OmpR and SsrB Regulation of Salmonella Virulence

OmpR 和 SsrB 对沙门氏菌毒力的调节

• 批准号: 8633083
• 负责人: Linda J. Kenney
• 金额: --
• 依托单位: JESSE BROWN VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8633083
• 关键词: Acute; Amino Acids; Autoimmune Process; Bacterial Chromosomes; Bacterial Infections; Binding; Binding Sites; Biochemistry; Biosensor; Cells; Chronic; Communities; Complex; Cytoplasm; Cytoplasmic Tail; DNase-I Footprinting; Defect; Deuterium; Digitonin; Disease; Disease Outbreaks; Drug resistance; Ensure; Enteral; Epithelial Cells; Escherichia coli; Event; Fibroblasts; Figs - dietary; Fluorescence Microscopy; Funding; Gastroenteritis; Gene Expression; Genes; HIV Seropositivity; Health; Hela Cells; Histidine; Hospitals; Hydrogen; Hydrogen Bonding; Immune; In Vitro; Incidence; Infection; Integration Host Factors; Intercistronic Region; Invaded; LacZ Genes; Lead; Mass Spectrum Analysis; Measures; Membrane; Military Personnel; Molecular; Molecular Biology; Molecular Conformation; Monitor; Morbidity - disease rate; Mus; Needles; Osmolalities; Pathogenesis; Pathogenicity Island; Pathway interactions; Patients; Peptides; Phagosomes; Phosphorylation; Play; Population Density; Process; Production; Progress Reports; Reactive Arthritis; Reagent; Regulation; Reiter Disease; Reporting; Research; Rest; Role; Salmonella; Salmonella infections; Septicemia; Signal Transduction; Source; Stimulus; Stress; System; Systemic infection; Testing; Time; Transcription Coactivator; Transcriptional Regulation; Typhoid Fever; Vacuole; Veterans; Virulence; acid stress; base; extracellular; in vivo; interest; macrophage; novel; pathogen; protein-histidine kinase; public health relevance; research study; response; tool

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.

项目概要 沙门氏菌感染是世界范围内的一个主要健康问题。沙门氏菌引起疾病 通过表达位于致病岛的基因。致病岛较大 一系列获得性基因，可促进毒力并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并侵入上皮细胞，而需要 SPI-2 基因 用于全身感染。称为三型分泌系统的专门分泌系统是 每个致病岛上都有编码，为沙门氏菌提供分泌手段 效应分子进入宿主，改变宿主功能并促进发病机制。现在的 提案重点关注 SPI-2 基因表达的控制。它是最关键的毒力之一 沙门氏菌的决定因素，但其转录调控的复杂分子生物学， 不明白。特别是体内基因表达途径的鉴定 仍然不明确。我们的研究重点是用分子术语定义这些途径。 SPI-2基因表达由双组分调控系统SsrA/B控制，其 表达反过来又受到其他调控网络的控制，包括 EnvZ/OmpR 两个网络 组成系统，转录激活子 SlyA 和全局阻遏子 H-NS。这 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。在这个提案中，我们将测试 EnvZ 感知酸化的假设 当沙门氏菌驻留在巨噬细胞液泡中时，会进入细胞质。使用氢-氘 交换质谱，我们将确定改变构象的 EnvZ 区域为 酸应激的结果。产生的 OmpR~P 水平将使用 phos-tag 测定 将确定试剂以及对 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被酸化至 pH 5.7，这 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查其对增强沙门氏菌的作用 在液泡中存活。了解目标为我们提供了抑制沙门氏菌的策略 在液泡中复制，这是传播和全身感染所必需的。这 控制效应分子分泌到宿主巨噬细胞中的门控机制 是有争议的，并且在不同的主持人背景中可能会有所不同。使用我们开发的工具 在之前的资助期间，我们将检查 HeLa 细胞、巨噬细胞和 3T3 成纤维细胞并识别感染期间改变沙门氏菌功能的宿主因素。作为一个 通过我们的研究结果，我们将对分子事件有更深入的了解 由于沙门氏菌感染而发生，以及如何在宿主中针对这些过程。