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的革兰氏阴性细菌，在世界上约50%的人口中定植于人类胃粘膜。它是目前已知唯一被世界卫生组织列为I型致癌物的细菌。幽门螺杆菌感染可能导致胃溃疡、胃黏膜相关淋巴组织淋巴瘤和胃腺癌的发展。幽门螺杆菌分泌的主要毒力因子之一是一种被称为VacA的成孔毒素。VacA以88kDa单体的形式分泌（能够形成大的寡聚复合物），能够结合到胃上皮细胞表面并寡聚在膜内形成孔。尽管VacA的毒性在于其寡聚和形成通道的能力，但VacA寡聚和形成孔的潜在机制尚不清楚。我的论文项目旨在结合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毒性对幽门螺杆菌发病机制的贡献提供机制框架。在目标1中，我将使用单粒子冷冻电镜生成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上形成孔隙。当两个目标的结果结合在一起时，这些结构快照将使我能够生成一个可测试的模型，用于研究VacA如何从可溶性毒素转变为膜插入毒素，从而提高我们对幽门螺杆菌发病机制的基本理解，并为开发新的治疗方法提供必要的平台，这些方法可以阻断VacA孔形成所需的转变。

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.