| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系 === 105 === Persistent infection of Helicobacter pylori increases the risk of developing gastroduodenal diseases, including peptic ulcer, duodenal ulcer, and gastric adenocarcinoma. Motility mediated by the flagella of H. pylori plays an important role for the cells to move toward the gastric mucus and facilitates further colonization onto the epithelium. Previous studies reported that global regulator CsrA is necessary for the full motility of H. pylori through controlling rpoN (σ54) expression with unknown mechanism. The aim of my study is to investigate the function of 5 putative genes (jhp0349, jhp0424, jhp0691, jhp1333, and jhp1431), which were found to be regulated by CsrA of H. pylori strain J99 by RNAseq. First, RNA levels of these genes in csrA-mutant J99 were checked by RT-qPCR. Online bioinformatics analysis suggested that four of these genes (jhp0349, jhp0424, jhp0691, and jhp1333) were associated to the flagella system. Then, we constructed insertion mutants of three genes (jhp0349, jhp0424, and jhp1431) and deletion mutant of jhp0691 and its revertant. By comparing with wild-type, all mutants resulted in varying degrees of motility lost (jhp0349, ~50%; jhp0424, 28~81%; jhp0691, 88%; jhp1431, 52%) in the soft-agar motility assay. To understand whether these genes are under rpoN regulation, RT-qPCR was conducted and 4 genes (jhp0349, jhp0424, jhp0691, and jhp1333) were found to be down-regulated in the rpoN-mutant J99. The potential rpoN boxes were found in the promoter regions of jhp0349 and jhp0691. We also investigated whether the unknown genes participate in virulence factors other than motility in H. pylori J99. All mutants and revertant were tested for urease activity, survival under oxidative stress, and CagA and VacA activity. The results showed that all strains had similar urease activity as wild-type. The viability of wild-type J99 under 200 mM H2O2 exposure decreased slowly within 2 h, all mutants and revertant showed the similar except for jhp0691 mutant whose viability merely dropped. AGS cells infected by each strain were all observed similar vacuolation (70~80%) as that of wild-type (76.35%). Finally, AGS cells infected by either strain expressed the hummingbird-phenotype in a similar degree (8~12%) with that of wild-type (9.93%). Taken together, we’ve identified four putative genes (jhp0349, jhp0424, jhp0691, and jhp1431) in this study and one putative gene (jhp1333) in previous study playing a role in Helicobacter pylori J99 motility under the CsrA control. Three of them (jhp0349, jhp0424, and jhp0691) were predicted to function as a member of the flagellar system. None of these genes were involved in urease activity, survival under oxidative stress, and VacA and CagA activity.
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