Role of the Y. pseudotuberculosis YscF protein in toxin delivery into host cells

描述(由申请人提供)： 革兰氏阴性肠病原菌包括耶尔森氏菌、沙门氏菌、志贺氏菌以及肠源性和肠出血性大肠杆菌，可引起人类多种胃肠道疾病。所有这些病原体都含有一种基本的毒力因子，称为III型分泌系统，它将细菌毒素直接输送到宿主细胞中。III型分泌系统在细菌膜上产生一个结构，该结构有一个从细菌表面延伸出来的针状附属物，毒素通过该附属物分泌。分泌系统还在宿主质膜上产生一个孔，称为转运子，允许毒素进入宿主细胞胞浆。目前尚不清楚细菌表面的针状物和宿主细胞膜中的转运子是如何协作实现毒素传递的。为了确定感染期间有效的针-转位相互作用的要求，我分离了假结核耶尔森菌(YscF)针蛋白的突变，这些突变不能将毒素输送到宿主细胞，但保留了将毒素从细菌分泌到细胞外空间的能力。在这个建议中，我的目标是使用WT耶尔森氏菌和yscF突变体：1)确定感染期间针头是否与转位子直接接触，2)确定针-转位子相互作用是否需要宿主细胞中的孔形成，以及3)定位YscF中针头-转位子相互作用所需的区域和配对蛋白。 利用WT和突变的耶尔森氏菌表征细菌与宿主细胞的相互作用，将有助于深入了解假结核杆菌向宿主细胞输送毒素的机制。这种毒力机制在其他肠道病原体中是保守的，因此这项工作将直接适用于沙门氏菌、志贺氏菌和大肠杆菌引起的疾病的研究，并将发现抗微生物药物的候选对象。

DESCRIPTION (provided by applicant): Gram-negative enteropathogenic bacteria, including Yersinia, Salmonella, Shigella and both enteropathogenic and enterohemorrhagic E. coli, cause a wide variety of gastero intestinal diseases in humans. All of these pathogens harbor an essential virulence factor called the Type III Secretion System which delivers bacterial toxins directly into host cells. The Type III Secretion System produces a structure in the bacterial membrane with a needle-like appendage extending out from the bacterial surface through which toxins are secreted. The secretion system also produces a pore in the host plasma membrane, termed the translocon, which allows access of the toxins to the host cell cytosol. It is not known how the needle on the bacterial surface and the translocon in the host cell membrane cooperate to enable toxin delivery. In an effort to determine the requirements for a productive needle-translocon interaction during infection I have isolated mutations in the needle protein of Yersinia pseudotuberculosis (YscF) that are incapable of delivering toxins into the host cell, but retain the ability to secrete toxins out of the bacteria into the extracellular space. In this proposal, I aim to use both WT Yersinia and the yscF mutants to: 1) determine if the needle is in direct contact with the translocon during infection, 2) determine if the needle-translocon interaction is required for pore formation in the host cell and 3) map the regions in YscF and the partner protein that are required for needle-translocon interactions. Characterization of the bacteria-host cell interactions using both WT and mutant Yersinia will provide insight into the mechanism of toxin delivery into host cells used by Y. pseudotuberculosis. This virulence mechanism is conserved amongst other enteric pathogens, and thus this work will be directly applicable to the study of disease caused by Salmonella, Shigella and E. coli, and will uncover candidates for targets of anti-microbial agents.