Regulation of Vibrio cholerae motility, stress response and detachment from intes

霍乱弧菌运动、应激反应和肠道脱离的调节

• 批准号: 8339044
• 负责人: ANISIA J SILVA-BENITEZ
• 金额: $ 31.84万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-25 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8339044
• 关键词: Acute; Address; Adherence; Adult; Affect; Agar; Attenuated; Bacteria sigma factor KatF protein; Binding Proteins; Biological Assay; Cell Density; Cells; Cholera; Cholera Toxin; Clinical; Complex; Cultured Cells; Cyclic AMP Receptor Protein; DNA Binding; Data; Diarrhea; Disease; Environment; Enzymes; Event; Exhibits; Exotoxins; Flagella; Gastric Acid; Goblet Cells; Histones; In Vitro; Infection; Intestinal Mucosa; Intestines; Laboratories; Link; Mastigophora; Metabolic; Microbial Genetics; Microscope; Molecular; Motor; Mucins; Mutagenesis; Oryctolagus cuniculus; Paralysed; Phase; Phenotype; Production; Regulation; Regulatory Pathway; Repression; Rice; Role; Second Messenger Systems; Signal Pathway; Small Intestines; Staging; Stress; Swimming; System; Testing; Vibrio; Vibrio cholerae; Vibrio cholerae O1; Virulence Factors; Water; base; biological adaptation to stress; bis(3' ,5' )-cyclic diguanylic acid; cell motility; chromatin immunoprecipitation; hemagglutinin-protease; in vitro Model; in vivo Model; inhibitor/antagonist; mutant; novel; promoter; protein structure; quorum sensing; second messenger; yeast two hybrid system

DESCRIPTION (provided by applicant): Cholera is an acute water-borne diarrheal disease caused by Vibrio cholerae of serogroups O1 and O139. Most studies on V. cholerae infection have focused on identifying factors that promote initial adherence and colonization of the intestinal mucosa and enterotoxicity. However, fewer studies have focused on those factors that promote detachment of Vibrios, their dissemination throughout the small intestine and return to the aquatic environment. Expression of hemagglutinin (HA)/protease and motility have been suggested to facilitate V. cholerae detachment from the intestinal mucosa when infective Vibrios reach high cell density. Studies conducted in our laboratory have revealed a complex interplay between the second messenger cyclic diguanylate (c-di-GMP), the general stress response regulator RpoS and quorum sensing in the coordinate regulation of HA/protease and motility. In addition, we have examined the role of RpoS and the histone-like nucleoid structuring protein (H-NS) in the regulation of motility. Specifically, we have demonstrated that (i) c-di-GMP negatively regulates HA/protease expression, (ii) RpoS diminishes the c-di-GMP pool to enhance HA/protease and motility, (iii) c-di-GMP negatively regulates RpoS expression when cells are not in quorum sensing mode, and (iv) RpoS enhances motility by both lowering the c-di-GMP pool and attenuating H-NS repression of the motility regulator FlrA. Here we propose to thoroughly characterize these new regulatory pathways and how they affect environmental stress response and detachment of V. cholerae from intestinal cells. In Aim 1 we will determine the signaling pathway connecting c-di-GMP to RpoS expression and investigate the role of quorum sensing in this regulation. Then, we will examine if the negative regulation of RpoS by c-di-GMP diminishes the capacity of V. cholerae to withstand environmental stresses. In Aim 2 we will determine the regulatory events involved in RpoS and H-NS regulation of motility. Briefly, we will determine the c-di- GMP metabolic enzymes regulated by RpoS and combine microbial genetics, DNA binding assays and chromatin immunoprecipitation (ChIP) to determine how expression of RpoS diminishes H-NS occupancy at the flrA promoter. Finally, we will exploit the yeast two-hybrid system and pull down assays to determine the molecular basis for the flagellated non-motile (Mot-) phenotype exhibited by hns mutants testing the hypothesis that H-NS directly interacts with the flagellar motor. In Aim 3 we will determine the role of c-di-GMP in V. cholerae detachment from the intestinal mucosa. To this end, we will exploit the use of strains in which the c-di-GMP pool can be artificially increased and examine the effect of this second messenger in V. cholerae adherence and detachment from intestinal cells using in vitro and in vivo models of infection. As an in vitro model we will use HT29-18N2 cultured cells which can be induced to differentiate into mucin-secreting goblet cells. As an in vivo model we will use adult rabbit ileal loops. PUBLIC HEALTH RELEVANCE: Most studies on V. cholerae infection have focused on identifying factors that promote bacterial colonization and enterotoxicity. However, the factors that promote detachment of Vibrios, their dissemination throughout the small intestine and return to the aquatic environment are relatively unknown. The purpose of our study is to determine the regulatory mechanisms that control bacterial stress response and detachment from the intestinal mucosa late in infection.

描述（由申请人提供）：霍乱是由血清群O1和O139引起的急性水传播腹泻疾病。大多数关于霍乱谷感染的研究都集中在识别促进肠粘膜和肠毒性初始粘附和定殖的因素上。但是，更少的研究集中在促进颤音脱离的因素上，它们在整个小肠中的传播并返回水生环境。当感染性颤音达到高细胞密度时，已经提出了血凝蛋白（HA）/蛋白酶和蛋白酶和运动性的表达，以促进霍乱谷链球菌脱离。在我们的实验室中进行的研究表明，第二信使环绕二甘氨酸酯（C-DI-GMP），一般应力响应调节剂RPOS和Quorum Sensing在HA/蛋白酶和运动性的坐标调节中都有复杂的相互作用。此外，我们研究了RPOS和组蛋白样的核苷结构蛋白（H-NS）在运动性调节中的作用。 Specifically, we have demonstrated that (i) c-di-GMP negatively regulates HA/protease expression, (ii) RpoS diminishes the c-di-GMP pool to enhance HA/protease and motility, (iii) c-di-GMP negatively regulates RpoS expression when cells are not in quorum sensing mode, and (iv) RpoS enhances motility by both lowering the c-di-GMP pool and attenuating H-NS对运动调节剂FLRA的抑制。在这里，我们建议彻底表征这些新的调节途径，以及它们如何影响环境应力反应和霍乱弧菌与肠细胞的脱离。在AIM 1中，我们将确定将C-DI-GMP连接到RPOS表达的信号通路，并研究法定感应在此调节中的作用。然后，我们将检查C-DI-GMP对RPO的负调控是否会降低霍乱弧菌以承受环境应力的能力。在AIM 2中，我们将确定RPOS和H-NS运动调节中涉及的调节事件。简而言之，我们将确定由RPO调节的C-DI-GMP代谢酶，并结合了微生物遗传学，DNA结合测定法和染色质免疫沉淀（CHIP），以确定RPOS的表达如何降低FLRA启动子的H-NS占用率。最后，我们将利用酵母的两杂化系统并下拉测定，以确定HNS突变体表现出的鞭毛非运动型（MOT-）表型的分子基础，该表型检验了H-NS直接与鞭毛运动相互作用的假设。在AIM 3中，我们将确定C-DI-GMP在肠道粘膜中的霍乱链球菌脱离中的作用。为此，我们将利用菌株的使用，其中C-DI-GMP可以人为地增加C-DI-GMP池，并检查第二嵌液在霍乱弧菌粘附和使用体外感染模型与肠道细胞中脱离肠细胞中的效果。作为一种体外模型，我们将使用HT29-18N2培养的细胞，可诱导将粘液分泌的杯状细胞分化。作为体内模型，我们将使用成年兔回肠环。 公共卫生相关性：大多数关于霍乱谷链球菌感染的研究都集中在识别促进细菌定植和肠毒性的因素上。但是，促进颤音脱离的因素，它们在整个小肠中的传播并返回水生环境是相对未知的。我们研究的目的是确定在感染后期控制细菌应激反应和与肠粘膜脱离的调节机制。