Regulation of the Vibrio cholerae intestinal exit plan

霍乱弧菌肠道排出计划的调节

基本信息

批准号：
7924037
负责人：
ANISIA J SILVA-BENITEZ
金额：
$ 17.5万
依托单位：
MOREHOUSE SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7924037
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

项目摘要

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）：霍乱是由血清群O1和O139引起的急性水传播腹泻疾病。细菌是通过粪便口服途径传播的人。感染颤动的颤音，从而克服胃酸屏障向朝向并粘附在肠道粘膜上并表达主要的毒力因子，例如毒素共同调节的毛皮细胞和霍乱毒素，这在很大程度上是这种疾病典型的水稻水性腹泻。在感染的后期，霍乱葡萄球菌下调了毒力因子的表达，并分离以返回环境。我们实验室中的先前研究表明，HAPA编码的RPOS依赖性的Zn-甲基蛋白酶血凝蛋白（HA）/蛋白酶可促进霍乱链球菌脱离培养的细胞和粘液。 RPO促进了在兔子回循环模型中进行的一项研究，表明霍乱链球菌脱离或粘膜逃脱促进了运动的表达。最近已经显示，霍乱粪便中新鲜脱落的弧菌处于一种超级感染状态，可以促进人与人之间的霍乱传播。因此，从肠粘膜中脱离超级感染性颤音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，该网络涉及环状AMP受体蛋白（CRP），主群传感调节剂HAPR，类似组蛋白的类核苷结构蛋白（H-NS）以及一般的应激反应调节剂RPO，这些调节剂RPOS协同起作用，可抑制病毒性和蛋白酶的产生和蛋白酶的产生。该研究计划的核心假设是，感染后期的HA/蛋白酶和运动的协调上调以协同的方式促进了霍乱链球菌脱离或粘膜逃脱。在这项研究中，我们建议通过整合各种方法，例如细菌遗传学，染色质免疫沉淀（CHIP），芯片序列，共聚焦显微镜和动物模型来严格检验这一假设。在AIM 1中，我们将确定感染后期触发V.霍乱脱离的调节相互作用。为此，我们计划研究H-NS和RPO如何调节RPON和FLRA的表达，以协调HA/蛋白酶生产的鞭毛运动的表达。我们将使用芯片来确定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Genathway（加利福尼亚州圣地亚哥），以确定V.霍乱基因组中的RPOS结合位点。该数据将用于识别将RPO与运动性，HA/蛋白酶和/或促进脱离的调节的中间调节器。在AIM 2中，我们将研究运动和HA/蛋白酶在体内脱离中的作用。为此，我们将使用兔回回环模型，共聚焦显微镜和荧光标记的等生突变体来定义贡献HA/蛋白酶和粘膜逃脱的运动。此外，我们将研究RPO是否通过比较缺乏FLAA（非运动，非鞭毛）和HAPA的RPOS阳性和负同源性菌株中的粘膜逃脱来增强其他分离因子的表达。最后，我们将使用婴儿小鼠过度感染性测定法来检查诱导性感染性和脱离之间的时间关系。我们预计，对抑制毒力和促进感染后期脱离的事件有更好的了解将为治疗霍乱提供新的途径，并提供重要的线索来解释这种人类病原体的流行潜力。公共卫生相关性：霍乱继续导致人口稠密地区的亚洲，非洲和拉丁美洲的季节性暴发。该疾病是由血清群O1和O139的纤维霍乱引起的，这些疾病是通过粪便口服途径传播的人。 V.霍乱将人类的小肠定居，分泌霍乱毒素，引起腹泻，并在感染后期脱离以返回水生环境。新鲜的棚（分离）颤音处于“超级感染”生理阶段，可以增强人的传播和霍乱传播。因此，感染后期与肠粘膜脱离肠粘膜是霍乱传播链的关键步骤。我们研究的总体目的是建立触发V.霍乱分离的监管相互作用以及蛋白酶生产和运动在此过程中的作用。这些研究可以开放新的途径，以治疗霍乱和阐明这种病原体的流行潜力。