Structural and functional characterisation of the type IV secretion system of Helicobacter pylori

Approximately 75 % of all Gastric adenocarcinoma cases are associated with H. pylori infection (Herrera et al., 2009). The precise mechanism by which H. pylori promotes gastric cancer remains unclear. In particular, the induction of IL-8 by H. pylori infection plays a significant role in tumour development (Waugh et al., 2008). Furthermore, IL-8 is a potent angiogenic factor (Petzelbauer et al., 1995). Thus, understanding the molecular mechanisms by which H. pylori induces IL-8 secretion is of crucial importance for understanding the molecular basis of H. pylori-associated gastric cancer. It has been shown that the type IV secretion system of H. pylori is involved in the induction of proinflammatory responses upon infection of gastric epithelial cells (Gorrell et al., 2012). CagL, a component of the H. pylori type IV secretion system, has been shown to be essential for the type IV-mediated secretion of CagA and induction of IL-8 through interaction with integrins on epithelial cells (Kwok et al., 2007). The aim of this study was to further elucidate the mechanisms involved in the above process. In the first Results chapter of my thesis, I addressed the structure-function of CagL and its significance in H. pylori pathogenesis. Construction of H. pylori cagL mutants with a mutated arginine-glycine-aspartate (RGD) motif (a motif important for interaction with integrins), and deletion of virD4 (a gene essential for substrate translocation) showed that CagL plays a direct role in triggering proinflammatory responses in infected epithelial cells in a manner independent of CagA translocation. H. pylori mutants expressing CagL fused to a C-terminal tetracysteine (TC) or hemagglutinin (HA) tag were also constructed. The data showed that the C-terminal tagging of CagL has a critical effect on its function. Mutants with substitutions in the conserved cysteine residues of CagL revealed that these conserved cysteine amino acids in CagL do not contribute significantly to CagL-mediated IL-8 induction or CagA translocation during infection. Additionally, polymorphisms at residue positions 58 and 59 of CagL, which are suggested to be associated with an increased risk of gastric cancer in H. pylori-infected patients, did not show a major effect on CagL function in terms of IL-8 induction and CagA translocation, up to 24 h post-infection. H. pylori has been detected in multiple studies in the submucosal surface or lamina propria, close to the erythrocytes and within blood vessels (Amieva et al., 2003, Aspholm et al., 2006, Necchi et al., 2007) It is highly likely that H. pylori, once gaining access to the basal side of the gastric epithelium, will come into contact with endothelial cells. These interesting observations support the notion of a possible interaction between H. pylori and endothelial cells. In the remaining Results chapters of my thesis, I address the molecular mechanisms by which H. pylori interacts with endothelial cells. Using Human Umbilical Vein Endothelial Cells (HUVECs) as a model, my results showed that endothelial cells are significantly more potent than gastric epithelial cells in secreting the proinflammatory cytokine IL-8 upon infection by H. pylori. Triggering such responses in H. pylori-infected HUVECs is type IV secretion system-dependent and CagL plays an important role in the induction of IL-8 in H. pylori-infected endothelial cells. CagA, (type IV translocation substrate) plays an important role in induction of IL-8 during H. pylori infection of epithelial cells. In contrast, CagA does not play a role in induction of IL-8 in during H. pylori infection of endothelial cells, nor does CagA translocate to endothelial cells during H. pylori infection. Scanning electron microscopy of H. pylori-infected HUVECs showed that the pili-like structures, which are easily detected at the interface of H. pylori and infected gastric epithelial cells (Rohde et al., 2003) were absent in H. pylori-infected HUVECs. Previous studies show that these pili-like structures are associated with the presence of a functional type IV secretion system in H. pylori. Taken together, my new findings therefore suggest that the H. pylori type IV secretion system is likely to interact differently with different host cell types during infection. Moreover, my results also demonstrated that H. pylori exploit different host cell receptors in interacting with endothelial cells and gastric epithelial cells. These novel data suggest that IL-8 induction by H. pylori in endothelial cells is independent of the integrins α5β1 and αvβ3, but dependent upon epithermal growth factor receptors (EGFRs). Given that both EGFRs and IL-8 are important players in cancer progression and angiogenesis, it was of interest to investigate whether H. pylori stimulates angiogenesis. A few controversial studies reported that H. pylori induced or inhibited angiogenesis, but the mechanisms by which H. pylori triggers or inhibits proliferation remain poorly understood. Using HUVECs as a model, results showed that H. pylori induces proliferation of endothelial cells. This effect was independent of the H. pylori type IV secretion system or the function of host integrin receptors. Interestingly, similar to IL-8 induction by H. pylori, the EGFR receptor also plays a role in the stimulation of proliferation of HUVECs by H. pylori. The results from this study suggest that the blocking of integrin receptors in order to inhibit proinflammatory responses during H. pylori infection is not an ideal approach, because it appears that integrins are not involved in the interaction of H. pylori with endothelial cells. Although CagL is involved in the induction of IL-8 in endothelial cells, surprisingly this pathway does not occur through interaction of CagL with integrins. Finally, this study shows that the EGFR plays an important role in IL-8 induction by H. pylori-infected endothelial cells, and that the EGFR is also involved in the stimulation of proliferation of endothelial cells by H. pylori. This is consistent with the established fact that that the EGFR plays a crucial role in cancer progression (Baker et al., 2002). This study provides new insight into the molecular mechanisms through which H. pylori could promote cancer development.