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B W Wren

Publications and source records attributed to B W Wren.

At least 19 recordsLinked to original sources
PubMed2006

Development of signature-tagged mutagenesis in Burkholderia pseudomallei to identify genes important in survival and pathogenesis.

Burkholderia pseudomallei, the causative agent of melioidosis, is an important human pathogen in Southeast Asia and northern Australia for which a vaccine is unavailable. A panel of 892 double signature-tagged mutants was screened for virulence using an intranasal BALB/c mouse model of infection. A novel DNA tag microarray identified 33 mutants as being attenuated in spleens, while 6 were attenuated in both lungs and spleens. The transposon insertion sites in spleen-attenuated mutants revealed genes involved in several stages of capsular polysaccharide biosynthesis and DNA replication and repair, a putative oxidoreductase, ABC transporters, and a lipoprotein that may be important in intercellular spreading. The six mutants identified as missing in both lungs and spleens were found to have insertions in recA involved in the SOS response and DNA repair; putative auxotrophs of leucine, threonine, p-aminobenzoic acid, and a mutant with an insertion in aroB causing auxotrophy for aromatic compounds were also found. Murine challenge studies revealed partial protection in BALB/c mice vaccinated with the aroB mutant. The refined signature-tagged mutagenesis approach developed in this study was used to efficiently identify attenuating mutants from this highly pathogenic species and could be applied to other organisms.

Animals↗
PubMed2006

Biofilm formation in Campylobacter jejuni.

The major gastrointestinal pathogen Campylobacter jejuni is shown to exist as three forms of monospecies biofilm in liquid culture. It attaches to a glass surface; forms an unattached aggregate (floc); and forms a pellicle at the liquid-gas interface. The three forms of biofilm resemble each other when examined by scanning electron microscopy. The biofilm mode of growth confers protection against environmental stress, the microaerobic bacteria in flocs surviving up to 24 days at ambient temperature and atmosphere compared to 12 days survival by planktonic bacteria. The wild-type strains C. jejuni 33106, 32799, 33084 and 31485 did not form flocs, and floc formation was reduced in strains mutant in a putative flagellar protein (FliS) and in a phosphate acetyltransferase (Cj0688). All other strains tested, including strains with mutations affecting capsular polysaccharide (kpsM), flagella (maf5), protein glycosylation (pglH) and lipo-oligosaccharide (neuB1) formed flocs. Similarly, all strains tested formed a pellicle and attached to glass except the aflagellate mutant maf5; pellicle formation was reduced in fliS and cj0688 mutants. Different mechanisms, therefore, may control formation of different forms of biofilm. It is proposed that these poorly characterized forms of growth are important for the persistence of C. jejuni in the environment and may in part explain the high incidence of Campylobacter-associated food borne disease.

Bacterial Adhesion↗
PubMed2006

Comparative phylogenomics of Clostridium difficile reveals clade specificity and microevolution of hypervirulent strains.

Clostridium difficile is the most frequent cause of nosocomial diarrhea worldwide, and recent reports suggested the emergence of a hypervirulent strain in North America and Europe. In this study, we applied comparative phylogenomics (whole-genome comparisons using DNA microarrays combined with Bayesian phylogenies) to model the phylogeny of C. difficile, including 75 diverse isolates comprising hypervirulent, toxin-variable, and animal strains. The analysis identified four distinct statistically supported clusters comprising a hypervirulent clade, a toxin A(-) B(+) clade, and two clades with human and animal isolates. Genetic differences among clades revealed several genetic islands relating to virulence and niche adaptation, including antibiotic resistance, motility, adhesion, and enteric metabolism. Only 19.7% of genes were shared by all strains, confirming that this enteric species readily undergoes genetic exchange. This study has provided insight into the possible origins of C. difficile and its evolution that may have implications in disease control strategies.

Animals↗
PubMed2005

Development and application of an insertional system for gene delivery and expression in Campylobacter jejuni.

The genetic investigation of Campylobacter jejuni, an important gastrointestinal pathogen, has been hampered by the lack of an efficient system for introduction of exogenous genetic information, as commonly used vectors designed for Escherichia coli and other bacteria cannot be maintained in Campylobacter cells. Additionally, gene expression in Campylobacter requires the presence of species-specific promoters. In this study we exploited the availability of several conserved copies of rRNA gene clusters for insertion of various genes into the chromosome by homologous recombination. The high conservation of the rRNA sequences means that the procedure can be applied to other Campylobacter strains. The presence of a Campylobacter-derived promoter in this vector ensures expression of exogenous genes in target cells. The efficiency of the procedure was demonstrated by complementation of mutations in two strains of Campylobacter. In addition, we applied the system for introduction and expression of a green fluorescent protein (GFP). GFP-expressing Campylobacter allowed visualization of sessile bacteria attached to a glass surface in stationary liquid culture. The study demonstrated that the attached bacteria contained an assemblage of coccoid and spiral forms with liquid channels preserving viable highly motile cells. We demonstrate a novel universal procedure for gene delivery and expression that can be used as an efficient tool to study this poorly understood pathogen. The principles developed in this study could be more widely applied for the manipulation of other bacteria that are refractory to genetic analysis.

Bacterial Proteins↗
PubMed2004

The Campylobacter jejuni general glycosylation system is important for attachment to human epithelial cells and in the colonization of chicks.

It has recently been shown that the enteropathogen Campylobacter jejuni has an N-linked general protein glycosylation pathway (Pgl) that modifies many of the organism's proteins. To determine the role of the N-linked general glycosylation in C jejuni, the authors studied the pglH gene, which shows high similarity to a family of sugar transferases. pglH mutants were constructed in strains 81116 and 11168H. Both mutants were shown to be deficient in their ability to glycosylate a number of C. jejuni proteins, but their lipooligosaccharide and capsule were unaffected. The pglH mutants had significantly reduced ability to adhere to and invade human epithelial Caco-2 cells. Additionally, the 81116 pglH mutant was severely affected in its ability to colonize chicks. These results suggest that glycosylation is important for the attachment of C. jejuni to human and chicken host cells and imply a role for glycoproteins in the pathogenesis of C. jejuni.

Animals↗
PubMed2003

A Caenorhabditis elegans model of Yersinia infection: biofilm formation on a biotic surface.

To investigate Yersinia pathogenicity and the evolutionary divergence of the genus, the effect of pathogenic yersiniae on the model organism Caenorhabditis elegans was studied. Three strains of Yersinia pestis, including a strain lacking pMT1, caused blockage and death of C. elegans; one strain, lacking the haemin storage (hms) locus, caused no effect. Similarly, 15 strains of Yersinia enterocolitica caused no effect. Strains of Yersinia pseudotuberculosis showed different levels of pathogenicity. The majority of strains (76 %) caused no discernible effect; 5 % caused a weak infection, 9.5 % an intermediate infection, and 9.5 % a severe infection. There was no consistent relationship between serotype and severity of infection; nor was there any relationship between strains causing infection of C. elegans and those able to form a biofilm on an abiotic surface. Electron microscope and cytochemical examination of infected worms indicated that the infection phenotype is a result of biofilm formation on the head of the worm. Seven transposon mutants of Y. pseudotuberculosis strain YPIII pIB1 were completely or partially attenuated; mutated genes included genes encoding proteins involved in haemin storage and lipopolysaccharide biosynthesis. A screen of 15 defined C. elegans mutants identified four where mutation caused (complete) resistance to infection by Y. pseudotuberculosis YPIII pIB1. These mutants, srf-2, srf-3, srf-5 and the dauer pathway gene daf-1, also exhibit altered binding of lectins to the nematode surface. This suggests that biofilm formation on a biotic surface is an interactive process involving both bacterial and invertebrate control mechanisms.

Animals↗
PubMed2002

Immunization with a DNA vaccine expressing a truncated form of varicella zoster virus glycoprotein E.

The gE glycoprotein of varicella zoster virus (VZV) is involved with cell entry and it is the most abundant glycoprotein produced in VZV-infected cells. It is also the first glycoprotein to be recognized by the immune system and induces neutralizing antibodies and cellular immunity. We have shown previously that immunization with a DNA vaccine encoding full length gE induces high antibody titres in BALB/c mice. In this study, we engineered a truncated form of gE to facilitate secretion of the glycoprotein, which is thought to increase the quantity of antigen available for B cells to mount an immune response. This hypothesis was tested by using inverse PCR mutagenesis (IPCRM) to engineer a mutated form of gE that was secreted from the cell. This construct was then evaluated as a potential DNA vaccine. Following immunization studies, the magnitude of the immune response induced with the mutant form of gE was found to be similar to that induced by membrane bound protein. This finding suggests that, in the case of VZV, a DNA vaccine expressing a secreted protein has no advantage over one expressing a membrane bound protein. However, mice immunized with the truncated form of gE (gED) displayed responses favouring IgG1 (Th2) in comparison with mice immunized with the full length gE construct, which generated an IgG2a (Th1) response. This observation indicates that immunization with a truncated form of a gene may induce immune modulation, a phenomenon that should be taken into account for the design of vaccines.

Animals↗
PubMed2001

Genome sequence of Yersinia pestis, the causative agent of plague.

The Gram-negative bacterium Yersinia pestis is the causative agent of the systemic invasive infectious disease classically referred to as plague, and has been responsible for three human pandemics: the Justinian plague (sixth to eighth centuries), the Black Death (fourteenth to nineteenth centuries) and modern plague (nineteenth century to the present day). The recent identification of strains resistant to multiple drugs and the potential use of Y. pestis as an agent of biological warfare mean that plague still poses a threat to human health. Here we report the complete genome sequence of Y. pestis strain CO92, consisting of a 4.65-megabase (Mb) chromosome and three plasmids of 96.2 kilobases (kb), 70.3 kb and 9.6 kb. The genome is unusually rich in insertion sequences and displays anomalies in GC base-composition bias, indicating frequent intragenomic recombination. Many genes seem to have been acquired from other bacteria and viruses (including adhesins, secretion systems and insecticidal toxins). The genome contains around 150 pseudogenes, many of which are remnants of a redundant enteropathogenic lifestyle. The evidence of ongoing genome fluidity, expansion and decay suggests Y. pestis is a pathogen that has undergone large-scale genetic flux and provides a unique insight into the ways in which new and highly virulent pathogens evolve.

Animals↗
PubMed2001

The failure of different strains of Yersinia pestis to produce lipopolysaccharide O-antigen under different growth conditions is due to mutations in the O-antigen gene cluster.

The lipopolysaccharide (LPS) from eight strains of Yersinia pestis which had been cultured at 28 degrees C appeared to be devoid of an O-antigen when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LPS isolated from three of these strains which had been cultured at 37 degrees C also appeared to be devoid of an O-antigen. When the LPS from Y. pestis strain CO92 was purified and analysed by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry, the observed signals were in the mass range predicted for molecules containing lipid A plus the core oligosaccharide but lacking an O-antigen. The nucleotide sequence of Y. pestis strain CO92 revealed the presence of a putative O-antigen gene cluster. However, frame-shift mutations in the ddhB, gmd, fcl and ushA genes are likely to prevent expression of the O-antigen thus explaining the loss of phenotype.

Genome, Bacterial↗
PubMed2001

Characterization of the lipopolysaccharide of Yersinia pestis.

Lipopolysaccharide (LPS) extracted from eight strains of Yersinia pestis, which had been cultured at 28 or 37 degrees C, reacted equally well, in Western blots, with four monoclonal antibodies generated against the LPS from a single strain of Y. pestis cultured at 28 degrees C. LPS was extracted and purified from Y. pestis strain GB, which had been cultured at 28 degrees C. When the LPS was analysed by SDS-PAGE and MALDI-TOF mass spectrometry it was found to be devoid of an O-antigen. The LPS possessed activity of 2.7 endotoxin units/ng in the Limulus amoebocyte lysate assay. The LPS stimulated the production of TNFalpha and IL-6 from mouse macrophages, but was less active in these assays than LPS isolated from Escherichia coli strain 0111. Y. pestis LPS, either alone or with cholera toxin B subunit, was used to immunize mice. Either immunization schedule resulted in the development of an antibody response to LPS. However, this response did not provide protection against 100 MLD of Y. pestis strain GB.

Animals↗
PubMed2001

Deciphering Campylobacter jejuni cell surface interactions from the genome sequence.

The completion of the Campylobacter jejuni genome sequence is a landmark in Campylobacter research. Discoveries directly arising from these data include the identification of a capsular polysaccharide, extensive capacity for phase variable gene expression and lipo-oligosaccharide structural phase variation. The recent identification of a unique system of general protein glycosylation in C. jejuni, a C. jejuni protein that is translocated into eukaryotic cells, and plasmid-encoded components of a putative type IV secretion system are likely to be significant in terms of the host-pathogen interaction.

Antigenic Variation↗
PubMed2001

Whole genome comparison of Campylobacter jejuni human isolates using a low-cost microarray reveals extensive genetic diversity.

Campylobacter jejuni is the leading cause of bacterial food-borne diarrhoeal disease throughout the world, and yet is still a poorly understood pathogen. Whole genome microarray comparisons of 11 C. jejuni strains of diverse origin identified genes in up to 30 NCTC 11168 loci ranging from 0.7 to 18.7 kb that are either absent or highly divergent in these isolates. Many of these regions are associated with the biosynthesis of surface structures including flagella, lipo-oligosaccharide, and the newly identified capsule. Other strain-variable genes of known function include those responsible for iron acquisition, DNA restriction/modification, and sialylation. In fact, at least 21% of genes in the sequenced strain appear dispensable as they are absent or highly divergent in one or more of the isolates tested, thus defining 1300 C. jejuni core genes. Such core genes contribute mainly to metabolic, biosynthetic, cellular, and regulatory processes, but many virulence determinants are also conserved. Comparison of the capsule biosynthesis locus revealed conservation of all the genes in this region in strains with the same Penner serotype as strain NCTC 11168. By contrast, between 5 and 17 NCTC 11168 genes in this region are either absent or highly divergent in strains of a different serotype from the sequenced strain, providing further evidence that the capsule accounts for Penner serotype specificity. These studies reveal extensive genetic diversity among C. jejuni strains and pave the way toward identifying correlates of pathogenicity and developing improved epidemiological tools for this problematic pathogen.

Campylobacter jejuni↗
PubMed2001

Application of high-density array-based signature-tagged mutagenesis to discover novel Yersinia virulence-associated genes.

Yersinia pestis, the causative agent of plague, and the enteropathogen Yersinia pseudotuberculosis have nearly identical nucleotide similarity yet cause markedly different diseases. To investigate this conundrum and to study Yersinia pathogenicity, we developed a high-density oligonucleotide array-based modification of signature-tagged mutagenesis (STM). Y. pseudotuberculosis YPIII mutants constructed with the tagged transposons were evaluated in the murine yersiniosis infection model. The DNA tags were amplified using biotinylated primers and hybridized to high-density oligonucleotide arrays containing DNA complementary to the tags. Comparison of the hybridization signals from input pools and output pools identified a mutant whose relative abundance was significantly reduced in the output pool. Sequence data from 31 transposon insertion regions was compared to the complete Y. pestis CO92 genome sequence. The 26 genes present in both species were found to be almost identical, but five Y. pseudotuberculosis genes identified through STM did not have counterparts in the Y. pestis genome and may contribute to the different tropisms in these closely related pathogens. Potential virulence genes identified include those involved in lipopolysaccharide biosynthesis, adhesion, phospholipase activity, iron assimilation, and gene regulation. The phospholipase A (PldA) mutant exhibited reduced phospholipase activity compared to the wild-type strain and in vivo attenuation of the mutant was confirmed. The combination of optimized double tag sequences and high-density array hybridization technology offers improved performance, efficiency, and reliability over classical STM and permits quantitative analysis of data.

Animals↗
PubMed2001

Helicobacter pylori pore-forming cytolysin orthologue TlyA possesses in vitro hemolytic activity and has a role in colonization of the gastric mucosa.

Hemolysins have been found to possess a variety of functions in bacteria, including a role in virulence. Helicobacter pylori demonstrates hemolytic activity when cultured on unlysed blood agar plates which is increased under iron-limiting conditions. However, the role of an H. pylori hemolysin in virulence is unclear. Scrutiny of the H. pylori 26695 genome sequence suggests the presence of at least two distinct hemolysins, HP1086 and HP1490, in this strain. Previous studies have shown that the in vitro hemolytic activity of H. pylori is reduced when it is coincubated with dextran 5000, suggesting the presence of a pore-forming cytolysin. HP1086 has homology to pore-forming cytolysins (TlyA) from other bacterial species, and the introduction of the cloned H. pylori tlyA gene into a nonhemolytic Escherichia coli strain conferred hemolytic activity. An H. pylori tlyA defined mutant showed reduced in vitro hemolytic activity, which appears to be due to pore formation, as the hemolytic activity of the wild-type strain is reduced to the same level as the tlyA mutant by the addition of dextran 5000. The mutant also showed reduced adhesion to human gastric adenocarcinoma cells and failed to colonize the gastric mucosa of mice. These data clearly suggest a role in virulence for H. pylori TlyA, contrary to the suggestion that hemolytic activity is an in vitro phenomenon for this pathogen.

Animals↗