(taken from the application) Helicobacter pylori infection is a significant risk factor for the development of peptic ulcer disease and gastric malignancies. The long-term objectives of this project are to elucidate pathogenic mechanisms whereby H. pylori causes disease, and to develop effective means for prevention and treatment of infection. This proposal focuses on an important virulence determinant of H. pylori: the vacuolating cytotoxin (VacA). In preliminary studies, we have purified the cytotoxin, cloned the vacA gene, demonstrated mosaicism in vacA alleles, demonstrated an association between type s1 vacA genotypes and peptic ulceration, and demonstrated that VacA binds and enters epithelial cells. Most recently, we have shown that VacA is a dodecamer of 90 kDa subunits that dissociates into functionally active monomers following exposure to acidic pH. The hypotheses of this proposal are (i) that VacA binds to a specific receptor on epithelial cells, (ii) that type m1 and m2 VacA proteins bind to different cell-surface receptors, (iii) that VacA oligomers enter cells via clathrin-mediated endocytosis, and (iv) that when acidified, VacA inserts into membranes to form ion-conductive channels.
The specific aims will be (i) to characterize the binding of VacA to the plasma membrane of epithelial cells, (ii) to identify and isolate the VacA receptor, and (iii) to investigate uptake of VacA by epithelial cells. In the first aim, we will develop methods for directly labelling VacA, characterize its binding to several different types of epithelial cells, compare the binding properties of VacA oligomers and monomers, and compare binding properties of type m1 and m2 VacA proteins. In the second aim, we will identify the putative VacA receptor by techniques involving cross-linking or immunoprecipitation methods, and seek to partially purify the receptor. In the third aim, we will use deep-etch electron microscopic imaging techniques to determine whether VacA inserts into membranes, and use pharmacologic agents and dynamin-mutant cell lines to determine the pathway whereby VacA enters cells. Understanding the mechanisms whereby VacA binds to and enters cells may ultimately result in the development of strategies to prevent and treat H. pylori-associated gastroduodenal illnesses.
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