Structural Studies of Bacterial Virulence Factors

描述(申请人提供)：大量医学上重要的细菌病原体利用III型蛋白分泌系统(T3SS)将大量毒力相关蛋白输送到宿主细胞中导致疾病。这些细菌中的T3SS以一种名为“分子注射器”的复杂纳米机器为中心，它跨越细菌的内膜和外膜，将丝状针状蛋白质投射到细胞外空间。T3SS的毒力因子底物具有生物化学多样性，操纵宿主细胞生物系统，如细胞骨架结构、信号转导、细胞周期进展和程序性细胞死亡，使细菌能够精确调节宿主组织和系统，从而有利于病原体的健康。该系统的底物被认为以部分非球状的状态通过分子注射器的内部通道，并在许多因素中包括与针结构对接的孔形成蛋白。在细菌内部，III型分泌器具有与底物相互作用并解开底物的ATPase，以及促进易位的特殊分泌伴侣。这项提案提出了一项计划，对这些毒力系统进行结晶学研究，特别是病原体鼠伤寒沙门氏菌。T3SS的三个具体方面将是研究的重点：(1)单个毒力因子在宿主中的作用；(2)细菌分泌伴侣与其底物之间的相互作用；(3)III型分泌系统ATPase及其相关元件与分泌伴侣及其底物的相互作用。这些元素及其生物络合物的晶体结构，结合后续的生化、细胞生物学和感染分析，将为这一广泛而重要的毒力系统的功能提供重要的新见解。由于利用这种毒力系统的细菌会导致广泛的人类、动物和植物疾病，了解它们是如何导致疾病的将为改善公共卫生提供重要工具。

DESCRIPTION (provided by applicant): A large number of medically important bacterial pathogens utilize a type III protein secretion system (T3SS) to deliver an arsenal of virulence-associated proteins into host cells to cause disease. The T3SS in these bacteria are centered on an intricate nano-machine termed a "molecular syringe" that spans both the inner and out membranes of the bacterium, projecting a filamentous needle-like protein into the extra-cellular space. The virulence factor substrates of T3SS are biochemically diverse, manipulating host cell biological systems such as cytoskeletal structure, signal transduction, cell cycle progression, and programmed cell death, allowing bacteria to precisely modulate host tissues and systems for the benefit of the pathogen. The substrates of the system are thought to travel in a partially non-globular state through the inner channel of the molecular syringe, and include among many factors pore forming proteins with which the needle structure docks. Within the bacterium, the type III secretion apparatus possesses an ATPase that interacts with and unfolds substrates, as well as specialized secretion chaperones that promote translocation. This proposal presents a plan to conduct crystallographic studies of these virulence systems, focused particularly on the pathogen Salmonella typhimurium. Three specific aspects of the T3SS will be the focus of the study: (1) individual virulence factor function in the host, (2) the interactions between bacterial secretion chaperones and their substrates, and (3) the interaction of the type III secretion system ATPase and associated elements with secretion chaperones and their substrates. Crystal structures of these elements and their biological complexes, in combination with follow-up biochemical, cell biological, and infection assays, will provide significant new insight into the functioning of this widespread and important virulence system. Because bacteria utilizing this virulence system cause widespread human, animal, and plant disease, this understanding how they cause disease will provide important tools for improving public health.