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

描述（由申请人提供）：幽门螺杆菌是一种螺旋形革兰氏阴性细菌，在约50%的世界人口中定植于人胃粘膜。它是目前已知的唯一被世界卫生组织列为I型致癌物的细菌。H.幽门螺杆菌感染可潜在地导致胃溃疡、胃粘膜相关淋巴组织（MALT）淋巴瘤和胃腺癌的发展。H.幽门螺杆菌是一种叫做VacA的成孔毒素。VacA作为88 kDa单体（能够形成大的寡聚复合物）分泌，其能够结合到胃上皮细胞的表面并寡聚化以在膜内产生孔。虽然VacA的毒性在于其寡聚化和形成通道的能力，但VacA如何寡聚化和形成孔的潜在机制尚不清楚。我的论文项目旨在使用单粒子冷冻电子显微镜，二维电子晶体学，脂质结合测定和细胞活力测定的组合来生成和测试基于结构的VacA功能模型。我的工作将为理解VacA毒性对H. pylori发病机制在目标1中，我将使用单粒子cryo-EM生成VacA六聚体和十二聚体的<10？分辨率结构。在目标2中，我将描述VacA如何与脂质结合并在膜中形成孔。当这两个目标的结果结合起来时，这些结构快照将使我能够生成一个可测试的模型，用于说明VacA如何从可溶性毒素转变为膜插入毒素，从而改善我们对H的基本理解。幽门螺杆菌的发病机制，并提供了一个必要的平台，为开发新的治疗方法，可以阻止VacA孔形成所需的转换。

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.