PROJECT SUMMARY (ABSTRACT) Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric mucosa of humans. Although most H. pylori-infected persons remain asymptomatic, potentially serious sequelae of infection include gastric adenocarcinoma, duodenal or gastric ulceration, and gastric lymphoma. Gastric cancer is the third leading cause of cancer-related death worldwide, and H. pylori has been classified as a type I carcinogen by the World Health Organization. One of the major secreted proteins of H. pylori is a toxin known as VacA. There is a high level of genetic variation among vacA alleles from unrelated H. pylori strains, and the encoded VacA proteins exhibit marked differences in their ability to cause alterations in human cells. A large body of literature indicates that H. pylori strains containing certain forms of vacA (termed s1, i1, or m1) are associated with a higher risk of gastric cancer or peptic ulcer disease than are strains containing other forms of vacA (termed s2, i2, or m2). Thus, VacA is considered to be an important H. pylori virulence factor. Most cellular effects of VacA are dependent on its ability to oligomerize and form anion-selective membrane channels, but relatively little is known about the molecular basis for these two critical steps in the VacA mechanism of action, and almost nothing is known about the roles of VacA oligomerization and pore formation in vivo. Therefore, the specific aims are to (i) elucidate the process by which VacA assembles into oligomeric structures; (ii) elucidate the process by which VacA inserts into membranes to form anion-selective channels, and (iii) define the consequences of VacA oligomerization and membrane channel formation in animal models. This work is relevant not only for the study of H. pylori-associated diseases, but will also increase our understanding of bacterial pore-forming toxins, chloride-conducting membrane channels, and β-helical passenger domains secreted by an autotransporter pathway.