OmpR and SsrB Regulation of Salmonella Virulence

项目摘要 沙门氏菌感染是世界范围内的主要健康问题。沙门氏菌引起疾病 通过表达位于致病岛上的基因。致病岛很大 获得性基因的片段，这些基因促进毒力，并且具有与其他基因不同的AT含量。 细菌染色体沙门氏菌致病岛-1（SPI-1）上的基因 使沙门氏菌能够粘附和侵入上皮细胞，而SPI-2基因是必需的， 全身性感染专门的分泌系统称为三型分泌系统， 在每个致病岛上编码，为沙门氏菌提供分泌 效应分子进入宿主，改变宿主功能并促进发病。本 该提案侧重于SPI-2基因表达的控制。它是最重要的毒力之一 沙门氏菌的决定因素，但其转录调控的复杂分子生物学， 不被理解。特别是，鉴定体内基因表达的途径 仍然定义不清。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， 表达反过来又受另外的调控网络控制，包括EnvZ/OmpR两个- 组成系统，转录激活因子SlyA和全局阻遏物H-NS。的 SPI-2的复杂调节需要集成多个环境信号，以确保 这些重要的毒力基因在巨噬细胞内的适当时间表达 吞噬体在这个提议中，我们将测试EnvZ感知酸化的 当沙门氏菌驻留在巨噬细胞空泡中时，细胞质中的细菌数量增加。使用氢-氘 交换质谱，我们将确定EnvZ的区域，改变构象， 是酸胁迫的结果OmpR~P产生的水平将使用荧光标记法测定。 将测定试剂的浓度和对SsrA/B表达和活化的影响。我们的初步 研究表明，沙门氏菌细胞质在液泡中被酸化至pH 5.7， 这个过程完全依赖于OmpR。我们将确定涉及的OmpR目标， 通过微阵列检测细胞内酸化并检测它们对增强沙门氏菌作用 在空泡中生存。了解这些目标为我们提供了抑制沙门氏菌的策略 在空泡中复制，这是传播和全身感染所必需的。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，可能会在不同的主机背景不同。使用我们在 在上一个资助期，我们将研究HeLa细胞，巨噬细胞和 3 T3成纤维细胞，并确定宿主因素，改变沙门氏菌感染过程中的功能。作为 通过我们的研究，我们将对分子事件有更深入的了解， 沙门氏菌感染的结果以及这些过程如何在宿主中靶向发生。

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 remain 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 an enhanced understanding of the molecular events that occur as a result of Salmonella infection and how these processes can be targeted in the host.