OmpR and SsrB Regulation of Salmonella Virulence

描述（由申请人提供）： 沙门氏菌感染是世界范围内的主要健康问题。沙门氏菌通过表达位于致病岛上的基因引起疾病。致病性岛是获得性基因的大片，其促进毒力并且具有与细菌染色体的其余部分不同的AT含量。沙门氏菌致病岛-1（SPI-1）上的基因使沙门氏菌能够粘附并侵入上皮细胞，而SPI-2基因是全身感染所必需的。被称为三型分泌系统的专门分泌系统在每个致病岛上编码，其为沙门氏菌提供将效应分子分泌到宿主中的手段，所述效应分子改变宿主功能并促进发病。目前的建议集中在SPI-2基因表达的控制。它是沙门氏菌最关键的毒力决定因子之一，但其转录调控的复杂分子生物学尚不清楚。特别是，在体内基因表达的途径的鉴定仍然很难确定。我们的研究集中在分子方面定义这些途径。SPI-2基因表达受双组分调控系统SsrA/B控制，其表达又受另外的调控网络控制，包括EnvZ/OmpR双组分系统、转录激活因子SlyA和全局阻遏物H-NS。SPI-2的复杂调控需要整合多种环境信号，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。在这个建议中，我们将测试的假设，EnvZ的感觉酸化的细胞质时，沙门氏菌驻留在巨噬细胞空泡。使用氢-氘交换质谱，我们将确定由于酸胁迫而改变构象的EnvZ区域。使用荧光标记试剂测定产生的OmpR~P水平，并测定对SsrA/B表达和活化的影响。我们的初步研究表明，沙门氏菌的细胞质被酸化到pH 5.7的液泡，这一过程是完全依赖于OmpR。我们将通过微阵列确定参与细胞内酸化的OmpR靶点，并研究它们对增强沙门氏菌在液泡中存活的影响。了解目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。控制效应分子分泌进入宿主巨噬细胞的门控机制是有争议的，并且可能在不同的宿主背景中有所不同。使用我们在上一个资助期开发的工具，我们将检查HeLa细胞，巨噬细胞和3 T3成纤维细胞中的第三型分泌，并确定在感染期间改变沙门氏菌功能的宿主因素。由于我们的研究，我们将有一个作为沙门氏菌感染的结果发生的分子事件，以及这些过程如何可以在宿主中的目标有了更深入的了解。

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.