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: 8515935
• 负责人: ANISIA J SILVA-BENITEZ
• 金额: $ 29.93万
• 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/8515935
• 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.

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。大多数关于霍乱弧菌感染的研究都集中在确定促进肠粘膜初始粘附和定植以及肠毒性的因素。然而，很少有研究关注那些促进弧菌脱离、其在小肠中传播并返回水生环境的因素。当感染性弧菌达到高细胞密度时，血凝素（HA）/蛋白酶的表达和运动性被认为有助于霍乱弧菌从肠粘膜脱离。我们实验室进行的研究揭示了第二信使环二鸟苷酸 (c-di-GMP)、一般应激反应调节剂 RpoS 和群体感应在 HA/蛋白酶和运动协调调节中存在复杂的相互作用。此外，我们还研究了 RpoS 和组蛋白样核结构蛋白 (H-NS) 在运动调节中的作用。具体来说，我们已经证明（i）c-di-GMP 负向调节 HA/蛋白酶表达，（ii）RpoS 减少 c-di-GMP 池以增强 HA/蛋白酶和运动性，（iii）c-di-GMP 负向调节当细胞不处于群体感应模式时调节 RpoS 表达，并且 (iv) RpoS 通过降低 c-di-GMP 库和减弱 H-NS 对运动的抑制来增强运动调节器 FlrA。在这里，我们建议彻底描述这些新的调控途径以及它们如何影响环境应激反应和霍乱弧菌与肠道细胞的分离。在目标 1 中，我们将确定连接 c-di-GMP 与 RpoS 表达的信号通路，并研究群体感应在此调节中的作用。然后，我们将研究 c-di-GMP 对 RpoS 的负调节是否会降低霍乱弧菌抵御环境胁迫的能力。在目标 2 中，我们将确定与 RpoS 和 H-NS 运动调节相关的调节事件。简而言之，我们将确定 RpoS 调节的 c-di-GMP 代谢酶，并结合微生物遗传学、DNA 结合测定和染色质免疫沉淀 (ChIP)，以确定 RpoS 的表达如何减少 flrA 启动子处的 H-NS 占据。最后，我们将利用酵母双杂交系统和下拉分析来确定 hns 突变体表现出的鞭毛非运动 (Mot-) 表型的分子基础，测试 H-NS 直接与鞭毛运动相互作用的假设。在目标 3 中，我们将确定 c-di-GMP 在霍乱弧菌从肠粘膜脱离中的作用。为此，我们将利用可人工增加 c-di-GMP 池的菌株，并使用体外和体内模型检查第二信使对霍乱弧菌粘附和与肠细胞分离的影响。感染。作为体外模型，我们将使用 HT29-18N2 培养细胞，该细胞可以被诱导分化为分泌粘蛋白的杯状细胞。作为体内模型，我们将使用成年兔回肠环。