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Characterisation of the conserved Helicobacter pyloriproteins AhpC and HpaA

Doktorsavhandling
Författare Anneli Maria Lundström
Datum för examination 2000-12-21
ISBN 91-628-4582-9
Publiceringsår 2000
Publicerad vid Institutionen för medicinsk mikrobiologi och immunologi
Språk en
Ämneskategorier Mikrobiologi inom det medicinska området

Sammanfattning

Background: Infection with Helicobacter pylori gives rise to gastritis and may cause duodenal and gastric ulcers and gastric carcinoma in humans. The infection may induce an immune response against different antigens, among them a 26 kDa protein (AhpC) and a 29 kDa protein (HpaA). Aims: The aims were to characterise the 26 kDa and 29 kDa H. pylori proteins in terms of genotypic and phenotypic expression including localisation of HpaA in relation to growth phase and morphology of H. pylori bacteria. Furthermore, to analyse the species-specificity and the biological function of the 26 kDa protein and to compare different properties of native HpaA from H. pylori with recombinant HpaA (rHpaA) produced in E. coli. Methods: The AhpC function was studied by complementation experiments using an ahpC-deficient E. coli mutant. Specific monoclonal antibodies (Mab:s) and PCR were used to identify the AhpC protein and the genes in different Helicobacter species. The ahpC promoter was mapped by primer extension experiments and the transcription of ahpC and hpaA was analysed by Northern blot and RT-PCR (hpaA). The HpaA protein was studied by immunoblot, semi-quantitative dot blot, colony blot, flow cytometry and immuno-electron microscopy using a HpaA specific Mab. The hpaA gene was cloned, sequenced and overexpressed in E. coli. The biological properties of purified native and rHpaA were compared in a fetuin binding assay and by immunisation experiments in mice. The relevance of HpaA for infection in mice was studied using an hpaA-defective H. pylori mutant.Results: The 26 kDa protein showed alkyl hydroperoxide reductase (AhpC) activity and may be of importance for the defense against oxidative stress in the human stomach. The AhpC protein or gene was found in eight other Helicobacter species. The ahpC gene was transcribed mono-cistronically, during the exponential and stationary growth phases, but not at acidic pH. The hpaA gene was transcribed in a putative operon during the exponential phase and stationary phase, together with a gene (omp18) encoding an outer membrane protein, and possibly also together with a second gene proposed to be involved in peptide secretion and trafficking (tig). The hpaA gene expression and protein production were similar in different H. pylori isolates but the number of bacteria with HpaA on the surface varied between strains. Coccoids had the largest proportion of bacteria with HpaA on the surface. Native and rHpaA had similar immunological properties and the binding properties to fetuin were also similar. Infection with an hpaA-defective H. pylori mutant in mice resulted in reduced numbers of bacteria in the stomach as compared to the wild type bacterium, suggesting that HpaA may be of importance for colonisation or survival in vivo. Conclusions: AhpC and HpaA are conserved proteins, which might be important for the establishment of H. pylori infection in the stomach. Their genes are expressed for a long time in vitro, in particular in bacillary bacteria and less in coccoids. The surface exposure of HpaA varied between H. pylori strains and was highest in coccoid bacteria.

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