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

假结核杆菌 YscF 蛋白在毒素递送至宿主细胞中的作用

• 批准号: 7264086
• 负责人: ALISON J DAVIS
• 金额: $ 10.47万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-15 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7264086
• 关键词: Bacteria; Bacterial Infections; Bacterial Toxins; Cell Communication; Cell membrane; Cells; Chemicals; Complex; Cultured Cells; Cytolysis; Cytosol; Defect; Digestive System Disorders; Disabled Persons; Disease; Distal; Electron Microscopy; Erythrocytes; Escherichia coli; Escherichia coli EHEC; Extracellular Space; Genetic Screening; Goals; Host Defense; Human; Infection; Intestinal Diseases; Link; Maps; Mass Spectrum Analysis; Membrane; Monitor; Mutagenesis; Mutation; Needles; Pasteurella pseudotuberculosis; Principal Investigator; Process; Protein Analysis; Protein translocation; Proteins; Research; Role; Salmonella; Shigella; Site; Structure; Suppressor Mutations; Surface; System; Testing; Toxin; Travel; Type III Secretion System Pathway; Virulence; Virulence Factors; Work; Yersinia; Yersinia pestis V antigen; antimicrobial drug; appendage; base; crosslink; enteric pathogen; insight; mutant; pathogen; prevent; protein protein interaction; size

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.

描述（由申请人提供）： 革兰氏阴性肠道致病菌，包括耶尔森氏菌、沙门氏菌、志贺氏菌以及致病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称为 III 型分泌系统的重要毒力因子，可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中产生一种结构，该结构具有从细菌表面延伸出来的针状附属物，通过该结构分泌毒素。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。目前尚不清楚细菌表面的针和宿主细胞膜中的易位子如何配合以实现毒素输送。为了确定感染期间有效的针-易位子相互作用的要求，我分离出了​​假结核耶尔森氏菌 (YscF) 针蛋白的突变，这些突变无法将毒素输送到宿主细胞中，但保留了将毒素分泌到宿主细胞中的能力。将细菌带入细胞外空间。在本提案中，我的目标是使用 WT 耶尔森氏菌和 yscF 突变体来：1）确定感染期间针是否与易位子直接接触，2）确定宿主中孔形成是否需要针-易位子相互作用细胞和 3) 绘制 YscF 中的区域以及针-易位子相互作用所需的伴侣蛋白。 使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有助于深入了解将毒素输送到假结核耶尔森氏菌使用的宿主细胞中的机制。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，并将揭示抗微生物剂的候选靶点。