Cholera Pathogenesis, mucinase activity and motility

霍乱发病机制、粘蛋白酶活性和运动性

• 批准号: 6861563
• 负责人: Jorge Antonio Benitez
• 金额: $ 11.47万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6861563
• 关键词: Vibrio cholerae; bacteria infection mechanism; bacterial cytopathogenic effect; cell motility; cholera; cholera toxin; endopeptidases; enzyme activity; genetic transcription; host organism interaction; laboratory mouse; laboratory rabbit; microorganism hemagglutinin; mutant; polymerase chain reaction; polysaccharide carbon oxygen lyase; quorum sensing; tissue /cell culture; transfection; virulence; western blottings

DESCRIPTION (provided by applicant): Cholera is a diarrheal disease caused by Vibrio cholerae that affects highly populated regions in Asia, Africa, and Latin America. In spite of significant efforts we still lack a vaccine that protects against both serogroups O1 and O139. Several live attenuated V. cholerae vaccine candidates still induced mild to moderate diarrhea in clinical trials. The reactogenicity of live vaccine candidates has been correlated with the capacity of attenuated vibrios to penetrate the protective mucus barrier. V. cholerae produces a metalloprotease: hemagglutinin/protease (Hap) encoded by hapA that has been associated with reactogenicity and promotes mucus penetration and detachment in vitro. We have demonstrated that expression of hapA requires the cyclic AMP receptor protein and the stationary phase sigma factor S. The aim our study is to determine the mechanism by which quorum sensing regulators and stationary phase factors control expression of hapA in responses to environmental stimuli and to characterize the role hapA in pathogenesis. To this end we will construct and analyze regulatory mutants at the mRNA, protein and functional level. This study will contribute to a better understanding of the coordination between global regulatory networks in vibrios and other enteric pathogens. Experiments with mutants lacking Hap mucinolytic activity using cell culture, animal models, and volunteers suggest that mucinase production could perturb the protective mucus barrier, promote detachment, and cooperate with motility to spread the infection. In order to prove this hypothesis, we will construct mutants lacking mucinase activity and motility as well as double mutants. We will examine the effect of these mutations on virulence and colonization. Finally, we will use in vivo expression technology to determine the effect of Hap and motility inactivation on the expression of V. cholerae main virulence factors: cholera toxin and toxin co-regulated in the infant mouse intestine. This knowledge will facilitate the development of improved cholera vaccines.

描述（由申请人提供）：霍乱是由福利奥霍乱引起的一种腹泻疾病，影响亚洲，非洲和拉丁美洲人口稠密的地区。尽管付出了巨大的努力，但我们仍然缺乏一种疫苗，可防止血清群O1和O139。在临床试验中，几种活体减弱的霍乱疫苗候选者仍会引起轻度至中度腹泻。活疫苗候选物的反应生成性与衰减的弧菌的能力相关，以穿透保护性粘液屏障。 V. Cholerae产生了金属蛋白酶：由HAPA编码的血凝素/蛋白酶（HAP），与反应生成性相关并促进体外粘液渗透和脱离。我们已经证明，HAPA的表达需要环状AMP受体蛋白和固定相的Sigma因子S。我们的研究的目的是确定群体传感调节剂和固定相因控制HAPA在对环境刺激反应中的表达的机制，并表征HAPA在病原体中的作用。为此，我们将在mRNA，蛋白质和功能水平上构建和分析调节突变体。这项研究将有助于更好地理解颤音中的全球监管网络与其他肠道病原体之间的协调。使用细胞培养，动物模型和志愿者缺乏HAP粘蛋白水解活性的突变体的实验表明，粘蛋白酶产生可以扰动保护性粘液屏障，促进脱离，并与运动能力一起散布以传播感染。为了证明这一假设，我们将构建缺乏粘液酶活性和运动性的突变体以及双突变体。我们将研究这些突变对毒力和定殖的影响。最后，我们将使用体内表达技术来确定HAP和运动灭活对霍乱弧菌主要毒力因子表达的影响：霍乱毒素和毒素在婴儿小鼠肠中共同调节。这些知识将促进改善的霍乱疫苗的发展。