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

描述（由申请人提供）： 革兰氏阴性肠致病菌包括耶尔森氏菌、沙门氏菌、志贺氏菌以及肠致病性和肠出血性大肠杆菌。大肠杆菌，导致各种各样的胃肠道疾病的人。所有这些病原体都具有一种称为III型分泌系统的基本毒力因子，该系统将细菌毒素直接递送到宿主细胞中。III型分泌系统在细菌膜中产生具有从细菌表面延伸出的针状附属物的结构，毒素通过该结构分泌。分泌系统还在宿主质膜上产生一个孔，称为易位子，它允许毒素进入宿主细胞胞质溶胶。细菌表面的针和宿主细胞膜中的易位子如何合作以实现毒素递送尚不清楚。为了确定在感染期间生产性针-易位子相互作用的要求，我分离了假结核耶尔森氏菌（YscF）针蛋白的突变，这些突变不能将毒素递送到宿主细胞中，但保留了将毒素从细菌分泌到细胞外空间的能力。在这个提议中，我的目标是使用野生型耶尔森氏菌和yscF突变体：1）确定在感染过程中针是否与易位子直接接触，2）确定针-易位子相互作用是否是宿主细胞中孔形成所需的，3）绘制YscF和配偶体蛋白中针-易位子相互作用所需的区域。 利用野生型和突变型耶尔森氏菌对细菌-宿主细胞相互作用的表征将提供对耶尔森氏菌将毒素递送到宿主细胞中的机制的深入了解。假结核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。大肠杆菌，并将发现候选人的目标抗菌剂。

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.