H. pylori is a remarkable microorganism that is uniquely adapted for survival in the human stomach, t infects, usually asymptomatically, over one half of the world's human population. However 15% of those infected will develop ulcer disease or gastric cancer. Our proposed research focuses on a single bacterial determinant, CagA, an effector protein translocated by a bacterial secretory apparatus into gastric epithelial cells. CagA is arguably the most important virulence determinant of this microbe, since ts presence is clearly correlated with ulcer disease and gastric malignancy, and its effects on host cell biology place it at the crux between microbial pathogenesis and cancer biology. We propose to address three fundamental questions related to how the initial encounter between the H. pylori CagA protein works to the advantage of the microorganism and to the detriment of the host. First, what are the molecular mechanisms by which CagA perturbs host epithelial cell biology? Second, what is the role of CagA for H. pylori survival in close association with the host cell surface? And finally, what are the in- vivo consequences of CagA delivery to the gastric mucosa in an animal model? Our previous research directs us to a clear focus on the interaction of the CagA protein and the host proteins of the apical junctional complex, a domain of the cell that controls cell polarity, provides the barrier function of the mucosa, and regulates the differentiation, growth and wound healing capacities of epithelia. CagA also triggers one, or possibly more, receptor tyrosine kinase signaling pathways. We predict that these are not unrelated functions, but rather that CagA usurps an intrinsic link between growth factor receptor signaling and the apical junctions. We have also hypothesized that H. pylori utilizes CagA to modify the host cell surface for colonization purposes, recruiting specific host proteins to the sites of bacterial attachment, disrupting cell polarity, and opening the epithelial junctions. We have proposed a hypothesis of CagA action that we believe can be tested both in-vitro and in-vivo. We have developed novel microscopic tools and an experimentally tractable model of chronic infection of polarized cells in culture to examine the cell biology of the CagA-host cell interaction. Moreover, we will also make extensive use of direct biochemical fractionation and transcriptional analysis to dissect the role of CagA at a molecular level. Finally we propose testing the observations we have gathered from precise in-vitro analysis in the more complex, but more relevant environment of the stomach in an animal host.
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