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.

项目摘要 沙门氏菌感染是全球主要的健康问题。沙门氏菌会引起疾病 通过表达位于致病岛上的基因。致病岛很大 促进毒力并在含量上不同的获得基因与其余部分不同 细菌染色体。位于沙门氏菌致病性岛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基因则需要 用于全身感染。专门的分泌系统称为三型分泌系统 在每个致病岛上编码，这些岛提供沙门氏菌的分泌方式 效应子分子进入宿主，以改变宿主功能并促进发病机理。现在 提案的重点是控制SPI-2基因表达。这是最关键的毒力之一 沙门氏菌的决定因素，是其转录调控的复杂分子生物学， 不了解。特别是，体内基因表达途径的识别 保持较差。我们的研究重点是用分子术语定义这些途径。 SPI-2基因表达由两个组件调节系统SSRA/B控制 表达又受其他调节网络的控制 组件系统，转录激活剂Slya和全局阻遏物H-NS。这 SPI-2的复杂调节需要整合多个环境信号，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。在此提案中，我们将测试ENVZ感应酸化的假设 当沙门氏菌驻留在巨噬细胞液泡中时，细胞质。使用氢驱虫 交换质谱法，我们将确定Envz的区域改变构象为 酸应激的结果。产生的OMPR 〜p的水平将使用PHOS标签确定 将确定试剂和对SSRA/B表达和激活的影响。我们的初步 研究表明，在液泡中将沙门氏菌细胞质酸化为pH 5.7，这是 过程完全取决于OMPR。我们将确定涉及的OMPR目标 通过微阵列进行细胞内酸化，并检查它们对增强沙门氏菌的影响 液泡中的生存。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 控制效应分子向宿主巨噬细胞分泌的门控机制 是有争议的，在不同的主机背景中可能会有所不同。使用我们在 以前的资金期，我们将检查Hela细胞，巨噬细胞和 3T3成纤维细胞并确定在感染过程中改变沙门氏菌功能的宿主因子。作为 我们的研究结果，我们将对分子事件有增强的理解 由于沙门氏菌感染以及如何将这些过程靶向宿主。