Understanding the molecular mechanisms of H. pylori VacA pore-formation

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DESCRIPTION (provided by applicant): Helicobacter pylori is a spiral shaped, gram-negative bacterium that colonizes the human gastric mucosa in ~50% of the world's population. It is currently the only known bacterium classified as a type I carcinogen by the World Health Organization. H. pylori infections can potentially lead to the development of gastric ulcers, gastric mucous associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma. One of the major virulence factors secreted by H. pylori is a pore-forming toxin known as VacA. VacA is secreted as an 88kDa monomer (capable of forming large oligomeric complexes) that is able to bind to the surface of gastric epithelial cells and oligomerize to create pores within the membrane. Although the toxicity of VacA lies in its ability to oligomerize and form channels, the underlying mechanism(s) for how VacA oligomerizes and forms pores are not understood. My thesis project is designed to use a combination of single particle cryo-electron microscopy, 2D electron crystallography, lipid binding assays, and cell viability assays to generate and test structure-based models of VacA function. My work will provide the mechanistic framework for understanding the contributions of VacA toxicity to H. pylori pathogenesis. In Aim 1, I will generate <10¿ resolution structures of VacA hexamers and dodecamers using single-particle cryo-EM. In Aim 2, I will characterize how VacA associates with lipids and forms pores in membranes. When results from both aims are combined, these structural snapshots will allow me to generate a testable model for how VacA transitions from a soluble to membrane-inserted toxin improving our basic understanding of H. pylori pathogenesis and providing a necessary platform for the development of new therapeutic approaches that can block the transitions required for VacA pore formation.