OmpR and SsrB Regulation of Salmonella Virulence

描述(由申请人提供)： 项目简介：沙门氏菌感染是世界范围内的一个主要健康问题。沙门氏菌通过表达位于致病岛上的基因来致病。致病岛是一大片促进毒力的获得性基因，其AT含量与细菌染色体的其余部分不同。沙门氏菌致病性岛-1(SPI-1)上的基因使沙门氏菌能够附着和入侵上皮细胞，而SPI-2基因是全身感染所必需的。沙门氏菌的每个致病岛上都编码有专门的分泌系统，称为三型分泌系统，它们为沙门氏菌提供了将效应分子分泌到宿主体内的手段，从而改变宿主功能并促进致病。本提案的重点是控制SPI-2基因的表达。它是沙门氏菌最关键的毒力决定因素之一，但其转录调控的复杂分子生物学尚不清楚。特别是，对体内基因表达途径的识别仍然缺乏明确的定义。我们的研究集中在从分子的角度定义这些途径。SPI-2基因的表达受一个双组分调控系统SsrA/B的控制，而SsrA/B系统的表达又受额外的调控网络控制，包括EnvZ/OmpR双组分系统、转录激活因子SlyA和全局抑制因子H-NS。SPI-2的复杂调控需要多种环境信号的整合，以确保这些重要的毒力基因在巨噬细胞吞噬体内的适当时间表达。在这项提议中，我们将检验这样的假设，即当沙门氏菌驻留在巨噬细胞空泡中时，EnvZ感觉到酸化的细胞质。利用氢-氚交换质谱仪，我们将确定由于酸胁迫而改变构象的EnvZ区域。将使用PHOS-Tag试剂测定产生的OmpR~P水平，并确定其对SsrA/B表达和激活的影响。我们的初步研究表明，沙门氏菌细胞质在液泡中酸化到pH 5.7，这一过程完全依赖于OmpR。我们将通过基因芯片识别参与细胞内酸化的OmpR靶点，并检测它们在促进沙门氏菌在空泡中存活的作用。了解这些靶标为我们提供了一种抑制沙门氏菌在空泡中复制的策略，这是传播和全身感染所必需的。门控控制效应分子分泌到宿主巨噬细胞的机制是有争议的，并且可能在不同的宿主背景下有所不同。使用我们在前一个资助期开发的工具，我们将检测HeLa细胞、巨噬细胞和3T3成纤维细胞的第三型分泌，并确定在感染期间改变沙门氏菌功能的宿主因素。作为我们研究的结果，我们将对沙门氏菌感染引起的分子事件以及如何在宿主中针对这些过程有更好的理解。

DESCRIPTION (provided by applicant): PROJECT SUMMARY: Salmonella infections are a major health problem worldwide. Salmonella causes disease by expressing genes that are located on pathogenicity islands. Pathogenicity islands are large tracts of acquired genes that promote virulence and have a different AT content from the rest of the bacterial chromosome. Genes that reside on Salmonella Pathogenicity Island-1 (SPI-1) enable Salmonella to adhere to and invade epithelial cells, whereas SPI-2 genes are required for systemic infection. Specialized secretory systems termed type three secretion systems are encoded on each pathogenicity island that provide Salmonella with the means to secrete effector molecules into the host that alter host functions and promote pathogenesis. The present proposal focuses on the control of SPI-2 gene expression. It is one of the most critical virulence determinants of Salmonella, yet the complex molecular biology of its transcriptional regulation, is not understood. In particular, the identification of the pathways for gene expression in vivo remains poorly defined. Our research is focused on defining these pathways in molecular terms. SPI-2 gene expression is controlled by a two-component regulatory system SsrA/B, whose expression is in turn controlled by additional regulatory networks, including the EnvZ/OmpR two- component system, the transcriptional activator SlyA and the global repressor H-NS. The complex regulation of SPI-2 requires integration of multiple environmental signals to ensure that these important virulence genes are expressed at the appropriate time within the macrophage phagosome. In this proposal, we will test the hypothesis that EnvZ senses the acidified cytoplasm when Salmonella resides in the macrophage vacuole. Using hydrogen-deuterium exchange mass spectrometry, we will identify the regions of EnvZ that change conformation as a result of acid stress. The levels of OmpR~P produced will be determined using phos-tag reagents and the effect on SsrA/B expression and activation will be determined. Our preliminary studies indicate that the Salmonella cytoplasm is acidified to pH 5.7 in the vacuole and that this process is completely dependent on OmpR. We will identify the OmpR targets involved in intracellular acidification by microarray and examine their effect on enhancing Salmonella survival in the vacuole. Knowing the targets provides us with a strategy to inhibit Salmonella replication in the vacuole, which is required for dissemination and systemic infection. The mechanism of gating that controls the secretion of effector molecules into the host macrophage is controversial and may vary in different host backgrounds. Using the tools we developed in the previous funding period, we will examine type three secretion in HeLa cells, macrophages and 3T3 fibroblasts and identify host factors that alter Salmonella function during infection. As a result of our studies, we will have a enhanced understanding of the molecular events that occur as a result of Salmonella infection and how these processes can be targeted in the host.