Biomedical subjects

Brendan W Wren

Publications and source records attributed to Brendan W Wren.

At least 19 recordsLinked to original sources

PubMed2006

The complete genome sequence and comparative genome analysis of the high pathogenicity Yersinia enterocolitica strain 8081.

The human enteropathogen, Yersinia enterocolitica, is a significant link in the range of Yersinia pathologies extending from mild gastroenteritis to bubonic plague. Comparison at the genomic level is a key step in our understanding of the genetic basis for this pathogenicity spectrum. Here we report the genome of Y. enterocolitica strain 8081 (serotype 0:8; biotype 1B) and extensive microarray data relating to the genetic diversity of the Y. enterocolitica species. Our analysis reveals that the genome of Y. enterocolitica strain 8081 is a patchwork of horizontally acquired genetic loci, including a plasticity zone of 199 kb containing an extraordinarily high density of virulence genes. Microarray analysis has provided insights into species-specific Y. enterocolitica gene functions and the intraspecies differences between the high, low, and nonpathogenic Y. enterocolitica biotypes. Through comparative genome sequence analysis we provide new information on the evolution of the Yersinia. We identify numerous loci that represent ancestral clusters of genes potentially important in enteric survival and pathogenesis, which have been lost or are in the process of being lost, in the other sequenced Yersinia lineages. Our analysis also highlights large metabolic operons in Y. enterocolitica that are absent in the related enteropathogen, Yersinia pseudotuberculosis, indicating major differences in niche and nutrients used within the mammalian gut. These include clusters directing, the production of hydrogenases, tetrathionate respiration, cobalamin synthesis, and propanediol utilisation. Along with ancestral gene clusters, the genome of Y. enterocolitica has revealed species-specific and enteropathogen-specific loci. This has provided important insights into the pathology of this bacterium and, more broadly, into the evolution of the genus. Moreover, wider investigations looking at the patterns of gene loss and gain in the Yersinia have highlighted common themes in the genome evolution of other human enteropathogens.

Evolution, Molecular↗

PubMed2006

The identification of two protective DNA vaccines from a panel of five plasmid constructs encoding Brucella melitensis 16M genes.

Five candidate genes from the Brucella melitensis 16M genome were selected. Eukaryotic expression plasmids encoding these antigens were constructed and expression was verified in vitro from transfected Cos7 cells. Each vaccine was assessed for protective efficacy in a BALB/c mouse brucellosis infection model. From these experiments two protective DNA vaccines were identified: p-omp25 and p-ialB. The Omp25 antigen (BMEI1249) has previously been studied in terms of Brucella virulence, serodiagnosis and as a protective antigen. However, this study represents the first report of a significant protective effect achieved against B. melitensis 16M challenge using the Omp25 antigen in a DNA vaccine approach. The other protective vaccine identified in this study was p-ialB. The ialB candidate (BMEI1584) was selected based upon its' putative function as an invasion protein which was assigned due to shared identity with the invasion protein B (ialB) of Bartonella bacilliformis. This candidate has not previously been investigated with regard to Brucella virulence or pathogenesis. This study is the first report to identify the Brucella invasion protein B (BMEI1584) as a novel protective antigen for brucellosis.

Acid Anhydride Hydrolases↗

PubMed2006

The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome.

We determined the complete genome sequence of Clostridium difficile strain 630, a virulent and multidrug-resistant strain. Our analysis indicates that a large proportion (11%) of the genome consists of mobile genetic elements, mainly in the form of conjugative transposons. These mobile elements are putatively responsible for the acquisition by C. difficile of an extensive array of genes involved in antimicrobial resistance, virulence, host interaction and the production of surface structures. The metabolic capabilities encoded in the genome show multiple adaptations for survival and growth within the gut environment. The extreme genome variability was confirmed by whole-genome microarray analysis; it may reflect the organism's niche in the gut and should provide information on the evolution of virulence in this organism.

Adaptation, Physiological↗

PubMed2006

Numerical analysis of DNA microarray data of Campylobacter jejuni strains correlated with survival, cytolethal distending toxin and haemolysin analyses.

Molecular epidemiological studies of the enteric pathogen Campylobacter jejuni have suggested that not all animal isolates are equally pathogenic to humans. We examined the use of numerical analysis of whole-genomotype data as a potential tool for evaluating C. jejuni virulence potential. Whole-genome microarray analysis was used to determine the gene-level complementarity of 12 Danish strains to the pathogenic, genome-sequenced strain NCTC 11168. Cytolethal distending toxin (CDT) and haemolysin activities, and survival characteristics under aerobic conditions at room temperature were also determined. Among the strains examined, 439 genes were polymorphic. Numerical analysis of these data by use of the squared Euclidean distance coefficient and Ward's clustering method clearly delineated strains into two clusters. CDT and haemolysin activities of cluster 1 strains were not statistically significantly different from cluster 2 strains. However, viability during aerobic incubation of cluster 1 strains was statistically significantly lower than corresponding estimates of cluster 2 strains. The number of missing or highly divergent genes in cluster 1 strains with respect to NCTC 11168 was also statistically significantly greater compared with those of cluster 2 strains. Sixty-seven genes present in NCTC 11168 were characteristically missing or divergent among cluster 1 strains. Of these, 53 genes were localised within 11 major gene clusters, of which eight were associated with surface structures and included flagellar, lipo-oligosaccharide, and membrane transport proteins. Our data indicate a correlation between C. jejuni genomic content, particularly in surface-coding regions, and its capacity for environmental survival, and may help explain why certain serotypes are more commonly reported in human disease.

Animals↗

PubMed2006

Mucosal immunization with a urease B DNA vaccine induces innate and cellular immune responses against Helicobacter pylori.

BACKGROUND: Helicobacter pylori is recognized as a major risk factor for recurrent gastroduodenal inflammatory diseases and gastric adenocarcinoma. The high prevalence of H. pylori infection worldwide, the risks of side-effects from antibiotic therapy, and increasing resistance to antibiotics are the main primers for the development of improved H. pylori vaccines. The antigenic potential of its urease enzyme, a critical virulence factor required for colonization of the gastric mucosa, has been demonstrated in animal and human studies. An important but controversial issue in H. pylori vaccine studies is the type of immune response required to control infection. A new approach in H. pylori vaccinology is the administration of DNA vaccines, which has included heat-shock protein and catalase DNA vaccines. MATERIALS AND METHODS: The H. pylori urease subunit B construct or vector alone was administered to mice via the intranasal route. Spleens and stomachs were examined on day 0 and weeks 3, 6, and 12 after immunization. Proliferation of spleen cells was assessed using the carboxyfluorescein diacetate succinimidyl ester-based flow cytometry assay and cytokine secretion from cultured spleen cells was detected by ELISA, after stimulation with the urease subunit B recombinant antigen. Total RNA was isolated from stomach and spleen tissue and the expression of beta-defensin and cytokine genes was monitored by reverse transcription followed by polymerase chain reaction (RT-PCR). Immunized mice were challenged with H. pylori and bacterial DNA quantified by TaqMan PCR. RESULTS: The urease B subunit DNA vaccine increased INF-gamma secretion and splenocyte proliferation without inducing adverse effects in the spleen. Increase in gastric beta-defensin 1 and marked induction in local IL-10 : IFN-gamma ratio up to 12 weeks post-immunization suggest a potential role for local innate immune responses in protection at the site of infection. Although significant bacterial reduction in the stomachs of urease B subunit DNA-immunized mice was observed, intermediate reduction was also noted in the vector group. Increased defensin expression and adjuvant effects of the cytosine preceding guanosine motifs may contribute to this phenomenon. Our data confirm that cytosine preceding guanosine motifs, even without coadministration with antigen, can reduce extracellular bacterial load. CONCLUSIONS: In this study, a DNA construct encoding the urease B subunit was assessed for its immune profile and its ability to reduce bacterial colonization in the murine stomach. Our studies suggest that local innate immune responses may play a greater role than previously supposed in limiting H. pylori colonization in the gastric mucosa.

Animals↗

PubMed2006

Application of comparative phylogenomics to study the evolution of Yersinia enterocolitica and to identify genetic differences relating to pathogenicity.

Yersinia enterocolitica, an important cause of human gastroenteritis generally caused by the consumption of livestock, has traditionally been categorized into three groups with respect to pathogenicity, i.e., nonpathogenic (biotype 1A), low pathogenicity (biotypes 2 to 5), and highly pathogenic (biotype 1B). However, genetic differences that explain variation in pathogenesis and whether different biotypes are associated with specific nonhuman hosts are largely unknown. In this study, we applied comparative phylogenomics (whole-genome comparisons of microbes with DNA microarrays combined with Bayesian phylogenies) to investigate a diverse collection of 94 strains of Y. enterocolitica consisting of 35 human, 35 pig, 15 sheep, and 9 cattle isolates from nonpathogenic, low-pathogenicity, and highly pathogenic biotypes. Analysis confirmed three distinct statistically supported clusters composed of a nonpathogenic clade, a low-pathogenicity clade, and a highly pathogenic clade. Genetic differences revealed 125 predicted coding sequences (CDSs) present in all highly pathogenic strains but absent from the other clades. These included several previously uncharacterized CDSs that may encode novel virulence determinants including a hemolysin, a metalloprotease, and a type III secretion effector protein. Additionally, 27 CDSs were identified which were present in all 47 low-pathogenicity strains and Y. enterocolitica 8081 but absent from all nonpathogenic 1A isolates. Analysis of the core gene set for Y. enterocolitica revealed that 20.8% of the genes were shared by all of the strains, confirming this species as highly heterogeneous, adding to the case for the existence of three subspecies of Y. enterocolitica. Further analysis revealed that Y. enterocolitica does not cluster according to source (host).

Animals↗

PubMed2006

Serotype differences and lack of biofilm formation characterize Yersinia pseudotuberculosis infection of the Xenopsylla cheopis flea vector of Yersinia pestis.

Yersinia pestis, the agent of plague, is usually transmitted by fleas. To produce a transmissible infection, Y. pestis colonizes the flea midgut and forms a biofilm in the proventricular valve, which blocks normal blood feeding. The enteropathogen Yersinia pseudotuberculosis, from which Y. pestis recently evolved, is not transmitted by fleas. However, both Y. pestis and Y. pseudotuberculosis form biofilms that adhere to the external mouthparts and block feeding of Caenorhabditis elegans nematodes, which has been proposed as a model of Y. pestis-flea interactions. We compared the ability of Y. pestis and Y. pseudotuberculosis to infect the rat flea Xenopsylla cheopis and to produce biofilms in the flea and in vitro. Five of 18 Y. pseudotuberculosis strains, encompassing seven serotypes, including all three serotype O3 strains tested, were unable to stably colonize the flea midgut. The other strains persisted in the flea midgut for 4 weeks but did not increase in numbers, and none of the 18 strains colonized the proventriculus or produced a biofilm in the flea. Y. pseudotuberculosis strains also varied greatly in their ability to produce biofilms in vitro, but there was no correlation between biofilm phenotype in vitro or on the surface of C. elegans and the ability to colonize or block fleas. Our results support a model in which a genetic change in the Y. pseudotuberculosis progenitor of Y. pestis extended its pre-existing ex vivo biofilm-forming ability to the flea gut environment, thus enabling proventricular blockage and efficient flea-borne transmission.

Animals↗

PubMed2005

Comparative phylogenomics of the food-borne pathogen Campylobacter jejuni reveals genetic markers predictive of infection source.

Campylobacter jejuni is the predominant cause of bacterial gastroenteritis worldwide, but traditional typing methods are unable to discriminate strains from different sources that cause disease in humans. We report the use of genomotyping (whole-genome comparisons of microbes using DNA microarrays) combined with Bayesian-based algorithms to model the phylogeny of this major food-borne pathogen. In this study 111 C. jejuni strains were examined by genomotyping isolates from humans with a spectrum of C. jejuni-associated disease (70 strains), chickens (17 strains), bovines (13 strains), ovines (5 strains), and the environment (6 strains). From these data, the Bayesian phylogeny of the isolates revealed two distinct clades unequivocally supported by Bayesian probabilities (P = 1); a livestock clade comprising 31/35 (88.6%) of the livestock isolates and a "nonlivestock" clade comprising further clades of environmental isolates. Several genes were identified as characteristic of strains in the livestock clade. The most prominent was a cluster of six genes (cj1321 to cj1326) within the flagellin glycosylation locus, which were confirmed by PCR analysis as genetic markers in six additional chicken-associated strains. Surprisingly these studies show that the majority (39/70, 55.7%) of C. jejuni human isolates were found in the nonlivestock clade, suggesting that most C. jejuni infections may be from nonlivestock (and possibly nonagricultural) sources. This study has provided insight into a previously unidentified reservoir of C. jejuni infection that may have implications in disease-control strategies. The comparative phylogenomics approach described provides a robust methodological prototype that should be applicable to other microbes.

Animals↗

PubMed2005

The complete genome sequence of Francisella tularensis, the causative agent of tularemia.

Francisella tularensis is one of the most infectious human pathogens known. In the past, both the former Soviet Union and the US had programs to develop weapons containing the bacterium. We report the complete genome sequence of a highly virulent isolate of F. tularensis (1,892,819 bp). The sequence uncovers previously uncharacterized genes encoding type IV pili, a surface polysaccharide and iron-acquisition systems. Several virulence-associated genes were located in a putative pathogenicity island, which was duplicated in the genome. More than 10% of the putative coding sequences contained insertion-deletion or substitution mutations and seemed to be deteriorating. The genome is rich in IS elements, including IS630 Tc-1 mariner family transposons, which are not expected in a prokaryote. We used a computational method for predicting metabolic pathways and found an unexpectedly high proportion of disrupted pathways, explaining the fastidious nutritional requirements of the bacterium. The loss of biosynthetic pathways indicates that F. tularensis is an obligate host-dependent bacterium in its natural life cycle. Our results have implications for our understanding of how highly virulent human pathogens evolve and will expedite strategies to combat them.

Base Sequence↗

PubMed2005

The Campylobacter jejuni glycome.

Microbial cell surface glycans in the form of glycolipids and glycoproteins frequently play important roles in cell-cell interaction and host immune responses. Given the likely importance of these surface structures in the survival and pathogenesis of Campylobacter jejuni, a concerted effort has been made to characterise these determinants genetically and structurally since the genome was sequenced in 2000. We review the considerable progress made in characterising the Campylobacter glycome including the lipooligosaccharide (LOS), the capsule and O- and N-linked protein glycosylation systems, and speculate on the roles played by glycan surface structures in the life-cycle of C. jejuni.

Animals↗

PubMed2005

Comparative phylogenomics of pathogenic bacteria by microarray analysis.

DNA microarrays represent a powerful technology that enables whole-scale comparison of bacterial genomes. This, coupled with new methods to model DNA microarray data, is facilitating the development of robust comparative phylogenomics analyses. Such studies have dramatically increased our ability to differentiate between bacteria, highlighting previously undetected genetic differences and population structures and providing new insight into virulence and evolution of bacterial pathogens. Recent results from such studies have generated insights into the evolution of bacterial pathogens, the levels of diversity and plasticity in the genome of a species, as well as the differences in virulence amongst pathogenic bacteria.

Bacteria↗

PubMed2005

Protein glycosylation in bacterial mucosal pathogens.

In eukaryotes, glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces. Their oligosaccharide moieties are implicated in a wide range of cell-cell and cell-matrix recognition events that are required for biological processes ranging from immune recognition to cancer development. Glycosylation was previously considered to be restricted to eukaryotes; however, through advances in analytical methods and genome sequencing, there have been increasing reports of both O-linked and N-linked protein glycosylation pathways in bacteria, particularly amongst mucosal-associated pathogens. Studying glycosylation in relatively less-complicated bacterial systems provides the opportunity to elucidate and exploit glycoprotein biosynthetic pathways. We will review the genetic organization, glycan structures and function of glycosylation systems in mucosal bacterial pathogens, and speculate on how this knowledge may help us to understand glycosylation processes in more complex eukaryotic systems and how it can be used for glycoengineering.

Bacteria↗

PubMed2005

Revised nomenclature of Clostridium difficile toxins and associated genes.

Several different nomenclatures have been applied to the Clostridium difficile toxins and their associated genes. This paper summarizes the new nomenclature that has been agreed to by the research groups currently active in the field. The revised nomenclature includes C. difficile toxins and other related large clostridial toxins produced by Clostridium sordellii and Clostridium novyi, and corresponding toxin genes, as well as toxin production types of C. difficile strains.

Bacterial Proteins↗

PubMed2005

Analysis of Campylobacter jejuni capsular loci reveals multiple mechanisms for the generation of structural diversity and the ability to form complex heptoses.

We recently demonstrated that Campylobacter jejuni produces a capsular polysaccharide (CPS) that is the major antigenic component of the classical Penner serotyping system distinguishing Campylobacter into >60 groups. Although the wide variety of C. jejuni serotypes are suggestive of structural differences in CPS, the genetic mechanisms of such differences are unknown. In this study we sequenced biosynthetic cps regions, ranging in size from 15 to 34 kb, from selected C. jejuni strains of HS:1, HS:19, HS:23, HS:36, HS:23/36 and HS:41 serotypes. Comparison of the determined cps sequences of the HS:1, HS:19 and HS:41 strains with the sequenced strain, NCTC11168 (HS:2), provides evidence for multiple mechanisms of structural variation including exchange of capsular genes and entire clusters by horizontal transfer, gene duplication, deletion, fusion and contingency gene variation. In contrast, the HS:23, HS:36 and HS:23/36 cps sequences were highly conserved. We report the first detailed structural analysis of 81-176 (HS:23/36) and G1 (HS:1) and refine the previous structural interpretations of the HS:19, HS:23, HS:36 and HS:41 serostrains. For the first time, we demonstrate the commonality and function of a second heptose biosynthetic pathway for Campylobacter CPS independent of the pathway for lipooligosaccharide (LOS) biosynthesis and identify a novel heptosyltransferase utilized by this alternate pathway. Furthermore, we show the retention of two functional heptose isomerases in Campylobacter and the sharing of a phosphatase for both LOS and CPS heptose biosynthesis.

Bacterial Capsules↗

PubMed2005

Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway.

We describe in this report the characterization of the recently discovered N-linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N-linked heptasaccharide. Furthermore, we show that C. jejuni- and E. coli-derived pathways can interact in the biosynthesis of N-linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP-GlcNAc: undecaprenylphosphate GlcNAc-1-phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N-linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni-derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N-linked glycan occurs on a lipid-linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N-linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.

Bacterial Proteins↗

PubMed2005

Explorative multifactor approach for investigating global survival mechanisms of Campylobacter jejuni under environmental conditions.

Explorative approaches such as DNA microarray experiments are becoming increasingly important in microbial research. Despite these major technical advancements, approaches to study multifactor experiments are still lacking. We have addressed this problem by using rotation testing and a novel multivariate analysis of variance (MANOVA) approach (50-50 MANOVA) to investigate interacting experimental factors in a complex experimental design. Furthermore, a new rotation testing based method was introduced to calculate false-discovery rates for each response. This novel analytical concept was used to investigate global survival mechanisms in the environment of the major food-borne pathogen C. jejuni. We simulated nongrowth environmental conditions by investigating combinations of the factors temperature (5 and 25 degrees C) and oxygen tension (anaerobic, microaerobic, and aerobic). Data were generated with DNA microarrays for information about gene expression patterns and Fourier transform infrared (FT-IR) spectroscopy to study global macromolecular changes in the cell. Microarray analyses showed that most genes were either unchanged or down regulated compared to the reference (day 0) for the conditions tested and that the 25 degrees C anaerobic condition gave the most distinct expression pattern with the fewest genes expressed. The few up-regulated genes were generally stress related and/or related to the cell envelope. We found, using FT-IR spectroscopy, that the amount of polysaccharides and oligosaccharides increased under the nongrowth survival conditions. Potential mechanisms for survival could be to down regulate most functions to save energy and to produce polysaccharides and oligosaccharides for protection against harsh environments. Basic knowledge about the survival mechanisms is of fundamental importance in preventing transmission of this bacterium through the food chain.

Bacterial Proteins↗

PubMed2005

Intestinal innate immunity to Campylobacter jejuni results in induction of bactericidal human beta-defensins 2 and 3.

Campylobacter jejuni is the most prevalent cause of bacterial diarrhea worldwide. Despite the serious health problems caused by this bacterium, human innate immune responses to C. jejuni infection remain poorly defined. Human beta-defensins, a family of epithelial antimicrobial peptides, are a major component of host innate defense at the gastrointestinal mucosal surface. In this study, the effect of two different C. jejuni wild-type strains on human intestinal epithelial innate responses was investigated. Up-regulation of beta-defensin gene and peptide expression during infection was observed and recombinant beta-defensins were shown to have a direct bactericidal effect against C. jejuni through disruption of cell wall integrity. Further studies using an isogenic capsule-deficient mutant showed that, surprisingly, the absence of the bacterial polysaccharide capsule did not change the innate immune responses induced by C. jejuni or the ability of C. jejuni to survive exposure to recombinant beta-defensins. This study suggests a major role for this family of antimicrobial peptides in the innate immune defense against this human pathogen.

Bacterial Capsules↗

PubMed2004

Invertebrates as a source of emerging human pathogens.

Despite their importance, little is known about the origins of many emerging human pathogens. However, given the age and current predominance of invertebrates, it is likely that bacteria-invertebrate interactions are not only a present source of human pathogens but have also shaped their evolution. Pathogens of invertebrate and unicellular organisms represent an extensive reservoir of bacterial strains equipped with virulence factors that evolved to overcome the innate immune responses of their hosts. This reservoir might represent a source of new human pathogenic strains and might also foster the spread of novel virulence factors into existing human commensal or pathogenic bacteria. This article examines the available evidence for this concept by examining pairs of closely related bacteria, one of which is benign, but insect associated, and one of which is a human pathogen.

Animals↗