Structural Studies of Bacterial Virulence Factors

描述（由申请人提供）：大量医学上重要的细菌病原体利用III型蛋白分泌系统（T3SS）将毒力相关蛋白库传递到宿主细胞中引起疾病。这些细菌中的T3SS集中在一个被称为“分子注射器”的复杂纳米机器上，该机器横跨细菌的内外膜，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、细胞周期进程和程序性细胞死亡，使细菌能够精确地调节宿主组织和系统，以使病原体受益。该系统的底物被认为以部分非球状状态通过分子注射器的内部通道，并包括与针结构对接的许多孔隙形成蛋白。在细菌中，III型分泌装置具有与底物相互作用并展开底物的atp酶，以及促进易位的专门分泌伴侣。这一建议提出了一个计划，进行晶体学研究这些毒力系统，特别是集中在病原体鼠伤寒沙门菌。T3SS的三个具体方面将是研究的重点：(1)个体毒力因子在宿主中的功能，(2)细菌分泌伴侣及其底物之间的相互作用，(3)III型分泌系统atp酶及其相关元件与分泌伴侣及其底物的相互作用。这些元素及其生物复合物的晶体结构，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。由于细菌利用这种毒力系统引起广泛的人类、动物和植物疾病，因此了解它们如何引起疾病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.