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B W Wren

Publications and source records attributed to B W Wren.

At least 37 records · Page 2Linked to original sources
PubMed2001

Demonstration of polysaccharide capsule in Campylobacter jejuni using electron microscopy.

Recently, we reported that Campylobacter jejuni, an important gastrointestinal pathogen, has the genetic determinants to produce a capsular polysaccharide (Karlyshev et al., Mol. Microbiol. 35:529-541, 2000). Despite these data, the presence of a capsule in these bacteria has remained controversial. In this study we stain C. jejuni cells with the cationic dye Alcian blue and demonstrate for the first time by electron microscopy that C. jejuni cells produce a polysaccharide capsule that is retained in the coccoid form but is absent in a kpsM mutant.

Alcian Blue↗
PubMed2001

Yersinia pestis pFra shows biovar-specific differences and recent common ancestry with a Salmonella enterica serovar Typhi plasmid.

Population genetic studies suggest that Yersinia pestis, the cause of plague, is a clonal pathogen that has recently emerged from Yersinia pseudotuberculosis. Plasmid acquisition is likely to have been a key element in this evolutionary leap from an enteric to a flea-transmitted systemic pathogen. However, the origin of Y. pestis-specific plasmids remains obscure. We demonstrate specific plasmid rearrangements in different Y. pestis strains which distinguish Y. pestis bv. Orientalis strains from other biovars. We also present evidence for plasmid-associated DNA exchange between Y. pestis and the exclusively human pathogen Salmonella enterica serovar Typhi.

Animals↗
PubMed2001

Detection and initial characterization of novel capsular polysaccharide among diverse Campylobacter jejuni strains using alcian blue dye.

We have recently demonstrated that most strains of Campylobacter jejuni produce capsular polysaccharide (CPS), which can be detected by immunoblotting with homologous Penner antisera on polyvinylidene difluoride membranes (A. V. Karlyshev, D. Linton, N. A. Gregson, A. J. Lastovica, and B. W. Wren, Mol. Microbiol. 35:529-541, 2000). In this report, we describe a universal and rapid staining procedure using Alcian blue for C. jejuni CPS, which does not rely on the availability of antisera and identifies CPS in untypeable strains. Furthermore, Alcian blue staining identified CPS in its lipid-free form directly on Tricine gels, and we demonstrate that CPS is thermostable and is accumulated in the culture supernatant in a lipid-free form. The identification of a newly described CPS and its lipid-free form in C. jejuni should prove invaluable in studying the pathogenesis and epidemiology of this important pathogen.

Alcian Blue↗
PubMed2000

Investigation into the role of the serine protease HtrA in Yersinia pestis pathogenesis.

The HtrA stress response protein has been shown to play a role in the virulence of a number of pathogens. For some organisms, htrA mutants are attenuated in the animal model and can be used as live vaccines. A Yersinia pestis htrA orthologue was identified, cloned and sequenced, showing 86% and 87% similarity to Escherichia coli and Salmonella typhimurium HtrAs. An isogenic Y. pestis htrA mutant was constructed using a reverse genetics approach. In contrast to the wild-type strain, the mutant failed to grow at an elevated temperature of 39 degrees C, but showed only a small increase in sensitivity to oxidative stress and was only partially attenuated in the animal model. However, the mutant exhibited a different protein expression profile to that of the wild-type strain when grown at 28 degrees C to simulate growth in the flea.

Animals↗
PubMed2000

Immunization with a DNA expression vector encoding the varicella zoster virus glycoprotein E (gE) gene via intramuscular and subcutaneous routes.

In this study we constructed a plasmid containing the gene encoding varicella-zoster virus transmembrane glycoprotein gE (VZV gE) and evaluated its utility for DNA immunization in mice. Our initial work demonstrates that intramuscular and subcutaneous injection of VZV gE DNA, without the use of costimulatory molecules or other adjuvant materials, results in the generation of antigen-specific antibodies of primarily the IgG2a subclass, indicating that this vaccine can stimulate Th1 type immunity. This is the first report of a prototype DNA vaccine for varicella-zoster virus.

Animals↗
PubMed2000

The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences.

Campylobacter jejuni, from the delta-epsilon group of proteobacteria, is a microaerophilic, Gram-negative, flagellate, spiral bacterium-properties it shares with the related gastric pathogen Helicobacter pylori. It is the leading cause of bacterial food-borne diarrhoeal disease throughout the world. In addition, infection with C. jejuni is the most frequent antecedent to a form of neuromuscular paralysis known as Guillain-Barré syndrome. Here we report the genome sequence of C. jejuni NCTC11168. C. jejuni has a circular chromosome of 1,641,481 base pairs (30.6% G+C) which is predicted to encode 1,654 proteins and 54 stable RNA species. The genome is unusual in that there are virtually no insertion sequences or phage-associated sequences and very few repeat sequences. One of the most striking findings in the genome was the presence of hypervariable sequences. These short homopolymeric runs of nucleotides were commonly found in genes encoding the biosynthesis or modification of surface structures, or in closely linked genes of unknown function. The apparently high rate of variation of these homopolymeric tracts may be important in the survival strategy of C. jejuni.

Amino Acid Sequence↗
PubMed2000

Helicobacter pylori vaccine strategies--triggering a gut reaction.

Helicobacter pylori causes peptic ulcer disease and some forms of gastric cancer; it is one of the most common chronic bacterial infections of humans. Although several prototype protein-based vaccines have shown promising results, they have not cleared infection and/or prevented reinfection. Nucleic acid vaccination offers a useful alternative to protein immunization, especially now that two complete H. pylori genome sequences are available, and facilitates the selection of antigenic targets.

Adjuvants, Immunologic↗
PubMed2000

Microbial genome analysis: insights into virulence, host adaptation and evolution.

Genome analysis of microbial pathogens has provided unique insights into their virulence, host adaptation and evolution. Common themes have emerged, including lateral gene transfer among enteric pathogens, genome decay among obligate intracellular pathogens and antigenic variation among mucosal pathogens. The advent of post-genomic approaches and the sequencing of the human genome will enable scientists to investigate the complex and dynamic interplay between host and pathogen. This wealth of information will catalyse the development of new intervention strategies to reduce the burden of microbial-related disease.

Adaptation, Physiological↗
PubMed2000

Genetic and biochemical evidence of a Campylobacter jejuni capsular polysaccharide that accounts for Penner serotype specificity.

Campylobacter jejuni, a Gram-negative spiral bacterium, is the most common bacterial cause of acute human gastroenteritis and is increasingly recognized for its association with the serious post-infection neurological complications of the Miller-Fisher and Guillain-Barré syndromes. C. jejuni lipopolysaccharide (LPS) is thought to be involved in the pathogenesis of both uncomplicated infection and more serious sequelae, yet the LPS remains poorly characterized. Current studies on C. jejuni suggest that all strains produce lipooligosaccharide (LOS), with about one-third of strains also producing high-molecular-weight LPS (referred to as O-antigen). In this report, we demonstrate the presence of the high-molecular-weight LPS in all C. jejuni strains tested. Furthermore, we show that this LPS is biochemically and genetically unrelated to LOS and is similar to group II and group III capsular polysaccharides. All tested kpsM, kpsS and kpsC mutants of C. jejuni lost the ability to produce O-antigen. Moreover, this correlated with serotype changes. We demonstrate for the first time that the previously described O-antigen of C. jejuni is a capsular polysaccharide and a common component of the thermostable antigen used for serotyping of C. jejuni.

ATP-Binding Cassette Transporters↗
PubMed2000

Multiple N-acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide.

N-acetyl neuraminic acid (NANA) is a common constituent of Campylobacter jejuni lipo-oligosaccharide (LOS). Such structures often mimic human gangliosides and are thought to be involved in the triggering of Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) following C. jejuni infection. Analysis of the C. jejuni NCTC 11168 genome sequence identified three putative NANA synthetase genes termed neuB1, neuB2 and neuB3. The NANA synthetase activity of all three C. jejuni neuB gene products was confirmed by complementation experiments in an Escherichia coli neuB-deficient strain. Isogenic mutants were created in all three neuB genes, and for one such mutant (neuB1) LOS was shown to have increased mobility. C. jejuni NCTC 11168 wild-type LOS bound cholera toxin, indicating the presence of NANA in a LOS structure mimicking the ganglioside GM1. This property was lost in the neuB1 mutant. Gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry analysis of LOS from wild-type and the neuB1 mutant strain demonstrated the lack of NANA in the latter. Expression of the neuB1 gene in E. coli confirmed that NeuB1 was capable of in vitro NANA biosynthesis through condensation of N-acetyl-D-mannosamine and phosphoenolpyruvate. Southern analysis demonstrated that the neuB1 gene was confined to strains of C. jejuni with LOS containing a single NANA residue. Mutagenesis of neuB2 and neuB3 did not affect LOS, but neuB3 mutants were aflagellate and non-motile. No phenotype was evident for neuB2 mutants in strain NCTC 11168, but for strain G1 the flagellin protein from the neuB2 mutant showed an apparent reduction in molecular size relative to the wild type. Thus, the neuB genes of C. jejuni appear to be involved in the biosynthesis of at least two distinct surface structures: LOS and flagella.

Base Sequence↗
PubMed2000

Phase variation of a beta-1,3 galactosyltransferase involved in generation of the ganglioside GM1-like lipo-oligosaccharide of Campylobacter jejuni.

Ganglioside mimicry by Campylobacter jejuni lipo-oligosaccharide (LOS) is thought to be a critical factor in the triggering of the Guillain-Barré and Miller-Fisher syndrome neuropathies after C. jejuni infection. The combination of a completed genome sequence and a ganglioside GM1-like LOS structure makes C. jejuni NCTC 11168 a useful model strain for the identification and characterization of the genes involved in the biosynthesis of ganglioside-mimicking LOS. Genome analysis identified a putative LOS biosynthetic cluster and, from this, we describe a putative gene (ORF Cj1139c), which we have termed wlaN, with a significant level of similarity to a number of bacterial glycosyltransferases. Mutation of this gene in C. jejuni NCTC 11168 resulted in a LOS molecule of increased electrophoretic mobility, which also failed to bind cholera toxin. Comparison of LOS structural data from wild type and the mutant strain indicated lack of a terminal beta-1,3-linked galactose residue in the latter. The wlaN gene product was demonstrated unambiguously as a beta-1,3 galactosyltransferase responsible for converting GM2-like LOS structures to GM1-like by in vitro expression. We also show that the presence of an intragenic homopolymeric tract renders the expression of a functional wlaN gene product phase variable, resulting in distinct C. jejuni NCTC 11168 cell populations with alternate GM1 or GM2 ganglioside-mimicking LOS structures. The distribution of wlaN among a number of C. jejuni strains with known LOS structure was determined and, for C. jejuni NCTC 12500, similar wlaN gene phase variation was shown to occur, so that this strain has the potential to synthesize a GM1-like LOS structure as well as the ganglioside GM2-like LOS structure proposed in the literature.

Amino Acid Sequence↗
PubMed2000

Sequencing of the Francisella tularensis strain Schu 4 genome reveals the shikimate and purine metabolic pathways, targets for the construction of a rationally attenuated auxotrophic vaccine.

Francisella tularensis is the etiological agent of tularemia, a serious disease in several Northern hemisphere countries. The organism has fastidious growth requirements and is very poorly understood at the genetic and molecular levels. Given the lack of data on this organism, we undertook the sample sequencing of its genome. A random library of DNA fragments from a highly virulent strain (Schu 4) of F. tularensis was constructed and the nucleotide sequences of 13,904 cloned fragments were determined and assembled into 353 contigs. A total of 1.83 Mb of nucleotide sequence was obtained that had a G+C content of 33.2%. Genes located on plasmids pOM1 and pNFL10, which had been previously isolated from low virulence strains of F. tularensis, were absent but all of the other known F. tularensis genes were represented in the assembled data. F. tularensis Schu4 was able to grow in the absence of aromatic amino acids and orthologues of genes which could encode enzymes in the shikimate pathway in other bacteria were identified in the assembled data. Genes that could encode all of the enzymes in the purine biosynthetic and most of the en- zymes in the purine salvage pathways were also identified. This data will be used to develop defined rationally attenuated mutants of F. tularensis, which could be used as replacements for the existing genetically undefined live vaccine strain.

Bacterial Vaccines↗
PubMed2000

Helicobacter pylori possesses two CheY response regulators and a histidine kinase sensor, CheA, which are essential for chemotaxis and colonization of the gastric mucosa.

Infection of the mucous layer of the human stomach by Helicobacter pylori requires the bacterium to be motile and presumably chemotactic. Previous studies have shown that fully functional flagella are essential for motility and colonization, but the role of chemotaxis remains unclear. The two-component regulatory system CheA/CheY has been shown to play a major role in chemotaxis in other enteric bacteria. Scrutiny of the 26695 genome sequence suggests that H. pylori has two CheY response regulators: one a separate protein (CheY1) and the other (CheY2) fused to the histidine kinase sensor CheA. Defined deletion mutations were introduced into cheY1, cheY2, and cheA in H. pylori strains N6 and SS1. Video tracking revealed that the wild-type H. pylori strain moves in short runs with frequent direction changes, in contrast to movement of cheY2, cheAY2, and cheAY2 cheY1 mutants, whose motion was more linear. The cheY1 mutant demonstrated a different motility phenotype of rapid tumbling. All mutants had impaired swarming and greatly reduced chemotactic responses to hog gastric mucin. Neither cheY1 nor cheAY2 mutants were able to colonize mice, but they generated a significant antibody response, suggesting that despite impaired chemotaxis, these mutants were able to survive in the stomach long enough to induce an immune response before being removed by gastric flow. Additionally, we demonstrated that cheY1 failed to colonize gnotobiotic piglets. This study demonstrates the importance of the roles of cheY1, cheY2, and cheA in motility and virulence of H. pylori.

Animals↗
PubMed2000

The response regulator PhoP is important for survival under conditions of macrophage-induced stress and virulence in Yersinia pestis.

The two-component regulatory system PhoPQ has been identified in many bacterial species. However, the role of PhoPQ in regulating virulence gene expression in pathogenic bacteria has been characterized only in Salmonella species. We have identified, cloned, and sequenced PhoP orthologues from Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. To investigate the role of PhoP in the pathogenicity of Y. pestis, an isogenic phoP mutant was constructed by using a reverse-genetics PCR-based strategy. The protein profiles of the wild-type and phoP mutant strains, grown at either 28 or 37 degrees C, revealed more than 20 differences, indicating that PhoP has pleiotrophic effects on gene expression in Y. pestis. The mutant showed a reduced ability to survive in J774 macrophage cell cultures and under conditions of low pH and oxidative stress in vitro. The mean lethal dose of the phoP mutant in mice was increased 75-fold in comparison with that of the wild-type strain, indicating that the PhoPQ system plays a key role in regulating the virulence of Y. pestis.

Animals↗
PubMed2000

Mutational analysis of genes encoding the early flagellar components of Helicobacter pylori: evidence for transcriptional regulation of flagellin A biosynthesis.

We investigated the roles of fliF, fliS, flhB, fliQ, fliG, and fliI of Helicobacter pylori, predicted by homology to encode structural components of the flagellar basal body and export apparatus. Mutation of these genes resulted in nonmotile, nonflagellate strains. Western blot analysis showed that all the mutants had considerably reduced levels of both flagellin subunits and of FlgE, the flagellar hook protein. RNA slot blot hybridization showed reduced levels of flaA mRNA, indicating that transcription of the major flagellin gene is inhibited in the absence of the early components of the flagellar-assembly pathway. This is the first demonstration of a checkpoint in H. pylori flagellar assembly.

Bacillus subtilis↗
PubMed1999

Nucleic acid immunization: concepts and techniques associated with third generation vaccines.

A radical change in vaccine methodology arrived nine years ago with the advent of nucleic acid immunization. Aspects such as plasmid design, gene selection, the use of immunostimulatory complexes and clinical trials are discussed in this review. Furthermore, concepts and protocols involved in the construction, evaluation and immunization of a DNA vaccine have been examined as new strategies to enhance this technology continues to grow.

Adjuvants, Immunologic↗
PubMed1999

Functional analysis of the roles of FliQ and FlhB in flagellar expression in Helicobacter pylori.

Expression of the two Helicobacter pylori flagellin proteins FlaA and FlaB is required for full motility and persistent infection of the gastric mucosa. The mechanisms and regulation of the biosynthesis and export of flagella in H. pylori are still poorly understood. Scrutiny of the H. pylori 26695 genome sequence revealed homologues of FliQ and FlhB. The roles of the fliQ and flhB genes in H. pylori were investigated by the construction and characterisation of defined isogenic mutants. The results indicate that these genes are involved in the flagellar expression, adhesion to and colonisation of the gastric mucosa.

Animals↗