Plasmid-Mediated Virulence in Salmonella

描述(由申请人提供)：该项目的长期目标是确定细菌毒力因素和宿主反应，以确定严重的播散性沙门氏菌感染的结果。在美国，非伤寒沙门氏菌菌株是食源性疾病的常见原因，在美国的一次生物恐怖主义袭击中得到了有效的利用。此外，沙门氏菌是研究宿主-病原菌相互作用的分子生物学和免疫学的重要研究工具。这个项目的重点是沙门氏菌SPV基因座，这是系统性疾病所需的一个关键毒力决定因素。Spv操纵子编码SpvB，这是一种ADP核糖转移酶，可以修饰感染细胞中的肌动蛋白单体，导致肌动蛋白细胞骨架的丢失。SpvB的活性NAD结合位点位于C-末端结构域，是毒力表型所必需的。SpvB是第一个ADP-核糖化毒素，它特异性地作用于细胞内的细菌，也是第一个肌动蛋白修饰毒素，被证明在细胞内的发病机制中起关键作用。该项目将侧重于SpvB分泌和运输的新方面，这些方面允许毒素从吞噬小体而不是从细胞外环境进入宿主细胞。SpvB介导的细胞毒性和细胞死亡的机制将被确定。其具体目的是：1)分析SpvB从吞噬小体分泌和转运到宿主细胞胞质的机制。假设这一过程既需要SPI2编码的细菌III型分泌系统，也需要SpvB蛋白的特定区域。2)明确SpvB N末端结构域在细胞内感染中的作用和功能。假设N-末端结构域包含指定从吞噬小体到宿主细胞细胞质的运输的区域。3)确定沙门氏菌细胞内感染过程中宿主细胞肌动蛋白解聚的病理生理后果。假说是，肌动蛋白解聚破坏了细胞功能的多个方面，促进了细胞内的生长，并触发了延迟的细胞死亡途径，从而在感染过程中加强了沙门氏菌的细胞间传播。

DESCRIPTION (provided by applicant): The long-term goal of this project is to define bacterial virulence factors and host responses that determine the outcome of serious, disseminated Salmonella infections. Non-typhoid Salmonella strains are common causes of food-borne illness in the United States and have been used effectively in a bioterrorism attack in this country. In addition, Salmonella represent an important research tool to study the molecular biology and immunology of the host-pathogen interaction. This project focuses on the Salmonella spv locus, a critical virulence determinant required for systemic disease. The spv operon encodes SpvB, an ADP-ribosyl transferase that modifies actin monomers in infected cells and leads to loss of the actin cytoskeleton. The active NAD-binding site of SpvB is located in the C-terminal domain and is required for the virulence phenotype. SpvB is the first ADP-ribosylating toxin that acts specifically from bacteria located intracellularly, and the first actin modifying toxin shown to be crucial for intracellular pathogenesis. This project will focus on the novel aspects of SpvB secretion and transport that allow the toxin to access the host cell from the phagosome rather than from the extracellular environment. The mechanisms of cytotoxicity and cell death mediated by SpvB will be determined. The Specific Aims are: 1) to analyze the mechanism of SpvB secretion and transport from the phagosome into the host cell cytoplasm. The hypothesis is that this process requires both the SPI2-encoded bacterial type III secretion system and specific regions of the SpvB protein. 2) to define the role and function of the N-terminal domain of SpvB in intracellular infection. The hypothesis is that the N-terminal domain contains regions that specify transport from the phagosome into the host-cell cytoplasm. 3) to determine the pathophysiologic consequences of actin depolymerization in the host cell during intracellular infection by Salmonella. The hypothesis is that actin depolymerization disrupts multiple aspects of cellular function, enhances intracellular growth, and triggers a delayed cell death pathway that enhances the cell-to cell spread of Salmonella during the infectious process.