Plasmid-Mediated Virulence in Salmonella

描述（由申请人提供）：该项目的长期目标是确定决定严重、播散性沙门氏菌感染结果的细菌毒力因素和宿主反应。非伤寒沙门氏菌菌株是美国食源性疾病的常见原因，并已被有效地用于该国的生物恐怖主义袭击。此外，沙门氏菌是研究宿主-病原体相互作用的分子生物学和免疫学的重要研究工具。该项目重点关注沙门氏菌 spv 基因座，这是全身性疾病所需的关键毒力决定因素。 spv操纵子编码SpvB，这是一种ADP-核糖基转移酶，可修饰受感染细胞中的肌动蛋白单体并导致肌动蛋白细胞骨架丢失。 SpvB 的活性 NAD 结合位点位于 C 端结构域，是毒力表型所必需的。 SpvB 是第一个专门作用于细胞内细菌的 ADP 核糖基化毒素，也是第一个被证明对细胞内发病机制至关重要的肌动蛋白修饰毒素。该项目将重点关注 SpvB 分泌和运输的新方面，这些方面允许毒素从吞噬体而不是从细胞外环境进入宿主细胞。将确定 SpvB 介导的细胞毒性和细胞死亡的机制。具体目标是：1）分析SpvB分泌和从吞噬体转运到宿主细胞细胞质的机制。假设该过程需要 SPI2 编码的 III 型细菌分泌系统和 SpvB 蛋白的特定区域。 2) 明确SpvB N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体转运到宿主细胞细胞质的区域。 3)确定沙门氏菌细胞内感染期间宿主细胞中肌动蛋白解聚的病理生理学后果。该假设认为，肌动蛋白解聚会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，从而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.