Plasmid-Mediated Virulence in Salmonella

描述（由申请人提供）：本项目的长期目标是确定细菌毒力因子和宿主反应，这些因子和宿主反应决定严重的播散性沙门氏菌感染的结果。非伤寒沙门氏菌菌株是美国食源性疾病的常见原因，并已在该国的生物恐怖主义袭击中得到有效利用。此外，沙门氏菌代表了研究宿主-病原体相互作用的分子生物学和免疫学的重要研究工具。该项目的重点是沙门氏菌spv基因座，一个关键的毒性决定因素所需的系统性疾病。spv操纵子编码SpvB，一种ADP-核糖基转移酶，其修饰感染细胞中的肌动蛋白单体并导致肌动蛋白细胞骨架的损失。SpvB的活性NAD结合位点位于C-末端结构域，并且是毒力表型所需的。SpvB是第一个ADP-核糖基化毒素，其特异性地作用于位于细胞内的细菌，并且第一个肌动蛋白修饰毒素被证明对细胞内发病机制至关重要。该项目将集中在SpvB分泌和运输的新方面，使毒素从吞噬体进入宿主细胞，而不是从细胞外环境。将确定由SpvB介导的细胞毒性和细胞死亡的机制。具体目的是：1）分析SpvB从吞噬体分泌和转运到宿主细胞质的机制。该假说认为，这一过程需要SPI 2编码的细菌III型分泌系统和SpvB蛋白的特定区域。2)确定SpvB的N端结构域在胞内感染中的作用和功能。假设是N-末端结构域包含指定从吞噬体运输到宿主细胞质的区域。3)确定沙门氏菌胞内感染过程中宿主细胞内肌动蛋白解聚的病理生理后果。假设是肌动蛋白解聚破坏细胞功能的多个方面，增强细胞内生长，并触发延迟细胞死亡途径，增强沙门氏菌在感染过程中的细胞间传播。

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.