuel pools at the Cofrentes Nuclear Power Plant (Valencia, Spain) were initiated to determine the microbial populations in the pools' water. Biofilm formation at the nuclear power plant facilities and the potential use of those microbial populations in the bioremediation of radioactive water were also studied. Biofilm formation was analyzed by immersing different austenitic stainless steel coupons (UNS S30400, UNS S30466, UNS S31600), as well as balls of stainless steel (UNS S44200) and titanium (99.9%) in a spent nuclear fuel pool (under static and dynamic conditions) for 34 months. Epifluorescence microscopy and scanning electron microscopy revealed that biofilm formed on the samples, in spite of the radioactive and oligotrophic conditions of the water. Based on standard culture methods and sequencing of 16S rDNA fragments, 57 bacteria belonging to alpha-, beta-, and gamma-Proteobacteria, Firmicutes and Actinobacteridae were identified in the biofilms. The radioactivity of the biofilm was measured using gamma-ray spectrometry, which revealed that biofilms were able to retain radionuclides, especially (60)Co. Using metallic materials to decontaminate radioactive water could become a new approach for bioremediation. [Abstract/Link to Full Text]

Cases I, de Lorenzo V
Genetically modified organisms for the environment: stories of success and failure and what we have learned from them.
Int Microbiol. 2005 Sep;8(3):213-22.
The expectations raised in the mid-1980s on the potential of genetic engineering for in situ remediation of environmental pollution have not been entirely fulfilled. Yet, we have learned a good deal about the expression of catabolic pathways by bacteria in their natural habitats, and how environmental conditions dictate the expression of desired catalytic activities. The many different choices between nutrients and responses to stresses form a network of transcriptional switches which, given the redundance and robustness of the regulatory circuits involved, can be neither unraveled through standard genetic analysis nor artificially programmed in a simple manner. Available data suggest that population dynamics and physiological control of catabolic gene expression prevail over any artificial attempt to engineer an optimal performance of the wanted catalytic activities. In this review, several valuable spin-offs of past research into genetically modified organisms with environmental applications are discussed, along with the impact of Systems Biology and Synthetic Biology in the future of environmental biotechnology. [Abstract/Link to Full Text]

Demnerová K, Mackova M, Spevákova V, Beranova K, Kochánková L, Lovecká P, Ryslavá E, Macek T
Two approaches to biological decontamination of groundwater and soil polluted by aromatics-characterization of microbial populations.
Int Microbiol. 2005 Sep;8(3):205-11.
As part of the EU project MULTIBARRIERS, six new endogenous aerobic bacterial isolates able to grow in the presence of BTmX (benzene, toluene, m-xylene) were characterized with respect to their growth specificities. Preliminary analysis included restriction fragment length polymorphism profiles and 16S rDNA sequencing. The diversity of these strains was confirmed by denaturing gradient gel electrophoresis. Additional aerobic bacterial strains were isolated from the rhizospheres of plants grown in polychlorinated biphenyl (PCB)-contaminated soils. Pot experiments were designed to show the beneficial effect of plants on the bacterial degradation of PCBs. The effect of PCB removal from soil was evaluated and bacteria isolated from three different plant species were examined for the presence of the bph operon. [Abstract/Link to Full Text]

Martínez AT, Speranza M, Ruiz-Dueńas FJ, Ferreira P, Camarero S, Guillén F, Martínez MJ, Gutiérrez A, del Río JC
Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin.
Int Microbiol. 2005 Sep;8(3):195-204.
Wood is the main renewable material on Earth and is largely used as building material and in paper-pulp manufacturing. This review describes the composition of lignocellulosic materials, the different processes by which fungi are able to alter wood, including decay patterns caused by white, brown, and soft-rot fungi, and fungal staining of wood. The chemical, enzymatic, and molecular aspects of the fungal attack of lignin, which represents the key step in wood decay, are also discussed. Modern analytical techniques to investigate fungal degradation and modification of the lignin polymer are reviewed, as are the different oxidative enzymes (oxidoreductases) involved in lignin degradation. These include laccases, high redox potential ligninolytic peroxidases (lignin peroxidase, manganese peroxidase, and versatile peroxidase), and oxidases. Special emphasis is given to the reactions catalyzed, their synergistic action on lignin, and the structural bases for their unique catalytic properties. Broadening our knowledge of lignocellulose biodegradation processes should contribute to better control of wood-decaying fungi, as well as to the development of new biocatalysts of industrial interest based on these organisms and their enzymes. [Abstract/Link to Full Text]

González JM, Sáiz-Jiménez C
Application of molecular nucleic acid-based techniques for the study of microbial communities in monuments and artworks.
Int Microbiol. 2005 Sep;8(3):189-94.
Microorganisms play critical roles in every kind of habitat on Earth, including those constructed by humans. Thus, our cultural heritage is affected by microbial colonization. While classical microbiological methods based on culturing procedures have provided important, but limited information on the microbial diversity of natural samples, novel molecular techniques have been extremely valuable in unraveling the diversity of microbiota involved in the biodeterioration of our monuments and artworks. The knowledge gained from these approaches has allowed the design of strategies for conserving and protecting monuments for the benefit of future generations. This review describes the state-of-the-art of the application of molecular methods to the analysis of cultural assets, and provides near-future perspectives on the subject. [Abstract/Link to Full Text]


Recent Articles in Retrovirology

Pando MA, Eyzaguirre LM, Carrion G, Montano SM, Sanchez JL, Carr JK, Avila MM
High genetic variability of HIV-1 in female sex workers from Argentina.
Retrovirology. 2007;458.
BACKGROUND: A cross-sectional study on 625 Female Sex Workers (FSWs) was conducted between 2000 and 2002 in 6 cities in Argentina. This study describes the genetic diversity and the resistance profile of the HIV-infected subjects. RESULTS: Seventeen samples from HIV positive FSWs were genotyped by env HMA, showing the presence of 9 subtype F, 6 subtype B and 2 subtype C. Sequence analysis of the protease/RT region on 16 of these showed that 10 were BF recombinants, three were subtype B, two were subtype C, and one sample presented a dual infection with subtype B and a BF recombinant. Full-length genomes of five of the protease/RT BF recombinants were also sequenced, showing that three of them were CRF12_BF. One FSW had a dual HIV-1 infection with subtype B and a BF recombinant. The B sections of the BF recombinant clustered closely with the pure B sequence isolated from the same patient. Major resistance mutations to antiretroviral drugs were found in 3 of 16 (18.8%) strains. CONCLUSION: The genetic diversity of HIV strains among FSWs in Argentina was extensive; about three-quarters of the samples were infected with diverse BF recombinants, near twenty percent had primary ART resistance and one sample presented a dual infection. Heterosexual transmission of genetically diverse, drug resistant strains among FSWs and their clients represents an important and underestimated threat, in Argentina. [Abstract/Link to Full Text]

Amarnath S, Dong L, Li J, Wu Y, Chen W
Endogenous TGF-beta activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected human CD4+CD25- T cells.
Retrovirology. 2007;457.
BACKGROUND: CD4+CD25+ T regulatory cells (Tregs) play an important role in regulating immune responses, and in influencing human immune diseases such as HIV infection. It has been shown that human CD4+CD25+ Tregs can be induced in vitro by TCR stimulation of CD4+CD25- T cells. However, the mechanism remains elusive, and intriguingly, similar treatment of murine CD4+CD25- cells did not induce CD4+CD25+Foxp3+ Tregs unless exogenous TGF-beta was added during stimulation. Thus, we investigated the possible role of TGF-beta in the induction of human Tregs by TCR engagement. We also explored the effects of TGF-beta on HIV-1 infection mediated induction of human Tregs since recent evidence has suggested that HIV-1 infection may also impact the generation of Tregs in infected patients. RESULTS: We show here that endogenous TGF-beta is key to TCR induction of Foxp3 in human CD4+CD25- T cells. These events involve, first, the production of TGF-beta by TCR and CD28 stimulation and the activation of latent TGF-beta by reactive oxygen species generated from the activated T cells. Biologically active TGF-beta then engages in the induction of Foxp3. Neutralization of active TGF-beta with anti-TGF-beta antibody or elimination of ROS with MnTBAP abrogated Foxp3 expression. HIV-1 infection enhanced Foxp3 expression in activated CD4+CD25- T cells; which was also abrogated by blockade of endogenous TGF-beta. CONCLUSION: Several conclusions can be drawn from this work: (1) TCR and CD28-induced Foxp3 expression is a late event following TCR stimulation; (2) TGF-beta serves as a link in Foxp3 induction in human CD4+CD25- T cells following TCR stimulation, which induces not only latent, but also active TGF-beta; (3) the activation of TGF-beta requires reactive oxygen species; (4) HIV infection results in an increase in Foxp3 expression in TCR-activated CD25- T cells, which is also associated with TGF-beta. Taken together, our findings reinforce a definitive role of TGF-beta not only in the generation of Tregs with respect to normal immune responses, but also is critical in immune diseases such as HIV-1 infection. [Abstract/Link to Full Text]

Coiras M, López-Huertas MR, Rullas J, Mittelbrunn M, Alcamí J
Basal shuttle of NF-kappaB/I kappaB alpha in resting T lymphocytes regulates HIV-1 LTR dependent expression.
Retrovirology. 2007;456.
BACKGROUND: In HIV-infected T lymphocytes, NF-kappaB/Rel transcription factors are major elements involved in the activation of LTR-dependent transcription from latency. Most NF-kappaB heterodimer p65/p50 is sequestered as an inactive form in the cytoplasm of resting T lymphocytes via its interaction with I kappaB inhibitors. In these cells, both absolute HIV latency and low level ongoing HIV replication have been described. These situations could be related to differences in the balance between NF-kappaB and I kappaB alpha ratio. Actually, control of I kappaB alpha by cellular factors such as Murr-1 plays a critical role in maintaining HIV latency in unstimulated T lymphocytes. Formerly, our group demonstrated the presence of nuclear I kappaB alpha in T cells after PMA activation. Now we attempt to determine the dynamics of NF-kappaB/I kappaB alpha nucleocytosolic transport in absence of activation as a mechanism to explain both the maintenance of latency and the existence of low level ongoing HIV replication in resting CD4+ T lymphocytes. RESULTS AND CONCLUSION: We show that the inhibition of the nuclear export by leptomycin B in resting CD4+ T cells resulted in nuclear accumulation of both I kappaB alpha and p65/RelA, as well as formation of NF-kappaB/I kappaB alpha complexes. This proves the existence of a rapid shuttling of I kappaB alpha between nucleus and cytosol even in absence of cellular activation. The nuclear accumulation of I kappaB alpha in resting CD4+ T lymphocytes results in inhibition of HIV-LTR dependent transcription as well as restrains HIV replication in CD4+ T lymphocytes. On the other hand, basal NF-kappaB activity detected in resting CD4+ T lymphocytes was related to low level HIV replication in these cells. [Abstract/Link to Full Text]

Melikyan GB, Platt EJ, Kabat D
The role of the N-terminal segment of CCR5 in HIV-1 Env-mediated membrane fusion and the mechanism of virus adaptation to CCR5 lacking this segment.
Retrovirology. 2007;455.
BACKGROUND: HIV-1 envelope glycoprotein (Env) induces membrane fusion as a result of sequential binding to CD4 and chemokine receptors (CCR5 or CXCR4). The critical determinants of CCR5 coreceptor function are the N-terminal domain (Nt) and the second extracellular loop. However, mutations in gp120 adapt HIV-1 to grow on cells expressing the N-terminally truncated CCR5(Delta 18) (Platt et al., J. Virol. 2005, 79: 4357-68). RESULTS: We have functionally characterized the adapted Env (designated Env(NYP)) using a quantitative cell-cell fusion assay. The rate of fusion with target cells expressing wild-type CCR5 and the resistance to fusion inhibitors was virtually identical for wild-type Env and Env(NYP), implying that the coreceptor affinity had not increased as a result of adaptation. In contrast, Env(NYP)-induced fusion with cells expressing CCR5(Delta 18) occurred at a slower rate and was extremely sensitive to the CCR5 binding inhibitor, Sch-C. Resistance to Sch-C drastically increased after pre-incubation of Env(NYP)- and CCR5(Delta 18)-expressing cells at a temperature that was not permissive to fusion. This indicates that ternary Env(NYP)-CD4-CCR5(Delta 18) complexes accumulate at sub-threshold temperature and that low-affinity interactions with the truncated coreceptor are sufficient for triggering conformational changes in the gp41 of Env(NYP) but not in wild-type Env. We also demonstrated that the ability of CCR5(Delta 18) to support fusion and infection mediated by wild-type Env can be partially reconstituted in the presence of a synthetic sulfated peptide corresponding to the CCR5 Nt. Pre-incubation of wild-type Env- and CCR5(Delta 18)-expressing cells with the sulfated peptide at sub-threshold temperature markedly increased the efficiency of fusion. CONCLUSION: We propose that, upon binding the Nt region of CCR5, wild-type Env acquires the ability to productively engage the extracellular loop(s) of CCR5 - an event that triggers gp41 refolding and membrane merger. The adaptive mutations in Env(NYP) enable it to more readily release its hold on gp41, even when it interacts weakly with a severely damaged coreceptor in the absence of the sulfopeptide. [Abstract/Link to Full Text]

Grigorov B, Décimo D, Smagulova F, Péchoux C, Mougel M, Muriaux D, Darlix JL
Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers.
Retrovirology. 2007;454.
BACKGROUND: The HIV-1 nucleocapsid protein (NC) is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT), gRNA dimerization and packaging, and virion assembly. RESULTS: We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. CONCLUSION: These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses. [Abstract/Link to Full Text]

Goffinet C, Michel N, Allespach I, Tervo HM, Hermann V, Kräusslich HG, Greene WC, Keppler OT
Primary T-cells from human CD4/CCR5-transgenic rats support all early steps of HIV-1 replication including integration, but display impaired viral gene expression.
Retrovirology. 2007;453.
BACKGROUND: In vivo studies on HIV-1 pathogenesis and testing of antiviral strategies have been hampered by the lack of an immunocompetent small animal model that is highly susceptible to HIV-1 infection. Since native rodents are non-permissive, we developed transgenic rats that selectively express the HIV-1 receptor complex, hCD4 and hCCR5, on relevant target cells. These animals display a transient low-level plasma viremia after HIV-1YU-2 infection, demonstrating HIV-1 susceptibility in vivo. However, unlike macrophages, primary CD4 T-cells from double-transgenic animals fail to support viral spread ex vivo. To identify quantitative limitations or absolute blocks in this rodent species, we quantitatively assessed the efficiency of key steps in the early phase of the viral replication cycle in a side-by-side comparison in infected cell lines and primary T-cells from hCD4/hCCR5-transgenic rats and human donors. RESULTS: Levels of virus entry, HIV-1 cDNA synthesis, nuclear import, and integration into the host genome were shown to be remarkably similar in cell lines and, where technically accessible, in primary T-cells from both species. In contrast, a profound impairment at the level of early HIV gene expression was disclosed at the single-cell level in primary rat T-cells and most other rat-derived cells. Macrophages were a notable exception, possibly reflecting the unique transcriptional milieu in this evolutionarily conserved target cell of all lentiviruses. Importantly, transient trans-complementation by ex vivo nucleofection with the Tat-interacting protein Cyclin T1 of human origin markedly elevated HIV gene expression in primary rat T-cells. CONCLUSION: This is the first study that has quantitatively determined the efficiency of consecutive steps in the HIV-1 replication cycle in infected primary HIV target cells from a candidate transgenic small animal and compared it to human cells. Unlike cells derived from mice or rabbits, rat cells complete all of the early steps in the HIV-1 replication cycle, including provirus integration in vivo, with high efficiency. A deficiency in gene expression was disclosed at the single cell level and could be counteracted by the human pTEFb transcription complex factor Cyclin T1. Collectively, these results provide the basis for the advancement of this transgenic rat model through strategies aimed at boosting HIV-1 gene expression in primary rat CD4 T-cells, including human Cyclin T1 transgenesis. [Abstract/Link to Full Text]

Lama J, Planelles V
Host factors influencing susceptibility to HIV infection and AIDS progression.
Retrovirology. 2007;452.
Transmission of HIV first results in an acute infection, followed by an apparently asymptomatic period that averages ten years. In the absence of antiretroviral treatment, most patients progress into a generalized immune dysfunction that culminates in death. The length of the asymptomatic period varies, and in rare cases infected individuals never progress to AIDS. Other individuals whose behavioral traits put them at high-risk of HIV transmission, surprisingly appear resistant and never succumb to infection. These unique cases highlight the fact that susceptibility to HIV infection and progression to disease are complex traits modulated by environmental and genetic factors. Recent evidence has indicated that natural variations in host genes can influence the outcome of HIV infection and its transmission. In this review we summarize the available literature on the roles of cellular factors and their genetic variation in modulating HIV infection and disease progression. [Abstract/Link to Full Text]

Merimi M, Klener P, Szynal M, Cleuter Y, Bagnis C, Kerkhofs P, Burny A, Martiat P, Van den Broeke A
Complete suppression of viral gene expression is associated with the onset and progression of lymphoid malignancy: observations in Bovine Leukemia Virus-infected sheep.
Retrovirology. 2007;451.
BACKGROUND: During malignant progression, tumor cells need to acquire novel characteristics that lead to uncontrolled growth and reduced immunogenicity. In the Bovine Leukemia Virus-induced ovine leukemia model, silencing of viral gene expression has been proposed as a mechanism leading to immune evasion. However, whether proviral expression in tumors is completely suppressed in vivo was not conclusively demonstrated. Therefore, we studied viral expression in two selected experimentally-infected sheep, the virus or the disease of which had features that made it possible to distinguish tumor cells from their nontransformed counterparts. RESULTS: In the first animal, we observed the emergence of a genetically modified provirus simultaneously with leukemia onset. We found a Tax-mutated (TaxK303) replication-deficient provirus in the malignant B-cell clone while functional provirus (TaxE303) had been consistently monitored over the 17-month aleukemic period. In the second case, both non-transformed and transformed BLV-infected cells were present at the same time, but at distinct sites. While there was potentially-active provirus in the non-leukemic blood B-cell population, as demonstrated by ex-vivo culture and injection into naďve sheep, virus expression was completely suppressed in the malignant B-cells isolated from the lymphoid tumors despite the absence of genetic alterations in the proviral genome. These observations suggest that silencing of viral genes, including the oncoprotein Tax, is associated with tumor onset. CONCLUSION: Our findings suggest that silencing is critical for tumor progression and identify two distinct mechanisms-genetic and epigenetic-involved in the complete suppression of virus and Tax expression. We demonstrate that, in contrast to systems that require sustained oncogene expression, the major viral transforming protein Tax can be turned-off without reversing the transformed phenotype. We propose that suppression of viral gene expression is a contributory factor in the impairment of immune surveillance and the uncontrolled proliferation of the BLV-infected tumor cell. [Abstract/Link to Full Text]

Laurén A, Vincic E, Hoshino H, Thorstensson R, Fenyö EM
CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates.
Retrovirology. 2007;450.
BACKGROUND: CD4-independence has been taken as a sign of a more open envelope structure that is more accessible to neutralizing antibodies and may confer altered cell tropism. In the present study, we analyzed SIVsm isolates for CD4-independent use of CCR5, mode of CCR5-use and macrophage tropism. The isolates have been collected sequentially from 13 experimentally infected cynomolgus macaques and have previously been shown to use CCR5 together with CD4. Furthermore, viruses obtained early after infection were neutralization sensitive, while neutralization resistance appeared already three months after infection in monkeys with progressive immunodeficiency. RESULTS: Depending whether isolated early or late in infection, two phenotypes of CD4-independent use of CCR5 could be observed. The inoculum virus (SIVsm isolate SMM-3) and reisolates obtained early in infection often showed a pronounced CD4-independence since virus production and/or syncytia induction could be detected directly in NP-2 cells expressing CCR5 but not CD4 (CD4-independent-HIGH). Conversely, late isolates were often more CD4-dependent in that productive infection in NP-2/CCR5 cells was in most cases weak and was revealed only after cocultivation of infected NP-2/CCR5 cells with peripheral blood mononuclear cells (CD4-independent-LOW). Considering neutralization sensitivity of these isolates, newly infected macaques often harbored virus populations with a CD4-independent-HIGH and neutralization sensitive phenotype that changed to a CD4-independent-LOW and neutralization resistant virus population in the course of infection. Phenotype changes occurred faster in progressor than long-term non-progressor macaques. The phenotypes were not reflected by macrophage tropism, since all isolates replicated efficiently in macrophages. Infection of cells expressing CCR5/CXCR4 chimeric receptors revealed that SIVsm used the CCR5 receptor in a different mode than HIV-1. CONCLUSION: Our results show that SIVsm isolates use CCR5 independently of CD4. While the degree of CD4 independence and neutralization sensitivity vary over time, the ability to productively infect monocyte-derived macrophages remains at a steady high level throughout infection. The mode of CCR5 use differs between SIVsm and HIV-1, SIVsm appears to be more flexible than HIV-1 in its receptor requirement. We suggest that the mode of CCR5 coreceptor use and CD4-independence are interrelated properties. [Abstract/Link to Full Text]

Datta A, Silverman L, Phipps AJ, Hiraragi H, Ratner L, Lairmore MD
Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival.
Retrovirology. 2007;449.
BACKGROUND: Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation. RESULTS: Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes. CONCLUSION: Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival. [Abstract/Link to Full Text]

Khan MA, Goila-Gaur R, Opi S, Miyagi E, Takeuchi H, Kao S, Strebel K
Analysis of the contribution of cellular and viral RNA to the packaging of APOBEC3G into HIV-1 virions.
Retrovirology. 2007;448.
BACKGROUND: Efficient incorporation of the cellular cytidine deaminase APOBEC3G (APO3G) into HIV-1 virions is necessary for its antiviral activity. Even though cellular RNAs are known to be non-specifically incorporated into virus particles, we have previously found that encapsidation of APO3G into HIV-1 virions is specifically enhanced by viral genomic RNA. Intracellularly, APO3G was found to form large RNA-protein complexes involving a variety of cellular RNAs. The goal of this study was to investigate the possible contribution of host RNAs recently identified in intracellular APO3G ribonucleoprotein complexes to APO3G's encapsidation into HIV-1 virions. RESULTS: Our results show that 7SL RNA, a component of signal recognition particles, and hY1, hY3, hY4, hY5 RNAs were present in intracellular APO3G complexes and were packaged into HIV-1 particles lacking viral genomic RNA unlike APO3G, which was not packaged in significant amounts into genomic RNA-deficient particles. These results indicate that packaging of 7SL or hY RNAs is not sufficient for the packaging of APO3G into HIV-1 virions. We also tested the encapsidation of several other cellular RNAs including beta-actin, GAPDH, alpha-tubulin, and small nuclear RNAs and determined their effect on the packaging of APO3G into nascent virions. Again, we were unable to observe any correlation between APO3G encapsidation and the packaging of any of these cellular RNAs. CONCLUSION: The results from this study support our previous conclusion that viral genomic RNA is a critical determinant for APO3G incorporation into HIV-1 virions. While most cellular RNAs tested in this study were packaged into viruses or virus-like particles we failed to identify a correlation between APO3G encapsidation and the packaging of these cellular RNAs. [Abstract/Link to Full Text]

Biglione S, Byers SA, Price JP, Nguyen VT, Bensaude O, Price DH, Maury W
Inhibition of HIV-1 replication by P-TEFb inhibitors DRB, seliciclib and flavopiridol correlates with release of free P-TEFb from the large, inactive form of the complex.
Retrovirology. 2007;447.
BACKGROUND: The positive transcription elongation factor, P-TEFb, comprised of cyclin dependent kinase 9 (Cdk9) and cyclin T1, T2 or K regulates the productive elongation phase of RNA polymerase II (Pol II) dependent transcription of cellular and integrated viral genes. P-TEFb containing cyclin T1 is recruited to the HIV long terminal repeat (LTR) by binding to HIV Tat which in turn binds to the nascent HIV transcript. Within the cell, P-TEFb exists as a kinase-active, free form and a larger, kinase-inactive form that is believed to serve as a reservoir for the smaller form. RESULTS: We developed a method to rapidly quantitate the relative amounts of the two forms based on differential nuclear extraction. Using this technique, we found that titration of the P-TEFb inhibitors flavopiridol, DRB and seliciclib onto HeLa cells that support HIV replication led to a dose dependent loss of the large form of P-TEFb. Importantly, the reduction in the large form correlated with a reduction in HIV-1 replication such that when 50% of the large form was gone, HIV-1 replication was reduced by 50%. Some of the compounds were able to effectively block HIV replication without having a significant impact on cell viability. The most effective P-TEFb inhibitor flavopiridol was evaluated against HIV-1 in the physiologically relevant cell types, peripheral blood lymphocytes (PBLs) and monocyte derived macrophages (MDMs). Flavopiridol was found to have a smaller therapeutic index (LD50/IC50) in long term HIV-1 infectivity studies in primary cells due to greater cytotoxicity and reduced efficacy at blocking HIV-1 replication. CONCLUSION: Initial short term studies with P-TEFb inhibitors demonstrated a dose dependent loss of the large form of P-TEFb within the cell and a concomitant reduction in HIV-1 infectivity without significant cytotoxicity. These findings suggested that inhibitors of P-TEFb may serve as effective anti-HIV-1 therapies. However, longer term HIV-1 replication studies indicated that these inhibitors were more cytotoxic and less efficacious against HIV-1 in the primary cell cultures. [Abstract/Link to Full Text]

Sřrensen AB, Lund AH, Kunder S, Quintanilla-Martinez L, Schmidt J, Wang B, Wabl M, Pedersen FS
Impairment of alternative splice sites defining a novel gammaretroviral exon within gag modifies the oncogenic properties of Akv murine leukemia virus.
Retrovirology. 2007;446.
BACKGROUND: Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus. RESULTS: By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous gag mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as de novo diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants. CONCLUSION: We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus. [Abstract/Link to Full Text]

Lech P, Somia NV
Isolation and characterization of human cells resistant to retrovirus infection.
Retrovirology. 2007;445.
BACKGROUND: Identification of host cell proteins required for HIV-1 infection will add to our knowledge of the life cycle of HIV-1 and in the development of therapeutics to combat viral infection. We and other investigators have mutagenized rodent cells and isolated mutant cell lines resistant to retrovirus infection. Since there are differences in the efficiency of single round infection with VSVG pseudotyped HIV-1 on cells of different species, we conducted a genetic screen to isolate human cells resistant to HIV-1 infection. We chemically mutagenized human HeLa cells and validated our ability to isolate mutants at test diploid loci. We then executed a screen to isolate HeLa cell mutants resistant to infection by an HIV-1 vector coding for a toxic gene product. RESULTS: We isolated two mutant cell lines that exhibit up to 10-fold resistance to infection by HIV-1 vectors. We have verified that the cells are resistant to infection and not defective in gene expression. We have confirmed that the resistance phenotype is not due to an entry defect. Fusion experiments between mutant and wild-type cells have established that the mutations conferring resistance in the two clones are recessive. We have also determined the nature of the block in the two mutants. One clone exhibits a block at or before reverse transcription of viral RNA and the second clone has a retarded kinetic of viral DNA synthesis and a block at nuclear import of the preintegration complex. CONCLUSION: Human cell mutants can be isolated that are resistant to infection by HIV-1. The mutants are genetically recessive and identify two points where host cell factors can be targeted to block HIV-1 infection. [Abstract/Link to Full Text]

Whitney JB, Wainberg MA
Recovery of fitness of a live attenuated simian immunodeficiency virus through compensation in both the coding and non-coding regions of the viral genome.
Retrovirology. 2007;444.
We have analyzed a SIV deletion mutant that was compromised both in viral replication and RNA packaging. Serial passage of this variant in two different T-cell lines resulted in compensatory reversion and the generation of independent groups of point mutations within each cell line. Within each group, single point mutations were shown to contribute to increased viral infectivity and the rescue of wild-type replication kinetics. The complete recovery of viral fitness ultimately correlated with the restoration of viral RNA packaging. Consistent with the latter finding was the rescue of Pr55 Gag processing, also restoring proper virus core morphology in mature virions. These seemingly independently arising groups of compensatory mutations were functionally interchangeable in regard to the recovery of wild type replication in rhesus PBMCs. These findings indicate that viral reversion that overcomes a genetic bottleneck is not limited to a single pathway, and illustrates the remarkable adaptability of lentiviruses. [Abstract/Link to Full Text]

Churchill MJ, Chiavaroli L, Wesselingh SL, Gorry PR
Persistence of attenuated HIV-1 rev alleles in an epidemiologically linked cohort of long-term survivors infected with nef-deleted virus.
Retrovirology. 2007;443.
BACKGROUND: The Sydney blood bank cohort (SBBC) of long-term survivors consists of multiple individuals infected with nef-deleted, attenuated strains of human immunodeficiency virus type 1 (HIV-1). Although the cohort members have experienced differing clinical courses and now comprise slow progressors (SP) as well as long-term nonprogressors (LTNP), longitudinal analysis of nef/long-terminal repeat (LTR) sequences demonstrated convergent nef/LTR sequence evolution in SBBC SP and LTNP. Thus, the in vivo pathogenicity of attenuated HIV-1 strains harboured by SBBC members is dictated by factors other than nef/LTR. Therefore, to determine whether defects in other viral genes contribute to attenuation of these HIV-1 strains, we characterized dominant HIV-1 rev alleles that persisted in 4 SBBC subjects; C18, C64, C98 and D36. RESULTS: The ability of Rev derived from D36 and C64 to bind the Rev responsive element (RRE) in RNA binding assays was reduced by approximately 90% compared to Rev derived from HIV-1NL4-3, C18 or C98. D36 Rev also had a 50-60% reduction in ability to express Rev-dependent reporter constructs in mammalian cells. In contrast, C64 Rev had only marginally decreased Rev function despite attenuated RRE binding. In D36 and C64, attenuated RRE binding was associated with rare amino acid changes at 3 highly conserved residues; Gln to Pro at position 74 immediately N-terminal to the Rev activation domain, and Val to Leu and Ser to Pro at positions 104 and 106 at the Rev C-terminus, respectively. In D36, reduced Rev function was mapped to an unusual 13 amino acid extension at the Rev C-terminus. CONCLUSION: These findings provide new genetic and mechanistic insights important for Rev function, and suggest that Rev function, not Rev/RRE binding may be rate limiting for HIV-1 replication. In addition, attenuated rev alleles may contribute to viral attenuation and long-term survival of HIV-1 infection in a subset of SBBC members. [Abstract/Link to Full Text]

Jeang KT
Impact factor, H index, peer comparisons, and Retrovirology: is it time to individualize citation metrics?
Retrovirology. 2007;442.
There is a natural tendency to judge a gift by the attractiveness of its wrapping. In some respect, this reflects current mores of measuring the gravitas of a scientific paper based on the journal cover in which the work appears. Most journals have an impact factor (IF) which some proudly display on their face page. Although historically journal IF has been a convenient quantitative shorthand, has its (mis)use contributed to inaccurate perceptions of the quality of scientific articles? Is now the time that equally convenient but more individually accurate metrics be adopted? [Abstract/Link to Full Text]

Agbottah ET, Traviss C, McArdle J, Karki S, St Laurent GC, Kumar A
Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1.
Retrovirology. 2007;441.
BACKGROUND: Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. We describe properties of a cellular double-stranded RNA binding protein with intrinsic affinity for HIV-1 TAR RNA that interferes with Tat/TAR interaction and inhibits viral gene expression. RESULTS: Utilizing TAR affinity fractionation, North-Western blotting, and mobility-shift assays, we show that the C-terminal variant of nuclear factor 90 (NF90ctv) with strong affinity for the TAR RNA, competes with Tat/TAR interaction in vitro. Analysis of the effect of NF90ctv-TAR RNA interaction in vivo showed significant inhibition of Tat-transactivation of HIV-1 LTR in cells expressing NF90ctv, as well as changes in histone H3 lysine-4 and lysine-9 methylation of HIV chromatin that are consistent with the epigenetic changes in transcriptionally repressed gene. CONCLUSION: Structural integrity of the TAR element is crucial in HIV-1 gene expression. Our results show that perturbation Tat/TAR RNA interaction by the dsRNA binding protein is sufficient to inhibit transcriptional activation of HIV-1. [Abstract/Link to Full Text]

Towers GJ
The control of viral infection by tripartite motif proteins and cyclophilin A.
Retrovirology. 2007;440.
The control of retroviral infection by antiviral factors referred to as restriction factors has become an exciting area in infectious disease research. TRIM5alpha has emerged as an important restriction factor impacting on retroviral replication including HIV-1 replication in primates. TRIM5alpha has a tripartite motif comprising RING, B-Box and coiled coil domains. The antiviral alpha splice variant additionally encodes a B30.2 domain which is recruited to incoming viral cores and determines antiviral specificity. TRIM5 is ubiquitinylated and rapidly turned over by the proteasome in a RING dependent way. Protecting restricted virus from degradation, by inhibiting the proteasome, rescues DNA synthesis, but not infectivity, indicating that restriction of infectivity by TRIM5alpha does not depend on the proteasome but the early block to DNA synthesis is likely to be mediated by rapid degradation of the restricted cores. The peptidyl prolyl isomerase enzyme cyclophilin A isomerises a peptide bond on the surface of the HIV-1 capsid and impacts on sensitivity to restriction by TRIM5alpha from Old World monkeys. This suggests that TRIM5alpha from Old World monkeys might have a preference for a particular capsid isomer and suggests a role for cyclophilin A in innate immunity in general. Whether there are more human antiviral TRIMs remains uncertain although the evidence for TRIM19's (PML) antiviral properties continues to grow. A TRIM5-like molecule with broad antiviral activity in cattle suggests that TRIM mediated innate immunity might be common in mammals. Certainly the continued study of restriction of viral infectivity by antiviral host factors will remain of interest to a broad audience and impact on a variety of areas including development of animal models for infection, development of viral vectors for gene therapy and the search for novel antiviral drug targets. [Abstract/Link to Full Text]

Flockerzi A, Maydt J, Frank O, Ruggieri A, Maldener E, Seifarth W, Medstrand P, Lengauer T, Meyerhans A, Leib-Mösch C, Meese E, Mayer J
Expression pattern analysis of transcribed HERV sequences is complicated by ex vivo recombination.
Retrovirology. 2007;439.
BACKGROUND: The human genome comprises numerous human endogenous retroviruses (HERVs) that formed millions of years ago in ancestral species. A number of loci of the HERV-K(HML-2) family are evolutionarily much younger. A recent study suggested an infectious HERV-K(HML-2) variant in humans and other primates. Isolating such a variant from human individuals would be a significant finding for human biology. RESULTS: When investigating expression patterns of specific HML-2 proviruses we encountered HERV-K(HML-2) cDNA sequences without proviral homologues in the human genome, named HERV-KX, that could very well support recently suggested infectious HML-2 variants. However, detailed sequence analysis, using the software RECCO, suggested that HERV-KX sequences were produced by recombination, possibly arising ex vivo, between transcripts from different HML-2 proviral loci. CONCLUSION: As RT-PCR probably will be instrumental for isolating an infectious HERV-K(HML-2) variant, generation of "new" HERV-K(HML-2) sequences by ex vivo recombination seems inevitable. Further complicated by an unknown amount of allelic sequence variation in HERV-K(HML-2) proviruses, newly identified HERV-K(HML-2) variants should be interpreted very cautiously. [Abstract/Link to Full Text]

Oh T, Bajwa A, Jia G, Park F
Lentiviral vector design using alternative RNA export elements.
Retrovirology. 2007;438.
BACKGROUND: Lentiviral vectors have been designed with complex RNA export sequences in both the integrating and packaging plasmids in order to co-ordinate efficient vector production. Recent studies have attempted to replace the existing complex rev/RRE system with a more simplistic RNA export system from simple retroviruses to make these vectors in a rev-independent manner. RESULTS: Towards this end, lentiviral transfer plasmids were modified with various cis-acting DNA elements that co-ordinate RNA export during viral production to determine their ability to affect the efficiency of vector titer and transduction in different immortalized cell lines in vitro. It was found that multiple copies of the constitutive transport element (CTE) originating from different simian retroviruses, including simian retrovirus type 1 (SRV-1) and type-2 (SRV-2) and Mason-Pfizer (MPV) could be used to eliminate the requirement for the rev responsive element (RRE) in the transfer and packaging plasmids with titers >106 T.U./mL (n = 4-8 preparations). The addition of multiple copies of the murine intracisternal type A particle, the woodchuck post-regulatory element (WPRE), or single and dual copies of the simian CTE had minimal effect on viral titer. Immortalized cell lines from different species were found to be readily transduced by VSV-G pseudotyped lentiviral vectors containing the multiple copies of the CTE similar to the findings in HeLa cells, although the simian-derived CTE were found to have a lower infectivity into murine cell lines compared to the other species. CONCLUSION: These studies demonstrated that the rev-responsive element (RRE) could be replaced with other constitutive transport elements to produce equivalent titers using lentivectors containing the RRE sequence in vitro, but that concatemerization of the CTE or the close proximity of RNA export sequences was needed to enhance vector production. [Abstract/Link to Full Text]

Rakotobe D, Tardy JC, André P, Hong SS, Darlix JL, Boulanger P
Human Polycomb group EED protein negatively affects HIV-1 assembly and release.
Retrovirology. 2007;437.
BACKGROUND: The human EED protein, a member of the superfamily of Polycomb group (PcG) proteins with WD-40 repeats, has been found to interact with three HIV-1 components, namely the structural Gag matrix protein (MA), the integrase enzyme (IN) and the Nef protein. The aim of the present study was to analyze the possible biological role of EED in HIV-1 replication, using the HIV-1-based vector HIV-Luc and EED protein expressed by DNA transfection of 293T cells. RESULTS: During the early phase of HIV-1 infection, a slight negative effect on virus infectivity occurred in EED-expressing cells, which appeared to be dependent on EED-MA interaction. At late times post infection, EED caused an important reduction of virus production, from 20- to 25-fold as determined by CAp24 immunoassay, to 10- to 80-fold based on genomic RNA levels, and this decrease was not due to a reduction of Gag protein synthesis. Coexpression of WTNef, or the non-N-myristoylated mutant NefG2A, restored virus yields to levels obtained in the absence of exogenous EED protein. This effect was not observed with mutant NefDelta57 mimicking the Nef core, or with the lipid raft-retargeted fusion protein LAT-Nef. LATAA-Nef, a mutant defective in the lipid raft addressing function, had the same anti-EED effect as WTNef. Cell fractionation and confocal imaging showed that, in the absence of Nef, EED mainly localized in membrane domains different from the lipid rafts. Upon co-expression with WTNef, NefG2A or LATAA-Nef, but not with NefDelta57 or LAT-Nef, EED was found to relocate into an insoluble fraction along with Nef protein. Electron microscopy of HIV-Luc producer cells overexpressing EED showed significant less virus budding at the cell surface compared to control cells, and ectopic assembly and clustering of nuclear pore complexes within the cytoplasm. CONCLUSION: Our data suggested that EED exerted an antiviral activity at the late stage of HIV-1 replication, which included genomic RNA packaging and virus assembly, resulting possibly from a mistrafficking of viral genomic RNA (gRNA) or gRNA/Gag complex. Nef reversed the EED negative effect on virus production, a function which required the integrity of the Nef N-terminal domain, but not its N-myristoyl group. The antagonistic effect of Nef correlated with a cellular redistribution of both EED and Nef. [Abstract/Link to Full Text]

Molle D, Maiuri P, Boireau S, Bertrand E, Knezevich A, Marcello A, Basyuk E
A real-time view of the TAR:Tat:P-TEFb complex at HIV-1 transcription sites.
Retrovirology. 2007;436.
HIV-1 transcription is tightly regulated: silent in long-term latency and highly active in acutely-infected cells. Transcription is activated by the viral protein Tat, which recruits the elongation factor P-TEFb by binding the TAR sequence present in nascent HIV-1 RNAs. In this study, we analyzed the dynamic of the TAR:Tat:P-TEFb complex in living cells, by performing FRAP experiments at HIV-1 transcription sites. Our results indicate that a large fraction of Tat present at these sites is recruited by Cyclin T1. We found that in the presence of Tat, Cdk9 remained bound to nascent HIV-1 RNAs for 71s. In contrast, when transcription was activated by PMA/ionomycin, in the absence of Tat, Cdk9 turned-over rapidly and resided on the HIV-1 promoter for only 11s. Thus, the mechanism of trans-activation determines the residency time of P-TEFb at the HIV-1 gene, possibly explaining why Tat is such a potent transcriptional activator. In addition, we observed that Tat occupied HIV-1 transcription sites for 55s, suggesting that the TAR:Tat:P-TEFb complex dissociates from the polymerase following transcription initiation, and undergoes subsequent cycles of association/dissociation. [Abstract/Link to Full Text]

Merling R, Chen C, Hong S, Zhang L, Liu M, Kuo YL, Giam CZ
HTLV-1 Tax mutants that do not induce G1 arrest are disabled in activating the anaphase promoting complex.
Retrovirology. 2007;435.
HTLV-1 Tax is a potent activator of viral transcription and NF-kappaB. Recent data indicate that Tax activates the anaphase promoting complex/cyclosome (APC/C) ahead of schedule, causing premature degradation of cyclin A, cyclin B1, securin, and Skp2. Premature loss of these mitotic regulators is accompanied by mitotic aberrations and leads to rapid senescence and cell cycle arrest in HeLa and S. cerevisiae cells. Tax-induced rapid senescence (tax-IRS) of HeLa cells is mediated primarily by a dramatic stabilization of p27KIP and is also accompanied by a great surge in the level of p21CIP1mRNA and protein. Deficiencies in p27KIP prevent Tax-IRS. A collection of tax point mutants that permit normal growth of S. cerevisiae have been isolated. Like wild-type tax, many of them (C23W, A108T, L159F, and L235F) transactivate both the HTLV-LTR and the NF-kappaB reporters. One of them, V19M, preferentially activates NF-kappaB, but is attenuated for LTR activation. None of the mutants significantly elevated the levels of p21CIP1and p27KIP1, indicating that the dramatic surge in p21CIP1/WAF1and p27KIP 1induced by Tax is brought about by a mechanism distinct from NF-kappaB or LTR activation. Importantly, the ability of these mutants to activate APC/C is attenuated or abrogated. These data indicate that Tax-induced rapid senescence is causally associated with APC/C activation. [Abstract/Link to Full Text]

Buonaguro L, Tagliamonte M, Tornesello ML, Buonaguro FM
Genetic and phylogenetic evolution of HIV-1 in a low subtype heterogeneity epidemic: the Italian example.
Retrovirology. 2007;434.
The Human Immunodeficiency Virus type 1 (HIV-1) is classified into genetic groups, subtypes and sub-subtypes which show a specific geographic distribution pattern. The HIV-1 epidemic in Italy, as in most of the Western Countries, has traditionally affected the Intra-venous drug user (IDU) and Homosexual (Homo) risk groups and has been sustained by the genetic B subtype. In the last years, however, the HIV-1 transmission rate among heterosexuals has dramatically increased, becoming the prevalent transmission route. In fact, while the traditional risk groups have high levels of knowledge and avoid high-risk practices, the heterosexuals do not sufficiently perceive the risk of HIV-1 infection. This misperception, linked to the growing number of immigrants from non-Western Countries, where non-B clades and circulating recombinant forms (CRFs) are prevalent, is progressively introducing HIV-1 variants of non-B subtype in the Italian epidemic. This is in agreement with reports from other Western European Countries.In this context, the Italian HIV-1 epidemic is still characterized by low subtype heterogeneity and represents a paradigmatic example of the European situation. The continuous molecular evolution of the B subtype HIV-1 isolates, characteristic of a long-lasting epidemic, together with the introduction of new subtypes as well as recombinant forms may have significant implications for diagnostic, treatment, and vaccine development. The study and monitoring of the genetic evolution of the HIV-1 represent, therefore, an essential strategy for controlling the local as well as global HIV-1 epidemic and for developing efficient preventive and therapeutic strategies. [Abstract/Link to Full Text]

Chen H, Xu X, Jones IM
Immunogenicity of the outer domain of a HIV-1 clade C gp120.
Retrovirology. 2007;433.
BACKGROUND: The possibility that a sub domain of a C clade HIV-1 gp120 could act as an effective immunogen was investigated. To do this, the outer domain (OD) of gp120CN54 was expressed and characterized in a construct marked by a re-introduced conformational epitope for MAb 2G12. The expressed sequence showed efficient epitope retention on the isolated ODCN54 suggesting authentic folding. To facilitate purification and subsequent immunogenicity ODCN54 was fused to the Fc domain of human IgG1. Mice were immunised with the resulting fusion proteins and also with gp120CN54-Fc and gp120 alone. RESULTS: Fusion to Fc was found to stimulate antibody titre and Fc tagged ODCN54 was substantially more immunogenic than non-tagged gp120. Immunogenicity appeared the result of Fc facilitated antigen processing as immunisation with an Fc domain mutant that reduced binding to the FcR lead to a reduction in antibody titre when compared to the parental sequence. The breadth of the antibody response was assessed by serum reaction with five overlapping fragments of gp120CN54 expressed as GST fusion proteins in bacteria. A predominant anti-inner domain and anti-V3C3 response was observed following immunisation with gp120CN54-Fc and an anti-V3C3 response to the ODCN54-Fc fusion. CONCLUSION: The outer domain of gp120CN54 is correctly folded following expression as a C terminal fusion protein. Immunogenicity is substantial when targeted to antigen presenting cells but shows V3 dominance in the polyvalent response. The gp120 outer domain has potential as a candidate vaccine component. [Abstract/Link to Full Text]

Mosca JD, Chang YN, Williams G
Antigen-presenting particle technology using inactivated surface-engineered viruses: induction of immune responses against infectious agents.
Retrovirology. 2007;432.
BACKGROUND: Developments in cell-based and gene-based therapies are emerging as highly promising areas to complement pharmaceuticals, but present day approaches are too cumbersome and thereby limit their clinical usefulness. These shortcomings result in procedures that are too complex and too costly for large-scale applications. To overcome these shortcomings, we described a protein delivery system that incorporates over-expressed proteins into viral particles that are non-infectious and stable at room temperature. The system relies on the biological process of viral egress to incorporate cellular surface proteins while exiting their host cells during lytic and non-lytic infections. RESULTS: We report here the use of non-infectious surface-engineered virion particles to modulate immunity against three infectious disease agents--human immunodeficiency virus type 1 (HIV-1), herpes simplex virus (HSV), and Influenza. Surface-engineering of particles are accomplished by genetic modification of the host cell surface that produces the egress budding viral particle. Human peripheral blood lymphocytes from healthy donors exposed to CD80/B7.1, CD86/B7.2, and/or antiCD3 single-chain antibody surface-engineered non-infectious HIV-1 and HSV-2 particles stimulate T cell proliferation, whereas particles released from non-modified host cells have no T cell stimulatory activity. In addition to T cell proliferation, HIV-based particles specifically suppress HIV-1 replication (both monocytotropic and lymphocytotropic strains) 55 to 96% and HSV-based particles specifically induce cross-reactive HSV-1/HSV-2 anti-herpes virus antibody production. Similar surface engineering of influenza-based particles did not modify the intrinsic ability of influenza particles to stimulate T cell proliferation, but did bestow on the engineered particles the ability to induce cross-strain anti-influenza antibody production. CONCLUSION: We propose that non-infectious viral particles can be surface-engineered to produce antigen-presenting particles that mimic antigen-presenting cells to induce immune responses in human peripheral blood lymphocytes. The viral particles behave as "biological carriers" for recombinant proteins, thereby establishing a new therapeutic paradigm for molecular medicine. [Abstract/Link to Full Text]

Kinet S, Swainson L, Lavanya M, Mongellaz C, Montel-Hagen A, Craveiro M, Manel N, Battini JL, Sitbon M, Taylor N
Isolated receptor binding domains of HTLV-1 and HTLV-2 envelopes bind Glut-1 on activated CD4+ and CD8+ T cells.
Retrovirology. 2007;431.
BACKGROUND: We previously identified the glucose transporter Glut-1, a member of the multimembrane-spanning facilitative nutrient transporter family, as a receptor for both HTLV-1 and HTLV-2. However, a recent report concluded that Glut-1 cannot serve as a receptor for HTLV-1 on CD4 T cells: This was based mainly on their inability to detect Glut-1 on this lymphocyte subset using the commercial antibody mAb1418. It was therefore of significant interest to thoroughly assess Glut-1 expression on CD4 and CD8 T cells, and its association with HTLV-1 and -2 envelope binding. RESULTS: As previously reported, ectopic expression of Glut-1 but not Glut-3 resulted in significantly augmented binding of tagged proteins harboring the receptor binding domains of either HTLV-1 or HTLV-2 envelope glycoproteins (H1RBD or H2RBD). Using antibodies raised against the carboxy-terminal peptide of Glut-1, we found that Glut-1 expression was significantly increased in both CD4 and CD8 cells following TCR stimulation. Corresponding increases in the binding of H1RBD as well as H2RBD, not detected on quiescent T cells, were observed following TCR engagement. Furthermore, increased Glut-1 expression was accompanied by a massive augmentation in glucose uptake in TCR-stimulated CD4 and CD8 lymphocytes. Finally, we determined that the apparent contradictory results obtained by Takenouchi et al were due to their monitoring of Glut-1 with a mAb that does not bind cells expressing endogenous Glut-1, including human erythrocytes that harbor 300,000 copies per cell. CONCLUSION: Transfection of Glut-1 directly correlates with the capacities of HTLV-1 and HTLV-2 envelope-derived ligands to bind cells. Moreover, Glut-1 is induced by TCR engagement, resulting in massive increases in glucose uptake and binding of HTLV-1 and -2 envelopes to both CD4 and CD8 T lymphocytes. Therefore, Glut-1 is a primary binding receptor for HTLV-1 and HTLV-2 envelopes on activated CD4 as well as CD8 lymphocytes. [Abstract/Link to Full Text]

Houzet L, Morichaud Z, Mougel M
Fully-spliced HIV-1 RNAs are reverse transcribed with similar efficiencies as the genomic RNA in virions and cells, but more efficiently in AZT-treated cells.
Retrovirology. 2007;430.
We have shown previously that HIV actively and selectively packages the spliced HIV RNAs into progeny virions. In the present study, by using a RT-QPCR and QPCR strategies, we show that spliced viral RNAs are present in infectious particles and consequently participate, along with the unspliced genomic RNA, to some of the early steps of infection such as the reverse transcription step. This work provides the first quantitative data on reverse transcription of the fully spliced viral RNAs, also called the early transcripts, in target cells but also inside virions. The latter results were obtained by measuring the natural endogenous reverse transcription activity directly on intact HIV-1 particles. Our study reveals that spliced HIV RNAs are reverse transcribed as efficiently as the genomic RNA, both in cells and virions. Interestingly, we also show that reverse transcription of spliced RNAs is 56-fold less sensitive to the inhibitor AZT than reverse transcription of the genomic RNA. Therefore, the selection mediated by inhibitors of reverse transcription used to treat patients could lead to increased representativeness of spliced forms of HIV, thus favoring recombination between the HIV DNA species and facilitating HIV recovery. [Abstract/Link to Full Text]

Jakobsen MR, Haasnoot J, Wengel J, Berkhout B, Kjems J
Efficient inhibition of HIV-1 expression by LNA modified antisense oligonucleotides and DNAzymes targeted to functionally selected binding sites.
Retrovirology. 2007;429.
BACKGROUND: A primary concern when targeting HIV-1 RNA by means of antisense related technologies is the accessibility of the targets. Using a library selection approach to define the most accessible sites for 20-mer oligonucleotides annealing within the highly structured 5'-UTR of the HIV-1 genome we have shown that there are at least four optimal targets available. RESULTS: The biological effect of antisense DNA and LNA oligonucleotides, DNA- and LNAzymes targeted to the four most accessible sites was tested for their abilities to block reverse transcription and dimerization of the HIV-1 RNA template in vitro, and to suppress HIV-1 production in cell culture. The neutralization of HIV-1 expression declined in the following order: antisense LNA > LNAzymes > DNAzymes and antisense DNA. The LNA modifications strongly enhanced the in vivo inhibitory activity of all the antisense constructs and some of the DNAzymes. Notably, two of the LNA modified antisense oligonucleotides inhibited HIV-1 production in cell culture very efficiently at concentration as low as 4 nM. CONCLUSION: LNAs targeted to experimentally selected binding sites can function as very potent inhibitors of HIV-1 expression in cell culture and may potentially be developed as antiviral drug in patients. [Abstract/Link to Full Text]


Recent Articles in Virology Journal

Dicarlo A, Moller P, Lander A, Kolesnikova L, Becker S
Nucleocapsid formation and RNA synthesis of Marburg virus is dependent on two coiled coil motifs in the nucleoprotein.
Virol J. 2007 Oct 24;4(1):105.
ABSTRACT: The nucleoprotein (NP) of Marburg virus (MARV) is responsible for the encapsidation of viral genomic RNA and the formation of the helical nucleocapsid precursors that accumulate in intracellular inclusions in infected cells. To form the large helical MARV nucleocapsid, NP needs to interact with itself and the viral proteins VP30, VP35 and L, which are also part of the MARV nucleocapsid. In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor. Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system. Transfer of the coiled coil motif to a reporter protein was sufficient to mediate interaction of the constructed fusion protein with the N-terminus of NP. The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker. [Abstract/Link to Full Text]

Elsheikh RM, Daak AA, Elsheikh MA, Karsany MS, Adam I
Hepatitis B virus and hepatitis C virus in pregnant Sudanese women.
Virol J. 2007;4104.
ABSTRACT: BACKGROUND: The epidemiology of viral hepatitis during pregnancy is essential for health planners and programme managers. While much data exist concerning viral hepatitis during pregnancy in many African countries, no proper published data are available in Sudan. AIM: The study aimed to investigate the sero-prevalance and the possible risk factors for hepatitis B virus (HBV) and hepatitis C virus (HCV) among antenatal care attendants in central Sudan. METHODS: During 3 months from March-June 2006, sera were collected from pregnant women at Umdurman Maternity Hospital in Sudan, and they were tested for markers of hepatitis B virus (HBVsAg) and HCV. RESULTS: HBVsAg was detected in 41 (5.6%) out 728 women, Anti-HCV was detected in 3 (0.6%) out of 423 women, all of them were not aware of their condition. Age, parity, gestational age, residence, history of blood transfusion, dental manipulations, tattooing and circumcision did not contribute significantly to increased HBVsAg sero-positivity. CONCLUSION: Thus 5.6% of pregnant women were positive for HBVsAg irrespective of their age, parity and socio-demographic characteristics. There was low prevalence of Anti-HCV. [Abstract/Link to Full Text]

Han T, Tang Y, Ugai H, Perry LE, Siegal GP, Contreras JL, Wu H
Genetic incorporation of the protein transduction domain of Tat into Ad5 fiber enhances gene transfer efficacy.
Virol J. 2007 Oct 24;4(1):103.
ABSTRACT: BACKGROUND: Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector for a variety of diseases. Many target cells, however, express low levels of Ad5 native receptor, the Coxsackie-Adenovirus Receptor (CAR), and thus are resistant to Ad5 infection. The Protein Transduction Domain of the HIV Tat protein, namely PTDtat, has been shown to mediate protein transduction in a wide range of cells. We hypothesize that re-targeting Ad5 vector via the PTDtat motif would improve the efficacy of Ad5-mediated gene delivery. RESULTS: In this study, we genetically incorporated the PTDtat motif into the knob domain of Ad5 fiber, and rescued the resultant viral vector, Ad5.PTDtat. Our data showed the modification did not interfere with Ad5 binding to its native receptor CAR, suggesting Ad5 infection via the CAR pathway is retained. In addition, we found that Ad5.PTDtat exhibited enhanced gene transfer efficacy in all of the cell lines that we have tested, which included both low-CAR and high-CAR decorated cells. Competitive inhibition assays suggested the enhanced infectivity of Ad5.PTDtat was mediated by binding of the positively charged PTDtat peptide to the negatively charged epitopes on the cells' surface. Furthermore, we investigated in vivo gene delivery efficacy of Ad5.PTDtat using subcutaneous tumor models established with U118MG glioma cells, and found that Ad5.PTDtat exhibited enhanced gene transfer efficacy compared to unmodified Ad5 vector as analyzed by a non-invasive fluorescence imaging technique. CONCLUSION: Genetic incorporation of the PTDtat motif into Ad5 fiber allowed Ad5 vectors to infect cells via an alternative PTDtat targeting motif while retaining the native CAR-mediated infection pathway. The enhanced infectivity was demonstrated in both cultured cells and in in vivo tumor models. Taken together, our study identifies a novel tropism expanded Ad5 vector that may be useful for clinical gene therapy applications. [Abstract/Link to Full Text]

Wang Z, Duke GM
Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells.
Virol J. 2007 Oct 23;4(1):102.
ABSTRACT: BACKGROUND: Recent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin-Darby canine kidney (MDCK) cells are the primary cell-substrate candidate for influenza virus production but an efficient system for the direct rescue of influenza virus from cloned influenza cDNAs in MDCK cells did not exist. The objective of this study was to develop a highly efficient method for direct rescue of influenza virus in MDCK cells. RESULTS: The eight-plasmid DNA transfection system for the rescue of influenza virus from cloned influenza cDNAs was adapted such that virus can be generated directly from MDCK cells. This was accomplished by cloning the canine RNA polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA pol I promoter in the eight plasmid rescue system. The adapted system retains bi-directional transcription of the viral cDNA template into both RNA pol I transcribed negative-sense viral RNA and RNA pol II transcribed positive-sense viral mRNA. The utility of this system was demonstrated by rescue in MDCK cells of 6:2 genetic reassortants composed of the six internal gene segments (PB1, PB2, PA, NP, M and NS) from either the cold-adapted (ca) influenza A vaccine strain (ca A/Ann Arbor/1/60) or the ca influenza B vaccine strain (ca B/Ann Arbor/1/66) and HA and NA gene segments from wild type influenza A and B strains. Representative 6:2 reassortants were generated for influenza A (H1N1, H3N2, H5N1, H6N1, H7N3 and H9N2) and for both the Victoria and Yamagata lineages of influenza B. The yield of infectious virus in the supernatant of transfected MDCK cells was 10E6 to 10E7 plaque forming units per ml by 5 to 7 days post-transfection. CONCLUSIONS: This rescue system will enable efficient production of both influenza A and influenza B vaccines exclusively in MDCK cells and therefore provides a tool for influenza pandemic preparedness. [Abstract/Link to Full Text]

Hammarstedt M, Ahlqvist J, Jacobson S, Garoff H, Fogdell-Hahn A
Purification of infectious human herpesvirus 6A virions and association of host cell proteins.
Virol J. 2007 Oct 19;4(1):101.
ABSTRACT: BACKGROUND: Viruses that are incorporating host cell proteins might trigger autoimmune diseases. It is therefore of interest to identify possible host proteins associated with viruses, especially for enveloped viruses that have been suggested to play a role in autoimmune diseases, like human herpesvirus 6A (HHV-6A) in multiple sclerosis (MS). RESULTS: We have established a method for rapid and morphology preserving purification of HHV-6A virions, which in combination with parallel analyses with background control material released from mock-infected cells facilitates qualitative and quantitative investigations of the protein content of HHV-6A virions. In our iodixanol gradient purified preparation, we detected high levels of viral DNA by real-time PCR and viral proteins by metabolic labelling, silver staining and western blots. In contrast, the background level of cellular contamination was low in the purified samples as demonstrated by the silver staining and metabolic labelling analyses. Western blot analyses showed that the cellular complement protein CD46, which is the receptor for HHV-6A is associated with the purified and infectious virions. Also, cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions. CONCLUSIONS: Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated. [Abstract/Link to Full Text]

Costin JM
Cytopathic Mechanisms of HIV-1.
Virol J. 2007 Oct 18;4(1):100.
ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) has been intensely investigated since its discovery in 1983 as the cause of acquired immune deficiency syndrome (AIDS). With relatively few proteins made by the virus, it is able to accomplish many tasks, with each protein serving multiple functions. The Envelope glycoprotein, composed of the two noncovalently linked subunits, SU (surface glycoprotein) and TM (transmembrane glycoprotein) is largely responsible for host cell recognition and entry respectively. While the roles of the N-terminal residues of TM is well established as a fusion pore and anchor for Env into cell membranes, the role of the C-terminus of the protein is not well understood and is fiercely debated. This review gathers information on TM in an attempt to shed some light on the functional regions of this protein. [Abstract/Link to Full Text]

Coura Rdos S, Nardi NB
The state of the art of adeno-associated virus-based vectors in gene therapy.
Virol J. 2007;499.
ABSTRACT: The adeno-associated virus (AAV) has rapidly gained popularity in gene therapy since the establishment of the first AAV2 infectious clone, in 1982, due to some of their distinguishing characteristics such as lack of pathogenicity, wide range of infectivity, and ability to establish long-term transgene expression. Notably over the past decade, this virus has attracted considerable interest as a gene therapy vector, and about 85% of the currently available 2,041 PubMed references on adeno-associated viruses have been published during this time. The exponential progress of AAV-based vectors has been made possible by the advances in the knowledge of the virology and biology of this virus, which allows great improvement in AAV vectors construction and a better comprehension of their operation. Moreover, with the recent discovery of novel AAV serotypes, there is virtually one preferred serotype for nearly every organ or tissue to target. Thus, AAV-based vectors have been successfully overcoming the main gene therapy challenges such as transgene maintenance, safety and host immune response, and meeting the desirable vector system features of high level of safety combined with clinical efficacy and versatility in terms of potential applications. Consequently, AAV is increasingly becoming the vector of choice for a wide range of gene therapy approaches. This report will highlight the state of the art of AAV-based vectors studies and the advances on the use of AAV vectors for several gene therapy approaches. [Abstract/Link to Full Text]

Shen Q, Zhang W, Cao X, Mou J, Cui L, Hua X
Cloning of full genome sequence of hepatitis E virus of Shanghai swine isolate using RACE method.
Virol J. 2007 Oct 9;4(1):98.
ABSTRACT: Genotype 4 hepatitis E virus (HEV) was reportedly transmitted freely between humans and swine in eastern China. The full-length genomic sequence of Shanghai swine isolate (SH-SW-zs1) recovered from feces sample of a pig at commercial swine farm in Shanghai suburb was determined using RT-PCR and RACE (Rapid Amplification of cDNA Ends) methods. The full genome of the SH-SW-zs1 isolate was 7265 nucleotides in length and phylogenetic analysis indicated that this isolate belonged to genotype 4. Comparison of the 3' UTR sequence with the corresponding regions of other 38 HEV strains from different region revealed that the Shanghai swine isolate is 21-49bp longer than the other stains. [Abstract/Link to Full Text]

Hardies SC, Thomas JA, Serwer P
Comparative genomics of Bacillus thuringiensis phage 0305phi8-36: defining patterns of descent in a novel ancient phage lineage.
Virol J. 2007 Oct 5;4(1):97.
ABSTRACT: BACKGROUND: The recently sequenced 218 kb genome of morphologically atypical Bacillus thuringiensis phage 0305phi8-36 exhibited only limited detectable homology to known bacteriophages. The only known relative of this phage is a string of phage-like genes called BtI1 in the chromosome of B. thuringiensis israelensis. The high degree of divergence and novelty of phage genomes pose challenges in how to describe the phage from its genomic sequences. RESULTS: Phage 0305phi8-36 and BtI1 are estimated to have diverged 2.0 - 2.5 billion years ago. Positionally biased Blast searches aligned 30 homologous sturcture or morphogenesis genes between 0305phi8-36 and BtI1 that have maintained the same gene order. Functional clustering of the genes helped identify additional gene functions. A conserved long tape measure gene indicates that a long tail is an evolutionarily stable property of this phage lineage. An unusual form of the tail chaperonin system split to two genes was characterized, as was a hyperplastic homologue of the T4gp27 hub gene. Within this region some segments were best described as encoding a conservative array of structure domains fused with a variable component of exchangeable domains. Other segments were best described as multigene units engaged in modular horizontal exchange. The non-structure genes of 0305phi8-36 appear to include the remnants of two replicative systems leading to the hypothesis that the genome plan was created by fusion of two ancestral viruses. The case for a member of the RNAi RNA-directed RNA polymerase family residing in 0305phi8-36 was strengthened by extending the hidden Markov model of this family. Finally, it was noted that prospective transcriptional promoters were distributed in a gradient of small to large transcripts starting from a fixed end of the genome. CONCLUSIONS: Genomic organization at a level higher than individual gene sequence comparison can be analyzed to aid in understanding large phage genomes. Methods of analysis include 1) applying a time scale, 2) augmenting blast scores with positional information, 3) categorizing genomic rearrangments into one of several processes with characteristic rates and outcomes, and 4) correlating apparent transcript sizes with genomic position, gene content, and promoter motifs. [Abstract/Link to Full Text]

McIntosh MT, Behan SC, Mohamed FM, Lu Z, Moran KE, Burrage TG, Neilan JG, Ward GB, Botti G, Capucci L, Metwally SA
A pandemic strain of calicivirus threatens rabbit industries in the Americas.
Virol J. 2007 Oct 2;4(1):96.
ABSTRACT: Rabbit Hemorrhagic Disease (RHD) is a severe acute viral disease specifically affecting the European rabbit Oryctolagus cuniculus. As the European rabbit is the predominant species of domestic rabbit throughout the world, RHD contributes towards significant losses to rabbit farming industries and endangers wild populations of rabbits in Europe and other predatory animals in Europe that depend upon rabbits as a food source. Rabbit Hemorrhagic Disease virus (RHDV) - a Lagovirus belonging to the family Caliciviridae is the etiological agent of RHD. Typically, RHD presents with sudden death in 70 to 95 percent of infected animals. There have been four separate incursions of RHDV in the USA, the most recent of which occurred in the state of Indiana in June of 2005. Animal inoculation studies confirmed the pathogenicity of the Indiana 2005 isolate, which caused acute death and pathological changes characterized by acute diffuse severe liver necrosis and pulmonary hemorrhages. Complete viral genome sequences of all USA outbreak isolates were determined and comparative genomics revealed that each outbreak was the result of a separate introduction of virus rather than from a single virus lineage. All of the USA isolates clustered with RHDV genomes from China, and phylogenetic analysis of the major capsid protein (VP60) revealed that they were related to a pandemic antigenic variant strain known as RHDVa. Rapid spread of the RHDVa pandemic suggests a selective advantage for this new subtype. Given its rapid spread, pathogenic nature, and potential to further evolve, possibly broadening its host range to include other genera native to the Americas, RHDVa should be regarded as a threat. [Abstract/Link to Full Text]

Taiwo MA, Kareem KT, Nsa IY, Hughes JD
Cowpea viruses: Effect of single and mixed infections on symptomatology and virus concentration.
Virol J. 2007 Sep 27;4(1):95.
ABSTRACT: Natural multiple viral infections of cultivated cowpeas have been reported in Nigeria. In this study, three Nigerian commercial cowpea cultivars ("Olo 11", "Oloyin" and "White") and two lines from the IITA (IT86D- 719 and TVU 76) were mechanically inoculated with Cowpea aphid-borne mosaic virus (CABMV), Bean southern mosaic virus (SBMV) and Cowpea mottle virus (CMeV) singly, as well as in all possible combinations at 10, 20 and 30 days after planting (DAP). Samples of leaves or stems were collected at 10, 20 and 30 days after inoculation (DAI) and analyzed for relative virus concentration by Enzyme-Linked Immunosrbent Assay. All the cultivars and lines {CVS/L} were susceptible to the viruses but the commercial CVS showed more severe symptoms and had relatively higher viral concentration. In single virus infections, CABMV which induced the most severe symptoms had absorbance values (at 405nm) of 0.11 to 0.46 while SBMV and CMeV which induced moderate symptoms had virus titre of 0.74 to 1.99 and 0.11 to 0.90 respectively. Plants inoculated 10 DAP had significantly higher virus concentration than those inoculated 30 DAP. In mixed infections involving CABMV (10 DAP) apical necrosis and death were observed in commercial cultivars "Olo 11" and "White". Enhancement of CMeV titers were observed in plants infected with CMeV + CABMV. Multiple viral infections of cowpeas may result in complete yield loss, hence, the availability of seeds of cultivars with a high level of multiple virus resistance is recommended as a means of control. [Abstract/Link to Full Text]

Pignolet B, Duteyrat JL, Allemandou A, Gelfi J, Foucras G, Bertagnoli S
In vitro permissivity of bovine cells for wild-type and vaccinal myxoma virus strains.
Virol J. 2007;494.
Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe, non-replicative vaccine vector for non-host species. However, there is very little information concerning infection of non-laboratory animals species cells with MYXV. In this study, we investigated interactions between bovine cells and respectively a wild type strain (T1) and a vaccinal strain (SG33) of MYXV. We showed that bovine KOP-R, BT and MDBK cell lines do not support MYXV production. Electron microscopy observations of BT-infected cells revealed the low efficiency of viral entry and the production of defective virions. In addition, infection of bovine peripheral blood mononuclear cells (PBMC) occurred at a very low level, even following non-specific activation, and was always abortive. We did not observe significant differences between the wild type strain and the vaccinal strain of MYXV, indicating that SG33 could be used for new bovine vaccination strategies. [Abstract/Link to Full Text]

Lian M, Zhou X, Wei L, Qiu S, Zhou T, Li L, Gu X, Luo M, Zheng X
Serum levels of preS antigen (HBpreSAg) in chronic hepatitis B virus infected patients.
Virol J. 2007;493.
BACKGROUND: Hepatitis B virus (HBV) infection is a serious health problem worldwide. Treatment recommendation and response are mainly indicated by viral load, e antigen (HBeAg) seroconversion, and ALT levels. The S antigen (HBsAg) seroconversion is much less frequent. Since HBeAg can be negative in the presence of high viral replication, preS antigen (HBpreSAg) might be a useful indicator in management of chronic HBV infection. RESULTS: A new assay of double antibody sandwich ELISA was established to detect preS antigens. Sera of 104 HBeAg-negative and 50 HBeAg-positive chronic hepatitis B patients have been studied and 23 HBeAg-positive patients were enrolled in a treatment follow-up study. 70% of the HBeAg-positive patients and 47% of the HBeAg-negative patients showed HBpreSAg positive. Particularly, in the HBeAg-negative patients, 30 out of 47 HBpreSAg positive patients showed no evidence of viral replication based on HBV DNA copies. A comparison with HBV DNA copies demonstrated that the overall accuracy of the HBpreSAg test could reach 72% for active HBV replication. HBpreSAg changes were well correlated with changes of HBsAg, HBV DNA and ALT levels during the course of IFN-alpha treatment and follow-up. HBeAg positive patients responded well to treatment when reduction of HBpreSAg levels was more pronounced. CONCLUSION: Our results suggested that HBpreSAg could be detected effectively, and well correlated with HBsAg and HBV DNA copies. The reduction of HBpreSAg levels in conjunction with the HBV DNA copies appears to be an improved predictor of treatment outcome. [Abstract/Link to Full Text]

Zhang Q, Wu G, Richards E, Jia S, Zeng C
Universal primers for HBV genome DNA amplification across subtypes: a case study for designing more effective viral primers.
Virol J. 2007;492.
BACKGROUND: The highly heterogenic characteristic of viruses is the major obstacle to efficient DNA amplification. Taking advantage of the large number of virus DNA sequences in public databases to select conserved sites for primer design is an optimal way to tackle the difficulties in virus genome amplification. RESULTS: Here we use hepatitis B virus as an example to introduce a simple and efficient way for virus primer design. Based on the alignment of HBV sequences in public databases and a program BxB in Perl script, our method selected several optimal sites for HBV primer design. Polymerase chain reaction showed that compared with the success rate of the most popular primers for whole genome amplification of HBV, one set of primers for full length genome amplification and four sets of walking primers showed significant improvement. These newly designed primers are suitable for most subtypes of HBV. CONCLUSION: Researchers can extend the method described here to design universal or subtype specific primers for various types of viruses. The BxB program based on multiple sequence alignment not only can be used as a separate tool but also can be integrated in any open source primer design software to select conserved regions for primer design. [Abstract/Link to Full Text]

Umareddy I, Pluquet O, Wang QY, Vasudevan SG, Chevet E, Gu F
Dengue virus serotype infection specifies the activation of the unfolded protein response.
Virol J. 2007;491.
BACKGROUND: Dengue and Dengue hemorrhagic fever have emerged as some of the most important mosquito-borne viral diseases in the tropics. The mechanisms of pathogenesis of Dengue remain elusive. Recently, virus-induced apoptosis mediated by the Unfolded Protein Response (UPR) has been hypothesised to represent a crucial pathogenic event in viral infection. In an attempt to evaluate the contribution of the UPR to virus replication, we have characterized each component of this signalling pathway following Dengue virus infection. RESULTS: We find that upon Dengue virus infection, A549 cells elicit an UPR which is observed at the level of translation attenuation (as visualized by the phosphorylation of eIF2alpha) and activation of specific pathways such as nuclear translocation of ATF-6 and splicing of XBP-1. Interestingly, we find that specific serotype of virus modulate the UPR with different selectivity. In addition, we demonstrate that perturbation of the UPR by preventing the dephosphorylation of the translation initiation factor eIF2alpha using Salubrinal considerably alters virus infectivity. CONCLUSION: This report provides evidence that Dengue infection induces and regulates the three branches of the UPR signaling cascades. This is a basis for our understanding of the viral regulation and conditions beneficial to the viral infection. Furthermore, modulators of UPR such as Salubrinal that inhibit Dengue replication may open up an avenue toward cell-protective agents that target the endoplasmic reticulum for anti-viral therapy. [Abstract/Link to Full Text]

Chironna M, Prato R, Sallustio A, Martinelli D, Germinario C, Lopalco P, Quarto M
Genetic characterization of measles virus strains isolated during an epidemic cluster in Puglia, Italy 2006-2007.
Virol J. 2007;490.
The genetic characterization of wild-type measles strains isolated during an epidemic cluster of measles occurred in Puglia (South Italy), between November 2006 and January 2007, was performed. Measles virus (MV) detection was carried out by a nested RT-PCR on 8 of 18 total cases. The viruses were analyzed using the standard genotyping protocols. The N gene sequences of the strains from outbreak were identical to each other, and sequence analysis revealed that the viruses belonged to genotype B3, subgroup B3.1, never identified before in Italy. An importation of measles B3.1 strains from Africa was hypothesized. Molecular surveillance will help to monitor the progress in measles elimination. [Abstract/Link to Full Text]

Hazari S, Taylor L, Haque S, Garry RF, Florman S, Luftig R, Regenstein F, Dash S
Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon.
Virol J. 2007;489.
ABSTRACT: BACKGROUND: Alpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines. RESULTS: HCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b). Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein. CONCLUSION: This in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones. [Abstract/Link to Full Text]

Luz-Madrigal A, Clapp C, Aranda J, Vaca L
In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter.
Virol J. 2007;488.
BACKGROUND: Endothelial cells are a target for gene therapy because they are implicated in a number of vascular diseases. Recombinant baculovirus have emerged as novel gene delivery vectors. However, there is no information available concerning the use of endothelial-specific promoters in the context of the baculovirus genome. In the present study, we have generated a recombinant baculovirus containing the human flt-1 promoter (BacFLT-GFP) driving the expression of the green fluorescent protein. Transcriptional gene targeting was analyzed in vitro in different mammalian cell lines and in vivo in adult rat retinal vasculature. RESULTS: BacFLT-GFP evoked the highest levels of expression in the endothelial cell line BUVEC-E6E7-1, similar to those reached by recombinant baculovirus carrying the CMV promoter (112% relative to BacCMV-GFP, n = 4). Interestingly, BacFLT-GFP directed high levels of expression in rat glioma C6 and in human glioblastoma CH235 cells (34.78% and 47.86% relative to BacCMV-GFP, respectively). Histone deacetylase inhibitors such as butyrate or trichostatin A enhanced the transcriptional activity of both BacCMV-GFP and BacFLT-GFP. Thus, in this study histone deacetylation appears to be a central mechanism for the silencing of baculovirus, independently of the promoter utilized. In vivo transcriptional targeting was demonstrated in adult rat retinal vasculature by intravitreal delivery of BacFLT-GFP and immunohistochemical staining with von Willebrand factor (vWF). Analysis by fluorescence microscopy and deconvolved three-dimensional confocal microscopy of retinal whole mounts obtained after 3 days of baculovirus injection showed that most GFP-expressing cells localized to the inner limiting membrane (ILM) and ganglion cell layer (GCL) and colocalize with vWF (70%, n = 10) in blood vessels, confirming the endothelial phenotype of the transduced cells. CONCLUSION: Taken together, our results indicate that the restricted expression in endothelial cells mediated by the flt-1 promoter is not affected by the context of the baculovirus genome and demonstrate the potential of using recombinant baculovirus for transcriptional targeted gene expression into the eye vasculature. [Abstract/Link to Full Text]

Shelby BD, LaMarca HL, McFerrin HE, Nelson AB, Lasky JA, Sun G, Myatt L, Offermann MK, Morris CA, Sullivan DE
Kaposi's sarcoma associated herpesvirus G-protein coupled receptor activation of cyclooxygenase-2 in vascular endothelial cells.
Virol J. 2007;487.
BACKGROUND: Kaposi's sarcoma associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), a highly vascularized neoplasm characterized by endothelial-derived spindle-shaped tumor cells. KSHV-infected microvascular endothelial cells demonstrate increased cyclooxygenase-2 (COX-2) expression and KS lesions have high levels of prostaglandin E2 (PGE2), a short-lived eicosanoid dependent on cyclooxygenase activity that has been linked to pathogenesis of other neoplasias. To determine whether increased COX-2 expression and PGE2 production is mediated by the angiogenic and tumorigenic KSHV-encoded G-protein coupled receptor (vGPCR), we developed a recombinant retrovirus to express vGPCR in Human Umbilical Vascular Endothelial Cells (HUVEC). RESULTS: In the present study, we show that vGPCR-expressing HUVEC exhibit a spindle-like morphology that is characteristic of KS endothelial cells and demonstrate selective induction of PGE2 and COX-2. By treating vGPCR-expressing HUVEC with selective and non-selective COX inhibitors, we show that vGPCR-induced PGE2 production is dependent on the expression of COX-2 but not COX-1. CONCLUSION: Taken together, these results demonstrate that vGPCR induces expression of COX-2 and PGE2 that may mediate the paracrine effects of this key viral protein in KS pathogenesis. [Abstract/Link to Full Text]

Menton JF, Kearney K, Morgan JG
Development of a real-time RT-PCR and Reverse Line probe Hybridisation assay for the routine detection and genotyping of Noroviruses in Ireland.
Virol J. 2007;486.
BACKGROUND: Noroviruses are the most common cause of non-bacterial gastroenteritis. Improved detection methods have seen a large increase in the number of human NoV genotypes in the last ten years. The objective of this study was to develop a fast method to detect, quantify and genotype positive NoV samples from Irish hospitals. RESULTS: A real-time RT-PCR assay and a Reverse Line Blot Hybridisation assay were developed based on the ORF1-ORF2 region. The sensitivity and reactivity of the two assays used was validated using a reference stool panel containing 14 NoV genotypes. The assays were then used to investigate two outbreaks of gastroenteritis in two Irish hospitals. 56 samples were screened for NoV using a real-time RT-PCR assay and 26 samples were found to be positive. Genotyping of these positive samples found that all positives belonged to the GII/4 variant of NoV. CONCLUSION: The combination of the Real-time assay and the reverse line blot hybridisation assay provided a fast and accurate method to investigate a NoV associated outbreak. It was concluded that the predominant genotype circulating in these Irish hospitals was GII/4 which has been associated with the majority of NoV outbreaks worldwide. The assays developed in this study are useful tools for investigating NoV infection. [Abstract/Link to Full Text]

Akl H, Badran B, Dobirta G, Manfouo-Foutsop G, Moschitta M, Merimi M, Burny A, Martiat P, Willard-Gallo KE
Progressive loss of CD3 expression after HTLV-I infection results from chromatin remodeling affecting all the CD3 genes and persists despite early viral genes silencing.
Virol J. 2007;485.
BACKGROUND: HTLV-I infected CD4+ T-cells lines usually progress towards a CD3- or CD3low phenotype. In this paper, we studied expression, kinetics, chromatin remodeling of the CD3 gene at different time-points post HTLV-I infection. RESULTS: The onset of this phenomenon coincided with a decrease of CD3gamma followed by the subsequent progressive reduction in CD3delta, then CD3epsilon and CD3zeta mRNA. Transient transfection experiments showed that the CD3gamma promoter was still active in CD3- HTLV-I infected cells demonstrating that adequate amounts of the required transcription factors were available. We next looked at whether epigenetic mechanisms could be responsible for this progressive decrease in CD3 expression using DNase I hypersensitivity (DHS) experiments examining the CD3gamma and CD3delta promoters and the CD3delta enhancer. In uninfected and cells immediately post-infection all three DHS sites were open, then the CD3gamma promoter became non accessible, and this was followed by a sequential closure of all the DHS sites corresponding to all three transcriptional control regions. Furthermore, a continuous decrease of in vivo bound transcription initiation factors to the CD3gamma promoter was observed after silencing of the viral genome. Coincidently, cells with a lower expression of CD3 grew more rapidly. CONCLUSION: We conclude that HTLV-I infection initiates a process leading to a complete loss of CD3 membrane expression by an epigenetic mechanism which continues along time, despite an early silencing of the viral genome. Whether CD3 progressive loss is an epiphenomenon or a causal event in the process of eventual malignant transformation remains to be investigated. [Abstract/Link to Full Text]

Prepens S, Kreuzer KA, Leendertz F, Nitsche A, Ehlers B
Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach.
Virol J. 2007;484.
Targeting the highly conserved herpes DNA polymerase (DPOL) gene with PCR using panherpes degenerate primers is a powerful tool to universally detect unknown herpesviruses. However, vertebrate hosts are often infected with more than one herpesvirus in the same tissue, and pan-herpes DPOL PCR often favors the amplification of one viral sequence at the expense of the others. Here we present two different technical approaches that overcome this obstacle: (i) Pan-herpes DPOL PCR is carried out in the presence of an oligonucleotide substituted with locked nucleic acids (LNA).This suppresses the amplification of a specific herpesvirus DPOL sequence by a factor of approximately 1000, thereby enabling the amplification of a second, different DPOL sequence. (ii) The less conserved glycoprotein B (gB) gene is targeted with several sets of degenerate primers that are restricted to gB genes of different herpesvirus subfamilies or genera. These techniques enable the amplification of gB and DPOL sequences of multiple viruses from a single specimen. The partial gB and DPOL sequences can be connected by long-distance PCR, producing final contiguous sequences of approximately 3.5 kbp. Such sequences include parts of two genes and therefore allow for a robust phylogenetic analysis. To illustrate this principle, six novel herpesviruses of the genera Rhadinovirus, Lymphocryptovirus and Cytomegalovirus were discovered in multi-infected samples of non-human primates and phylogenetically characterized. [Abstract/Link to Full Text]

Ren J, Ding T, Zhang W, Song J, Ma W
Does Japanese encephalitis virus share the same cellular receptor with other mosquito-borne flaviviruses on the C6/36 mosquito cells?
Virol J. 2007;483.
ABSTRACT: Japanese encephalitis virus (JEV) is a member of mosquito-borne Flaviviridae. To date, the mechanisms of the early events of JEV infection remain poorly understood, and the cellular receptors are unidentified. There are evidences that the structure of the virus attachment proteins (VAP), envelope glycoprotein of mosquito-borne flaviviruses is very similar, and the vector-virus interaction of mosquito-borne flaviviruses is also very similar. Based on the studies previously demonstrated that the similar molecules present on the mosquito cells involved in the uptake process of JEV, West Nile virus (WNV) and Dengue virus (DV), it is proposed that the same receptor molecules for mosquito-borne flaviviruses (JEV, WNV and DV) may present on the surface of C6/36 mosquito cells. By co-immunoprecipitation assay, we investigated a 74-KDa protein on the C6/36 cells binds JEV, and the mass spectrometry results indicated it may be heat shock cognate protein 70(HSC70) from Aedes aegypti. Based upon some other viruses use of heat shock protein 70 (HSP70) family proteins as cell receptors, its possible HSC70's involvement in the fusion of the JEV E protein with the C6/36 cells membrane, and known form of cation channels in the interaction of HSC70 with the lipid bilayer, it will further be proposed that HSC70 as a penetration receptor mediates JEV entry into C6/36 cells. [Abstract/Link to Full Text]

Hammamieh R, Barmada M, Ludwig G, Peel S, Koterski N, Jett M
Blood genomic profiles of exposures to Venezuelan equine encephalitis in Cynomolgus macaques (Macaca fascicularis).
Virol J. 2007;482.
BACKGROUND: Lymphocytes provide invaluable whistle blowers of changes due to infections. We use the information registered by these cells using their mRNAs as they encounter the pathogen to develop patterns of expression that correspond to that specific pathogen.Venezuelan equine encephalitis (VEE) is a mosquito-borne viral disease characterized by fever and one or more of the following: severe headache, back pain, myalgias, prostration, chills, nausea, vomiting, weakness and other flu-like symptoms.Screening for host mRNA obtained from blood samples after exposure to VEEV may provide the means for early detection of surrogate markers of the impending illness and provide appropriate strategies for treatment. RESULTS: We have been carrying out gene expression analysis of PBMC exposed to VEEV to extract signatures and diagnostic markers of early exposure to be used in non invasive blood analysis methods.In this study, we used high throughput gene expression analysis to identify markers of early and late exposures to VEEV in vivo in Cynomolgus macaques (Macaca fascicularis). We carried out cDNA microarrays and real time PCR on blood samples obtained from the NHP model resulting in a panel of host genes that are altered in response to VEEV. CONCLUSION: Screening for host mRNA obtained from blood samples after exposure to VEEV may provide the means for early detection of surrogate markers of the impending illness and provide appropriate strategies for treatment. [Abstract/Link to Full Text]

Hussain A, Das SR, Tanwar C, Jameel S
Oligomerization of the human immunodeficiency virus type 1 (HIV-1) Vpu protein--a genetic, biochemical and biophysical analysis.
Virol J. 2007;481.
BACKGROUND: The human immunodeficiency virus type 1(HIV-1) is a complex retrovirus and the causative agent of acquired immunodeficiency syndrome (AIDS). The HIV-1 Vpu protein is an oligomeric integral membrane protein essential for particle release, viral load and CD4 degradation. In silico models show Vpu to form pentamers with an ion channel activity. RESULTS: Using Vpu proteins from a primary subtype C and the pNL4-3 subtype B isolates of HIV-1, we show oligomerization of the full-length protein as well as its transmembrane (TM) domain by genetic, biochemical and biophysical methods. We also provide direct evidence of the presence of Vpu pentamers in a stable equilibrium with its monomers in vitro. This was also true for the TM domain of Vpu. Confocal microscopy localized Vpu to the endoplasmic reticulum and Golgi regions of the cell, as well as to post-Golgi vesicles. In fluorescence resonance energy transfer (FRET) experiments in live cells we show that Vpu oligomerizes in what appears to be either the Golgi region or intracellular vesicles, but not in the ER. CONCLUSION: We provide here direct evidence that the TM domain, is critical for Vpu oligomerization and the most favourable channel assembly is a pentamer. The Vpu oligomerization appears to be either the Golgi region or intracellular vesicles, but not in the ER. [Abstract/Link to Full Text]

Yadav PD, Vincent MJ, Nichol ST
Thottapalayam virus is genetically distant to the rodent-borne hantaviruses, consistent with its isolation from the Asian house shrew (Suncus murinus).
Virol J. 2007;480.
Thottapalayam (TPM) virus belongs to the genus Hantavirus, family Bunyaviridae. The genomes of hantaviruses consist of three negative-stranded RNA segments (S, M and L) encoding the virus nucleocapsid (N), glycoprotein (Gn, Gc), and polymerase (L) proteins, respectively. The genus Hantavirus contains predominantly rodent-borne viruses, with the prominent exception of TPM virus which was isolated in India in 1964 from an insectivore, Suncus murinus, commonly referred to as the Asian house shrew or brown musk shrew. Analysis of the available TPM virus S (1530 nt) RNA genome segment sequence and the newly derived M (3621 nt) and L (6581 nt) segment sequences demonstrate that the entire TPM virus genome is very unique. Remarkably high sequence differences are seen at the nucleotide (up to S - 47%, M - 49%, L - 38%) and protein (up to N - 54%, Gn/Gc - 57% and L - 39%) levels relative to the rodent-borne hantaviruses, consistent with TPM virus having a unique host association. [Abstract/Link to Full Text]

Moratorio G, Martínez M, Gutiérrez MF, González K, Colina R, López-Tort F, López L, Recarey R, Schijman AG, Moreno MP, García-Aguirre L, Manascero AR, Cristina J
Evolution of naturally occurring 5'non-coding region variants of Hepatitis C virus in human populations of the South American region.
Virol J. 2007;479.
BACKGROUND: Hepatitis C virus (HCV) has been the subject of intense research and clinical investigation as its major role in human disease has emerged. Previous and recent studies have suggested a diversification of type 1 HCV in the South American region. The degree of genetic variation among HCV strains circulating in Bolivia and Colombia is currently unknown. In order to get insight into these matters, we performed a phylogenetic analysis of HCV 5' non-coding region (5'NCR) sequences from strains isolated in Bolivia, Colombia and Uruguay, as well as available comparable sequences of HCV strains isolated in South America. METHODS: Phylogenetic tree analysis was performed using the neighbor-joining method under a matrix of genetic distances established under the Kimura-two parameter model. Signature pattern analysis, which identifies particular sites in nucleic acid alignments of variable sequences that are distinctly representative relative to a background set, was performed using the method of Korber & Myers, as implemented in the VESPA program. Prediction of RNA secondary structures was done by the method of Zuker & Turner, as implemented in the mfold program. RESULTS: Phylogenetic tree analysis of HCV strains isolated in the South American region revealed the presence of a distinct genetic lineage inside genotype 1. Signature pattern analysis revealed that the presence of this lineage is consistent with the presence of a sequence signature in the 5'NCR of HCV strains isolated in South America. Comparisons of these results with the ones found for Europe or North America revealed that this sequence signature is characteristic of the South American region. CONCLUSION: Phylogentic analysis revealed the presence of a sequence signature in the 5'NCR of type 1 HCV strains isolated in South America. This signature is frequent enough in type 1 HCV populations circulating South America to be detected in a phylogenetic tree analysis as a distinct type 1 sub-population. The coexistence of distinct type 1 HCV subpopulations is consistent with quasispecies dynamics, and suggests that multiple coexisting subpopulations may allow the virus to adapt to its human host populations. [Abstract/Link to Full Text]

Yang SJ
Characterization of vaccinia virus A12L protein proteolysis and its participation in virus assembly.
Virol J. 2007;478.
Vaccinia virus (VV) undergoes a proteolytic processing to evolve from immature virus particles into intracellular mature virus particles. Most of structural core protein precursors such as p4a, p4b, and p25K are assembled into previrions and then proteolytically processed to yield core proteins, 4a, 4b, and 25 K, which become components of a mature virus particle. These structural rearrangements take place at a conserved cleavage motif, Ala-Gly-X (where X is any amino acid) and catalyzed by a VV encoded proteinase, the I7L gene product. The VV A12L gene product, a 25 kDa protein synthesized at late times during infection is cleaved at an N-terminal AG/A site, resulting in a 17 kDa cleavage product. However, due to the distinct characteristics of A12L proteolysis such as the localization of both the A12L full-length protein and its cleavage product in mature virions and two putative cleavage sites (Ala-Gly-Lys) located at internal and C-terminal region of A12L ORF, it was of interest to examine the A12L proteolysis for better understanding of regulation and function of VV proteolysis. Here, we attempted to examine the in vivo A12L processing by: determining the kinetics of the A12L proteolysis, the responsible viral protease, and the function of the A12L protein and its cleavage events. Surprisingly, the A12L precursor was cleaved into multiple peptides not only at an N-terminal AG/A but also at both an N- and a C-terminus. Despite the involvement of I7L proteinase for A12L proteolysis, its incomplete processing with slow kinetics and additional cleavages not at the two AG/K sites demonstrate unique regulation of VV proteolysis. An immunoprecipitation experiment in concert with N-terminal sequencing analyses and mass spectrometry led to the identification of VV core and membrane proteins, which may be associated with the A12L protein and suggested possible involvement of A12L protein and its cleavage products in multiple stages in virus morphogenesis. [Abstract/Link to Full Text]

Evseenko VA, Bukin EK, Zaykovskaya AV, Sharshov KA, Ternovoi VA, Ignatyev GM, Shestopalov AM
Experimental infection of H5N1 HPAI in BALB/c mice.
Virol J. 2007;477.
BACKGROUND: In 2005 huge epizooty of H5N1 HPAI occurred in Russia. It had been clear that territory of Russia becoming endemic for H5N1 HPAI. In 2006 several outbreaks have occurred. To develop new vaccines and antiviral therapies, animal models had to be investigated. We choose highly pathogenic strain for these studies. RESULTS: A/duck/Tuva/01/06 belongs to Quinghai-like group viruses. Molecular markers-cleavage site, K627 in PB2 characterize this virus as highly pathogenic. This data was confirmed by direct pathogenic tests: IVPI = 3.0, MLD50 = 1,4Log10EID50. Also molecular analysis showed sensitivity of the virus to adamantanes and neuraminidase inhibitors. Serological analysis showed wide cross-reactivity of this virus with sera produced to H5N1 HPAI viruses isolated earlier in South-East Asia. Mean time to death of infected animals was 8,19+/-0,18 days. First time acute delayed hemorrhagic syndrome was observed in mice lethal model. Hypercytokinemia was determined by elevated sera levels of IFN-gamma, IL-6, IL-10. CONCLUSION: Assuming all obtained data we can conclude that basic model parameters were characterized and virus A/duck/Tuva/01/06 can be used to evaluate anti-influenza vaccines and therapeutics. [Abstract/Link to Full Text]

Butler-Cole C, Wagner MJ, Da Silva M, Brown GD, Burke RD, Upton C
An ectromelia virus profilin homolog interacts with cellular tropomyosin and viral A-type inclusion protein.
Virol J. 2007;476.
BACKGROUND: Profilins are critical to cytoskeletal dynamics in eukaryotes; however, little is known about their viral counterparts. In this study, a poxviral profilin homolog, ectromelia virus strain Moscow gene 141 (ECTV-PH), was investigated by a variety of experimental and bioinformatics techniques to characterize its interactions with cellular and viral proteins. RESULTS: Profilin-like proteins are encoded by all orthopoxviruses sequenced to date, and share over 90% amino acid (aa) identity. Sequence comparisons show highest similarity to mammalian type 1 profilins; however, a conserved 3 aa deletion in mammalian type 3 and poxviral profilins suggests that these homologs may be more closely related. Structural analysis shows that ECTV-PH can be successfully modelled onto both the profilin 1 crystal structure and profilin 3 homology model, though few of the surface residues thought to be required for binding actin, poly(L-proline), and PIP2 are conserved. Immunoprecipitation and mass spectrometry identified two proteins that interact with ECTV-PH within infected cells: alpha-tropomyosin, a 38 kDa cellular actin-binding protein, and the 84 kDa product of vaccinia virus strain Western Reserve (VACV-WR) 148, which is the truncated VACV counterpart of the orthopoxvirus A-type inclusion (ATI) protein. Western and far-western blots demonstrated that the interaction with alpha-tropomyosin is direct, and immunofluorescence experiments suggest that ECTV-PH and alpha-tropomyosin may colocalize to structures that resemble actin tails and cellular protrusions. Sequence comparisons of the poxviral ATI proteins show that although full-length orthologs are only present in cowpox and ectromelia viruses, an ~ 700 aa truncated ATI protein is conserved in over 90% of sequenced orthopoxviruses. Immunofluorescence studies indicate that ECTV-PH localizes to cytoplasmic inclusion bodies formed by both truncated and full-length versions of the viral ATI protein. Furthermore, colocalization of ECTV-PH and truncated ATI protein to protrusions from the cell surface was observed. CONCLUSION: These results suggest a role for ECTV-PH in intracellular transport of viral proteins or intercellular spread of the virus. Broader implications include better understanding of the virus-host relationship and mechanisms by which cells organize and control the actin cytoskeleton. [Abstract/Link to Full Text]


Recent Articles in Microbial Cell Factories

Tsantili IC, Karim MN, Klapa MI
Quantifying the metabolic capabilities of engineered Zymomonas mobilis using linear programming analysis.
Microb Cell Fact. 2007;68.
BACKGROUND: The need for discovery of alternative, renewable, environmentally friendly energy sources and the development of cost-efficient, "clean" methods for their conversion into higher fuels becomes imperative. Ethanol, whose significance as fuel has dramatically increased in the last decade, can be produced from hexoses and pentoses through microbial fermentation. Importantly, plant biomass, if appropriately and effectively decomposed, is a potential inexpensive and highly renewable source of the hexose and pentose mixture. Recently, the engineered (to also catabolize pentoses) anaerobic bacterium Zymomonas mobilis has been widely discussed among the most promising microorganisms for the microbial production of ethanol fuel. However, Z. mobilis genome having been fully sequenced in 2005, there is still a small number of published studies of its in vivo physiology and limited use of the metabolic engineering experimental and computational toolboxes to understand its metabolic pathway interconnectivity and regulation towards the optimization of its hexose and pentose fermentation into ethanol. RESULTS: In this paper, we reconstructed the metabolic network of the engineered Z. mobilis to a level that it could be modelled using the metabolic engineering methodologies. We then used linear programming (LP) analysis and identified the Z. mobilis metabolic boundaries with respect to various biological objectives, these boundaries being determined only by Z. mobilis network's stoichiometric connectivity. This study revealed the essential for bacterial growth reactions and elucidated the association between the metabolic pathways, especially regarding main product and byproduct formation. More specifically, the study indicated that ethanol and biomass production depend directly on anaerobic respiration stoichiometry and activity. Thus, enhanced understanding and improved means for analyzing anaerobic respiration and redox potential in vivo are needed to yield further conclusions for potential genetic targets that may lead to optimized Z. mobilis strains. CONCLUSION: Applying LP to study the Z. mobilis physiology enabled the identification of the main factors influencing the accomplishment of certain biological objectives due to metabolic network connectivity only. This first-level metabolic analysis model forms the basis for the incorporation of more complex regulatory mechanisms and the formation of more realistic models for the accurate simulation of the in vivo Z. mobilis physiology. [Abstract/Link to Full Text]

Galindo E, Peńa C, Núńez C, Segura D, Espín G
Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii.
Microb Cell Fact. 2007;67.
Several aspects of alginate and PHB synthesis in Azotobacter vinelandii at a molecular level have been elucidated in articles published during the last ten years. It is now clear that alginate and PHB synthesis are under a very complex genetic control. Genetic modification of A. vinelandii has produced a number of very interesting mutants which have particular traits for alginate production. One of these mutants has been shown to produce the alginate with the highest mean molecular mass so far reported. Recent work has also shed light on the factors determining molecular mass distribution; the most important of these being identified as; dissolved oxygen tension and specific growth rate. The use of specific mutants has been very useful for the correct analysis and interpretation of the factors affecting polymerization. Recent scale-up/down work on alginate production has shown that oxygen limitation is crucial for producing alginate of high molecular mass, a condition which is optimized in shake flasks and which can now be reproduced in stirred fermenters. It is clear that the phenotypes of mutants grown on plates are not necessarily reproducible when the strains are tested in lab or bench scale fermenters. In the case of PHB, A. vinelandii has shown itself able to produce relatively large amounts of this polymer of high molecular weight on cheap substrates, even allowing for simple extraction processes. The development of fermentation strategies has also shown promising results in terms of improving productivity. The understanding of the regulatory mechanisms involved in the control of PHB synthesis, and of its metabolic relationships, has increased considerably, making way for new potential strategies for the further improvement of PHB production. Overall, the use of a multidisciplinary approach, integrating molecular and bioengineering aspects is a necessity for optimizing alginate and PHB production in A. vinelandii. [Abstract/Link to Full Text]

Wittmann C
Fluxome analysis using GC-MS.
Microb Cell Fact. 2007;66.
Fluxome analysis aims at the quantitative analysis of in vivo carbon fluxes in metabolic networks, i. e. intracellular activities of enzymes and pathways. It allows investigating the effects of genetic or environmental modifications and thus precisely provides a global perspective on the integrated genetic and metabolic regulation within the intact metabolic network. The experimental and computational approaches developed in this area have revealed fascinating insights into metabolic properties of various biological systems. Most of the comprehensive approaches for metabolic flux studies today involve isotopic tracer studies and GC-MS for measurement of the labeling pattern of metabolites. Initially developed and applied mainly in the field of biomedicine these GC-MS based metabolic flux approaches have been substantially extended and optimized during recent years and today display a key technology in metabolic physiology and biotechnology. [Abstract/Link to Full Text]

Karhumaa K, Garcia Sanchez R, Hahn-Hägerdal B, Gorwa-Grauslund MF
Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae.
Microb Cell Fact. 2007;65.
BACKGROUND: Two heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i) the xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway and ii) the xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3). The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. RESULTS: In defined mineral medium, the xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. CONCLUSION: Despite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed. [Abstract/Link to Full Text]

Jaluria P, Konstantopoulos K, Betenbaugh M, Shiloach J
A perspective on microarrays: current applications, pitfalls, and potential uses.
Microb Cell Fact. 2007;64.
With advances in robotics, computational capabilities, and the fabrication of high quality glass slides coinciding with increased genomic information being available on public databases, microarray technology is increasingly being used in laboratories around the world. In fact, fields as varied as: toxicology, evolutionary biology, drug development and production, disease characterization, diagnostics development, cellular physiology and stress responses, and forensics have benefiting from its use. However, for many researchers not familiar with microarrays, current articles and reviews often address neither the fundamental principles behind the technology nor the proper designing of experiments. Although, microarray technology is relatively simple, conceptually, its practice does require careful planning and detailed understanding of the limitations inherently present. Without these considerations, it can be exceedingly difficult to ascertain valuable information from microarray data. Therefore, this text aims to outline key features in microarray technology, paying particular attention to current applications as outlined in recent publications, experimental design, statistical methods, and potential uses. Furthermore, this review is not meant to be comprehensive, but rather substantive; highlighting important concepts and detailing steps necessary to conduct and interpret microarray experiments. Collectively, the information included in this text will highlight the versatility of microarray technology and provide a glimpse of what the future may hold. [Abstract/Link to Full Text]

Al-Hasani K, Boyce J, McCarl VP, Bottomley S, Wilkie I, Adler B
Identification of novel immunogens in Pasteurella multocida.
Microb Cell Fact. 2007;63.
P. multocida is a Gram-negative pathogen responsible for causing diseases in animals of economic significance to livestock industries throughout the world. Current vaccines include bacterins, which provide only limited protection against homologous serotypes. Therefore there is a need for more effective vaccines to control diseases caused by P. multocida. As a step towards developing vaccines against fowl cholera, a genomics based approach was applied for the identification of novel immunogens. RESULTS: Bioinformatics analysis of the P. multocida genome predicted 129 proteins as secreted, located in the outer membrane, or lipoproteins. 105 of the genes encoding these proteins were cloned and recombinant protein expressed in Escherichia coli. Polyclonal serum from P. multocida-infected chickens reacted with a subset of these proteins. CONCLUSION: These data show the range of bacterial immunogens recognized by the chicken immune system, including 6 novel immunoreactive proteins. [Abstract/Link to Full Text]

Jordan E, Hust M, Roth A, Biedendieck R, Schirrmann T, Jahn D, Dübel S
Production of recombinant antibody fragments in Bacillus megaterium.
Microb Cell Fact. 2007;62.
BACKGROUND: Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments. RESULTS: The lysozyme specific single chain Fv (scFv) fragment D1.3 was successfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41 degrees C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 microg of recombinant His6-tagged antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli. CONCLUSION: High yields of functional scFv antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli. [Abstract/Link to Full Text]

Cardoso LS, Araujo MI, Góes AM, Pacífico LG, Oliveira RR, Oliveira SC
Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis.
Microb Cell Fact. 2007;61.
BACKGROUND: Recombinant proteins expressed in Escherichia coli vectors are generally contaminated with endotoxin. In this study, we evaluated the ability of Polymyxin B to neutralize the effect of LPS present as contaminant on Schistosoma mansoni recombinant proteins produced in E. coli in inducing TNF-alpha and IL-10. Peripheral blood mononuclear cells from individuals chronically infected with S. mansoni were stimulated in vitro with recombinant Sm22.6, Sm14 and P24 antigens (10 microg/mL) in the presence of Polymyxin B (10 microg/mL). RESULTS: The levels of cytokines were measured using ELISA. There was greater than 90% reduction (p < 0.05) in the levels of TNF-alpha and IL-10 when Polymyxin B was added to the cultures stimulated with LPS. In cultures stimulated with S. mansoni recombinant proteins in the presence of Polymyxin B, a reduction in the levels of TNF-alpha and IL-10 was also observed. However, the percentage of reduction was lower when compared to the cultures stimulated with LPS, probably because these proteins are able to induce the production of these cytokines by themselves. CONCLUSION: This study showed that Polymyxin B was able to neutralize the effect of endotoxin, as contaminant in S. mansoni recombinant antigens produced in E. coli, in inducing TNF-alpha and IL-10 production. [Abstract/Link to Full Text]

Rodríguez AP, Leiro RF, Trillo MC, Cerdán ME, Siso MI, Becerra M
Secretion and properties of a hybrid Kluyveromyces lactis-Aspergillus niger beta-galactosidase.
Microb Cell Fact. 2006;541.
BACKGROUND: The beta-galactosidase from Kluyveromyces lactis is a protein of outstanding biotechnological interest in the food industry and milk whey reutilization. However, due to its intracellular nature, its industrial production is limited by the high cost associated to extraction and downstream processing.The yeast-system is an attractive method for producing many heterologous proteins. The addition of a secretory signal in the recombinant protein is the method of choice to sort it out of the cell, although biotechnological success is not guaranteed. The cell wall acting as a molecular sieve to large molecules, culture conditions and structural determinants present in the protein, all have a decisive role in the overall process.Protein engineering, combining domains of related proteins, is an alternative to take into account when the task is difficult. In this work, we have constructed and analyzed two hybrid proteins from the beta-galactosidase of K. lactis, intracellular, and its Aspergillus niger homologue that is extracellular. In both, a heterologous signal peptide for secretion was also included at the N-terminus of the recombinant proteins. One of the hybrid proteins obtained has interesting properties for its biotechnological utilization. RESULTS: The highest levels of intracellular and extracellular beta-galactosidase were obtained when the segment corresponding to the five domain of K. lactis beta-galactosidase was replaced by the corresponding five domain of the A. niger beta-galactosidase. Taking into account that this replacement may affect other parameters related to the activity or the stability of the hybrid protein, a thoroughly study was performed. Both pH (6.5) and temperature (40 degrees C) for optimum activity differ from values obtained with the native proteins. The stability was higher than the corresponding to the beta-galactosidase of K. lactis and, unlike this, the activity of the hybrid protein was increased by the presence of Ni2+. The affinity for synthetic (ONPG) or natural (lactose) substrates was higher in the hybrid than in the native K. lactis beta-galactosidase. Finally, a structural-model of the hybrid protein was obtained by homology modelling and the experimentally determined properties of the protein were discussed in relation to it. CONCLUSION: A hybrid protein between K. lactis and A. niger beta-galactosidases was constructed that increases the yield of the protein released to the growth medium. Modifications introduced in the construction, besides to improve secretion, conferred to the protein biochemical characteristics of biotechnological interest. [Abstract/Link to Full Text]

Cusano AM, Parrilli E, Marino G, Tutino ML
A novel genetic system for recombinant protein secretion in the Antarctic Pseudoalteromonas haloplanktis TAC125.
Microb Cell Fact. 2006;540.
BACKGROUND: The final aim of recombinant protein production is both to have a high specific production rate and a high product quality. It was already shown that using cold-adapted bacteria as host vectors, some "intractable" proteins can be efficiently produced at temperature as low as 4 degrees C. RESULTS: A novel genetic system for the production and secretion of recombinant proteins in the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 was set up. This system aims at combining the low temperature recombinant product production with the advantages of extra-cellular protein targeting.The psychrophilic alpha-amylase from Pseudoalteromonas haloplanktis TAB23 was used as secretion carrier. Three chimerical proteins were produced by fusing intra-cellular proteins to C-terminus of the psychrophilic alpha-amylase and their secretion was analysed. Data reported in this paper demonstrate that all tested chimeras were translocated with a secretion yield always higher than 80%. CONCLUSION: Data presented here demonstrate that the "cold" gene-expression system is efficient since the secretion yield of tested chimeras is always above 80%. These secretion performances place the alpha-amylase derived secretion system amongst the best heterologous secretion systems in Gram-negative bacteria reported so far. As for the quality of the secreted passenger proteins, data presented suggest that the system also allows the correct disulphide bond formation of chimera components, secreting a fully active passenger. [Abstract/Link to Full Text]

Hartner FS, Glieder A
Regulation of methanol utilisation pathway genes in yeasts.
Microb Cell Fact. 2006;539.
Methylotrophic yeasts such as Candida boidinii, Hansenula polymorpha, Pichia methanolica and Pichia pastoris are an emerging group of eukaryotic hosts for recombinant protein production with an ever increasing number of applications during the last 30 years. Their applications are linked to the use of strong methanol-inducible promoters derived from genes of the methanol utilisation pathway. These promoters are tightly regulated, highly repressed in presence of non-limiting concentrations of glucose in the medium and strongly induced if methanol is used as carbon source. Several factors involved in this tight control and their regulatory effects have been described so far. This review summarises available data about the regulation of promoters from methanol utilisation pathway genes. Furthermore, the role of cis and trans acting factors (e.g. transcription factors, glucose processing enzymes) in the expression of methanol utilisation pathway genes is reviewed both in the context of the native cell environment as well as in heterologous hosts. [Abstract/Link to Full Text]

Kemmer C, Neubauer P
Antisense RNA based down-regulation of RNaseE in E. coli.
Microb Cell Fact. 2006;538.
BACKGROUND: Messenger RNA decay is an important mechanism for controlling gene expression in all organisms. The rate of the mRNA degradation directly affects the steady state concentration of mRNAs and therefore influences the protein synthesis. RNaseE has a key importance for the general mRNA decay in E. coli. While RNaseE initiates the degradation of most mRNAs in E. coli, it is likely that the enzyme is also responsible for the degradation of recombinant RNAs. As RNaseE is essential for cell viability and knockout mutants cannot be cultured, we investigated the possibility for a down-regulation of the intracellular level of RNaseE by antisense RNAs. During this study, an antisense RNA based approach could be established which revealed a strong reduction of the intracellular level of RNaseE in E. coli. RESULTS: Despite the autoregulation of rne mRNA by its gene product, significant antisense downregulation of RNaseE is possible. The expression of antisense RNAs did not effect the cell growth negatively. The amount of antisense RNA was monitored quantitatively by a fluorescence based sandwich hybridisation assay. Induction by anhydrotetracycline was followed by a 25-fold increase of the detectable antisense RNA molecules per cell. The antisense RNA level was maintained above 400 molecules per cell until the stationary phase, which caused the level of expressed antisense RNAs to decrease markedly. Western blot experiments revealed the strongest reduction in the RNaseE protein level 90 min after antisense RNA induction. The cellular level of RNaseE could be decreased to 35% of the wild type level. When the growth entered the stationary phase, the RNaseE level was maintained still at 50 to 60% of the wild type level. CONCLUSION: In difference to eukaryotic cells, where the RNAi technology is widely used, this technology is rather unexplored in bacteria, although different natural systems use antisense RNA-based silencing, and a few studies have earlier indicated the potential of this technology also in prokaryotes. Our results show that even complicated self-regulatory systems such as RNaseE may be controlled by antisense RNA technology, indicating that systems based on antisense RNA expression may have a potential for controlling detrimental factors with plasmid-based constructs in arbitrary strains while maintaining their beneficial characteristics. The study also proved that the RNA sandwich hybridisation technique is directly applicable to quantify small RNA molecules in crude cell extracts, which may have a broader application potential as a monitoring tool in RNA inhibition applications. [Abstract/Link to Full Text]

Maurer M, Kühleitner M, Gasser B, Mattanovich D
Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris.
Microb Cell Fact. 2006;537.
BACKGROUND: Secretion of heterol