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Subject: search.filters.subject.Helicobacter pylori

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    The genetic characterisation of a carbon starvation gene of Helicobacter pylori : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 1998) Beddek, Amanda Jane
    The carbon starvation gene (csg) of the gastric pathogen Helicobacter pylori was studied to elucidate the role of the gene product during the carbon starvation response. Studies were also conducted to investigate the relationship of csg to the glutamine permease operon (glnP1, glnP2, glnQ, and glnH), which is located directly upstream of csg on the H. pylori chromosome. csg and glnP1 were sequenced to determine the length of the genes, the base composition, and the location of putative promoters and regulator binding sites. The sequence information was then used to construct a csg mutant using a deletion insertion strategy, whereby the promoter regions of csg and glnP1 were removed and replaced with the KanΩ antibiotic resistance marker. Phenotypic analysis was carried out on this mutant in complex, defined, and semi-defined media. The plasmid carrying the csg mutation was also transformed into H. pylori Sydney strain to allow colonisation studies in mice. A H. pylori strain was constructed, which would allow the study of the expression of csg under various environmental conditions. The promoterless chloramphenicol acetyl transferase (CAT) cassette from pCM4 was used as the reporter gene and cloned into an engineered BglII site directly downstream of csg to disrupt the transcription terminator. The kanamycin resistance cassette from pILL600 was also cloned into this site in a three way ligation, to allow selection of transformants in H. pylori. Sequence of the csg mutant showed putative σ 70 responsive -10 and -35 sequences, and a putative cAMP receptor protein (CRP) binding sequence for csg in the 5' sequence of glnP1. The CRP site implied that csg may be regulated by cAMP. The location of the CRP binding site also suggested that csg and glnP1 were linked, which was confirmed by PCR amplification of a region containing the csg and glnP1 transcription start points and the intergenic region from ten H. pylori strains. The same PCR amplification failed for a H. mustelae strain, suggesting that this conservation of sequence is specific to H. pylori strains. Phenotypic analysis of the csg mutant showed no difference compared to the wild type when grown in complex and semi-defined medium, both of which contained peptides. However, the csg mutant grew more slowly than the wild type in the defined medium, which contained glucose and amino acids as the sole carbon sources. The role of csg could not be determined with certainty. The results suggested that Csg may scavenge for carbon sources to help the cell escape carbon starvation. This thesis work has shown csg to be non-essential in vitro. However, a non-functional csg gene in vivo may impair transmission, when carbon sources are lacking, but the mutation would be silent in the stomach where peptides are abundant.
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    Identification of an immunogenic 18 kDa protein of Helicobacter pylori using alkaline phosphatase gene fusions : a thesis presented in partial fulfillment of the requirement for the Doctor of Philosophy in Molecular Microbiology, Massey University, Palmerston North, NZ
    (Massey University, 1999) Oliaro, Jane
    Secreted or surface-associated proteins play an important role in the immunopathogenesis of Helicobacter pylori infection. The aim of this study was to identify, using a genetic approach, H. pylori exported proteins and assess their role in the host immune response to infection. As part of this work, an H. pylori expression library was constructed and screened with a monoclonal antibody raised to a component of outer membrane vesicles from H. pylori, identified and characterised in a separate study. The screening strategy identified a locus of the genome containing two genes encoding exported proteins. Subsequent expression studies identified the gene product detected by the antibody as Lpp20, which encodes a well characterised lipoprotein from H. pylori. In addition, the use of alkaline phosphatase (AP) gene fusion methodology enabled the identification of a large number of other H. pylori exported proteins. Immunoscreening of a selection of enzymatically active H. pylori AP fusion proteins was carried out by Western blot analysis with patient sera and lymphocyte proliferation assays using peripheral blood mononuclear cells from H. pylori infected individuals. These assays identified a novel H. pylori exported antigen which was recognised by antibody derived from H. pylori infected individuals. Southern blot analysis revealed that the gene encoding the protein was absent in other Helicobacter species tested and sequence analysis of the gene demonstrated that it is highly conserved among H. pylori isolates. In order to obtain pure recombinant protein, the gene encoding the protein was cloned and expressed as a Beta-galactosidase (β-gal) fusion in Escherichia coli and the protein purified by affinity chromatography. The size of the recombinant protein released (18 kDa) was consistent with the calculated molecular mass of the polypeptide deduced from the DNA sequence. In Western blot assays, the purified protein was recognised by 71% of sera taken from patients infected with H. pylori, but by only 16% of sera taken from patients with unrelated or with no gastrointestinal disease. These results indicated that the 18 kDa protein from H. pylori was immunogenic and expressed in vivo. In other experiments, it was found that oral administration of this antigen did not protect mice against H. pylori colonisation following challenge with H. pylori.
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    Analysis of a Helicobacter pylori operon incorporating flagellar export genes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Institute of Molecular Biosciences at Massey University, New Zealand
    (Massey University, 1999) Porwollik, Steffan
    Motility of Helicobacter species has been shown to be essential for successful colonization of the host. Previous studies indicated that the regulation of flagellar biosynthesis in the human gastric pathogen Helicobacter pylori differs from the suggested model for Gram-negative Enterobacteriaeceae. In this study, the organization of two H. pylori genes involved in export of flagellar structural proteins was investigated. A 7 kb fragment of the H. pylori 17874 genome was cloned. Sequence determination and analysis revealed a putative operon comprising an ORF of unknown function (ORF03), and genes for the isoleucyl-tRNA synthetase (ileS), an Agrobacterium tumefaciens VirB11 homolog (virB11), an ATPase involved in flagellum-specific protein export (flil), a presumptive flagellar export channel component (fliQ), and a homolog of an enzyme necessary for cell wall biosynthesis (murB). The genetic organization of this region was found to be conserved in a panel of clinical H. pylori isolates, and in H. pylori 915 and SS1. The locus was also identified in the genome sequences of the H. pylori strains J99 and 26695. Cotranscription of ORF03, ileS, virB11, fliI, fliQ and murB was demonstrated by RT-PCR. Primer extension experiments identified the major transcription start site, which coincided with the A residue of the initiation codon of ORF03. A promoter element was inferred that resembled the E. coli ơ70 consensus sequence. In addition, a minor transcription start site was detected upstream from ileS. Non-polar mutation of virB11, fliI and fliQ was generated by an allele replacement strategy. Engineered H. pylori fliI and fliQ mutant strains were completely aflagellate and nonmotile, whereas a virB11 mutant still produced flagella and displayed slightly greater motility. The fliI and fliQ mutant strains produced severely reduced levels of flagellin and the hook protein FlgE, although reduction was less stable in the fliI mutant. Production of OMP4, a member of the outer membrane protein family identified in H. pylori 26695, was diminished in both the virB11 and the fliI mutant. This suggested related functions of the putative virulence factor transport protein (VirB11) and the flagellar export component (FliI).