Regulation of the Vibrio cholerae intestinal exit plan

霍乱弧菌肠道排出计划的调节

• 批准号: 7739331
• 负责人: ANISIA J SILVA-BENITEZ
• 金额: $ 21万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7739331
• 关键词: Acute; Adherence; Africa; Animal Model; Asia; Bacteria; Bacteria sigma factor KatF protein; Binding Sites; Biological Assay; Cholera; Cholera Toxin; Complex; Confocal Microscopy; Coupled; Cultured Cells; Cyclic AMP Receptor Protein; Data; Data Set; Diarrhea; Disease; Disease Outbreaks; Ecosystem; Environment; Epidemic; Epidemiology; Event; Feces; Fluorescence; Gastric Acid; Genes; Genetic Transcription; Genome; Hemagglutinin; Histones; Human; Infant; Infection; Intestinal Mucosa; Intestines; Knowledge; Label; Laboratories; Latin America; Life Cycle Stages; Light; Link; Mastigophora; Metalloproteases; Modeling; Mucous body substance; Mus; Oral; Oryctolagus cuniculus; Peptide Hydrolases; Persons; Phenotype; Physiological; Pilum; Process; Production; Regulation; Research; Rice; Role; Route; Shipping; Ships; Small Intestines; Staging; Swimming; Testing; Toxin; Up-Regulation; Vibrio; Vibrio cholerae; Vibrio cholerae O1; Virulence; Virulence Factors; Water; bacterial genetics; base; biological adaptation to stress; cell motility; chromatin immunoprecipitation; defined contribution; fitness; hemagglutinin-protease; in vivo; mutant; oligomycin sensitivity-conferring protein; pathogen; programs; protein structure; public health relevance; quorum sensing; transmission process

DESCRIPTION (provided by applicant): Cholera is an acute water-borne diarrheal disease caused by Vibrio cholerae of serogroups O1 and O139. The bacterium is transmitted person-to-person by the fecal oral route. Infecting Vibrios that overcome the gastric acid barrier swim toward and adhere to the intestinal mucosa and express major virulence factors such as the toxin co-regulated pilus and cholera toxin, which is largely responsible for the profuse rice-watery diarrhea typical of this disease. At a late stage of infection, V. cholerae down-regulates the expression of virulence factors and detaches to return to the environment. Previous studies in our laboratory showed that the RpoS- dependent Zn-metalloprotease hemagglutinin (HA)/protease encoded by hapA promote detachment of V. cholerae from cultured cells and mucus. A study conducted in the rabbit ileal loop model subsequently demonstrated that V. cholerae detachment or mucosal escape is facilitated by RpoS which enhances the expression of motility. It has been recently shown that freshly shed Vibrios in cholera stool are in a hyper infective state that could promote person-to-person cholera transmission. Therefore, detachment of hyper infective Vibrios from the intestinal mucosa is a key step in the cholera transmission chain. We have studied the regulatory circuitry that controls the expression of HA/protease. These studies have unveiled a complex regulatory network involving the cyclic AMP receptor protein (CRP), the master quorum sensing regulator HapR, the histone-like nucleoid structuring protein (H-NS), and the general stress response regulator RpoS that act in concert to inhibit the expression of virulence and to enhance the production of HA/protease and motility. The central hypothesis of this research plan is that the coordinated up-regulation of HA/protease and motility late in infection promotes V. cholerae detachment or mucosal escape in a synergistic manner. In this study we propose to rigorously test this hypothesis by integrating a variety of approaches such as bacterial genetics, chromatin immunoprecipitation (ChIP), ChiP-sequencing, confocal microscopy and animal models. In Aim 1, we will determine the regulatory interactions that trigger V. cholerae detachment late in infection. To this end, we plan to examine how H-NS and RpoS regulate the expression of RpoN and FlrA to coordinate the expression of flagellar motility with HA/protease production. We will use ChIP to determine if RpoS (CS) directly participates in transcription of rpoN, flrA and hapA. In addition, immunoprecipitates will shipped to GenPathway (San Diego, CA) to determine the RpoS binding sites in the V. cholerae genome. This data will be used to putatively identify intermediate regulators linking RpoS with the regulation of motility, HA/protease and/or promoting detachment. In Aim 2, we will examine the role of motility and HA/protease in detachment in vivo. To this end, we will use the rabbit ileal loop model, confocal microscopy and fluorescently-labeled isogenic mutants to define the contribution HA/protease and motility to mucosal escape. In addition, we will investigate if RpoS enhances the expression of additional detachment factors by comparing mucosal escape in RpoS-positive and negative isogenic strains that lack flaA (non-motile, non-flagellated) and hapA. Finally, we will use an infant mouse hyper infectivity assay to examine the temporal relationship between induction of hyper infectivity and detachment. We expect that a better understanding of the events that inhibit virulence and promote detachment late in infection will open new avenues to treat cholera and provide important clues to explain the epidemic potential of this human pathogen. PUBLIC HEALTH RELEVANCE: Cholera continues to cause seasonal outbreaks in highly populated regions in Asia, Africa and Latin America. This disease is caused by Vibrio cholerae of serogroups O1 and O139 which are transmitted person-to-person by the fecal oral route. V. cholerae colonizes the human small intestine, secretes cholera toxin to cause diarrhea and detaches late in infection to return to the aquatic environment. Freshly shed (detached) Vibrios are in a "hyper infective" physiological stage that could enhance person-to person transmission and cholera spread. Therefore, detachment of hyper infective bacteria from the intestinal mucosa late in infection is a critical step of the cholera transmission chain. The general aim of our research is to establish the regulatory interactions that trigger V. cholerae detachment and the role of protease production and motility in this process. These studies could open new avenues to treat cholera and shed light on the epidemic potential of this pathogen.

描述（由申请人提供）：霍乱是一种由 O1 和 O139 血清群霍乱弧菌引起的急性水源性腹泻病。该细菌通过粪便口腔途径在人与人之间传播。克服胃酸屏障的感染弧菌游向并粘附在肠粘膜上，并表达主要毒力因子，例如毒素共同调节的菌毛和霍乱毒素，这在很大程度上导致了这种疾病典型的大量米水样腹泻。在感染后期，霍乱弧菌下调毒力因子的表达并脱离并返回环境。我们实验室之前的研究表明，hapA 编码的 RpoS 依赖性锌金属蛋白酶血凝素 (HA)/蛋白酶可促进霍乱弧菌从培养细胞和粘液中脱离。随后在兔回肠环模型中进行的一项研究表明，RpoS 可增强运动性的表达，从而促进霍乱弧菌脱离或粘膜逃逸。最近的研究表明，霍乱粪便中新脱落的弧菌处于高度感染状态，可能会促进人与人之间的霍乱传播。因此，高度感染性弧菌从肠粘膜脱离是霍乱传播链中的关键步骤。我们研究了控制 HA/蛋白酶表达的调节电路。这些研究揭示了一个复杂的调节网络，涉及环 AMP 受体蛋白 (CRP)、主要群体感应调节器 HapR、组蛋白样核结构蛋白 (H-NS) 和一般应激反应调节器 RpoS，这些调节器协同作用抑制毒力的表达并增强HA/蛋白酶的产生和运动。该研究计划的中心假设是感染后期HA/蛋白酶和运动性的协调上调以协同方式促进霍乱弧菌脱离或粘膜逃逸。在这项研究中，我们建议通过整合细菌遗传学、染色质免疫沉淀 (ChIP)、ChiP 测序、共聚焦显微镜和动物模型等多种方法来严格检验这一假设。在目标 1 中，我们将确定在感染后期触发霍乱弧菌脱离的调控相互作用。为此，我们计划研究 H-NS 和 RpoS 如何调节 RpoN 和 FlrA 的表达，以协调鞭毛运动的表达与 HA/蛋白酶的产生。我们将使用 ChIP 来确定 RpoS (CS) 是否直接参与 rpoN、flrA 和 hapA 的转录。此外，免疫沉淀物将运送到 GenPathway（圣地亚哥，加利福尼亚州）以确定霍乱弧菌基因组中的 RpoS 结合位点。该数据将用于推定确定将 RpoS 与运动性、HA/蛋白酶和/或促进分离的调节联系起来的中间调节因子。在目标 2 中，我们将研究运动性和 HA/蛋白酶在体内分离中的作用。为此，我们将使用兔回肠环模型、共聚焦显微镜和荧光标记的同基因突变体来定义 HA/蛋白酶和运动性对粘膜逃逸的贡献。此外，我们将通过比较缺乏 flaA（非运动、非鞭毛）和 hapA 的 RpoS 阳性和阴性等基因菌株的粘膜逃逸来研究 RpoS 是否增强其他脱离因子的表达。最后，我们将使用婴儿小鼠高感染性测定来检查高感染性诱导和分离之间的时间关系。我们期望更好地了解感染后期抑制毒力和促进分离的事件将为治疗霍乱开辟新途径，并为解释这种人类病原体的流行潜力提供重要线索。公共卫生相关性：霍乱继续在亚洲、非洲和拉丁美洲人口稠密地区造成季节性疫情。该疾病由 O1 和 O139 血清群的霍乱弧菌引起，通过粪便、口腔途径在人与人之间传播。霍乱弧菌定植于人类小肠，分泌霍乱毒素引起腹泻，并在感染后期分离返回水生环境。新鲜脱落（分离）的弧菌处于“高度感染性”生理阶段，可能会增强人与人之间的传播和霍乱传播。因此，感染后期高度感染性细菌从肠粘膜脱离是霍乱传播链的关键步骤。我们研究的总体目标是确定触发霍乱弧菌脱离的调控相互作用以及蛋白酶产生和运动在此过程中的作用。这些研究可能开辟治疗霍乱的新途径，并揭示这种病原体的流行潜力。