Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958

Helicobacter pylori is a gastric pathogen which infects ~50% of the global population and can lead to the development of gastritis, gastric and duodenal ulcers and carcinoma. Genome sequencing of H. pylori revealed high levels of genetic variability; this pathogen is known for its adaptability due to mechanisms including phase variation, recombination and horizontal gene transfer. Motility is essential for efficient colonisation by H. pylori. The flagellum is a complex nanomachine which has been studied in detail in E. coli and Salmonella. In H. pylori, key differences have been identified in the regulation of flagellum biogenesis, warranting further investigation. In this study, the genomes of two H. pylori strains (CCUG 17874 and P79) were sequenced and published as draft genome sequences. Comparative studies identified the potential role of restriction modification systems and the comB locus in transformation efficiency differences between these strains. Core genome analysis of 43 H. pylori strains including 17874 and P79 defined a more refined core genome for the species than previously published. Comparative analysis of the genome sequences of strains isolated from individuals suffering from H. pylori related diseases resulted in the identification of “disease-specific” genes. Structure-function analysis of the essential motility protein HP0958 was performed to elucidate its role during flagellum assembly in H. pylori. The previously reported HP0958-FliH interaction could not be substantiated in this study and appears to be a false positive. Site-directed mutagenesis confirmed that the coiled-coil domain of HP0958 is involved in the interaction with RpoN (74-284), while the Zn-finger domain is required for direct interaction with the full length flaA mRNA transcript. Complementation of a non-motile hp0958-null derivative strain of P79 with site-directed mutant alleles of hp0958 resulted in cells producing flagellar-type extrusions from non-polar positions. Thus, HP0958 may have a novel function in spatial localisation of flagella in H. pylori

Molecular analysis of inflammatory mechanisms associated with Escherichia coli and inflammatory bowel diseases

Inflammatory bowel diseases (IBD), encompasses a range of chronic, immune-mediated inflammatory disorders that are usually classified under two major relapsing conditions, Crohn’s Disease (CD) and ulcerative colitis (UC). Extensive studies in the last decades have suggested that the etiology of IBD involves environmental and genetic factors that lead to dysfunction of epithelial barrier with consequent deregulation of the mucosal immune system and inadequate responses to gut microbiota.Over the last decade, the microbial species that has attracted the most attention, with respect to CD etiology, is Eschericia coli. In CD tissue, E. coli antigens have also been identified in macrophages within the lamina propria, granulomas, and in the germinal centres of mesenteric lymph nodes of patients. They have been shown to adhere to and invade intestinal epithelial cells whilst also being able to extensively replicate within macrophages. Through the work of genome-wide association studies (GWAS), there is growing evidence to suggest that the microbial imbalance between commensal and pathogenic bacteria in the gut is aided by a defect in the innate immune system. Autophagy represents a recently investigated pathway that is believed to contribute to the pathogenesis of CD, with studies identified a variant of the autophagy gene, ATG16L1, as a susceptibility gene. The aim of my thesis was to study the cellular and molecular mechanism promoted by E.coli strains in epithelial cells and to assess their contribution to IBD pathology. To achieve this we focused on developing both an in vitro and in vivo model of AIEC infection. This allowed us to further our knowledge on possible mechanisms utilised by AIEC that promoted their survival, as well as developing a better understanding of host reactions. We demonstrate a new survival mechanism promoted by E.coli HM605, whereby it induces the expression of the anti-apoptotic proteins Bcl-XL and BCL2, all of which is exacerbated in an autophagy deficient system. We have also demonstrated the presence of AIEC-induced inflammasome responses in epithelial cells which are exacerbated in an autophagy deficient system and expression of NOD-like receptors (NLRs) which might mediate inflammasome responses in vivo. Finally, we used the Citrobacter rodentium model of infectious colitis to identify Pellino3 as an important mediator in the NOD2 pathway and regulator of intestinal inflammation. In summary, we have developed robust and versatile models of AIEC infection as well as provide new insights into AIEC mediated survival pathways. The collected data provides a new perception into why AIEC bacteria are able to prosper in conditions associated with Crohn’s disease patients with a defect in autophagy.