Study of transmissible forms of Vibrio cholerae

霍乱弧菌传播形式的研究

• 批准号: 7048469
• 负责人: Andrew Camilli
• 金额: $ 34.82万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7048469
• 关键词: SDS polyacrylamide gel electrophoresis; Vibrio cholerae; bacteria; bacteria infection mechanism; bacterial proteins; bacterial toxicology; chemotaxis; cholera; cholera toxin; communicable disease transmission; diarrhea; feces; host organism interaction; laboratory mouse; microarray technology; polymerase chain reaction; proteomics; western blottings

DESCRIPTION (provided by applicant): Vibrio cholerae, which lives in association with plankton in brackish, temperate waters the world over, is the causative agent of endemic and epidemic cholera. Hallmarks of the disease include prodigious watery diarrhea resulting from the action of secreted cholera toxin (CT), and infrequent but deadly explosive epidemics. The strong link between explosive epidemics and human crowding accompanied with untreated drinking water suggests a very efficient mode of fecal-oral transmission. We have discovered a heightened state of transmissibility of stool V. cholerae (referred to simply as "hyperinfectivity"), which persists even after shedding into water reservoirs. Knowledge of the molecular basis for this phenotype, and a general characterization of this transmissible form of V. cholerae, would contribute to the design of vaccines to prevent cholera at the initial stage of infection. Aim 1 of this proposal will use transcriptional profiling and proteomics to help define this transmissible form. Spotted DNA microarrays will be used to determine the transcriptome of stool V. cholerae incubated in pond water, and this will be compared to that of fresh stool V. cholerae to identify potential differences. The results will be validated by quantitatively assaying the steady state mRNA and protein levels from select genes. Microscopy and transcriptome data on stool V. cholerae predict a bacterial state of motility working in the absence of chemotactic signaling. This counterintuitive state is hypothesized to be responsible, at least in part, for the hyperinfective phenotype. In Aim 2 of this proposal, quantitative immunodetection using paralog-specific antisera will be used to test for reduced expression of all three CheW linker proteins and all three CheR methytransferases in fresh and pond water-incubated stool V. cholerae, as is predicted by current transcriptome data. In addition, capillary tube chemotaxis assays will be performed directly on V. cholerae from these samples to substantiate this hypothesis. Aim 2 will also test a second hypothesis, that ToxR regulated factors, which are essential for pathogenesis, are not playing a role in the hyperinfectious state. Finally, Aim 3 will use mutation and infectivity analyses to determine if other metabolic, physiologic or phenotypic properties of the bacteria contribute to the hyperinfective phenotype or, alternatively, to colonization of an environmental planktonic host, Anabaena variabilis. These studies will establish a basis for understanding the hyperinfective phenotype, and the properties in general, that are exhibited by fresh and pond water-incubated stool V. cholerae. In turn, this knowledge will enhance our understanding of transmission of this and perhaps other water-borne pathogens, it will aid in the development of new cholera vaccines that target the antigens of 'incoming' vibrios, and it may suggest new approaches for the prevention of the dissemination of this lethal organism.

描述（由申请人提供）：霍乱弧菌与世界各地咸水、温带水域的浮游生物共同生活，是地方性和流行性霍乱的病原体。该疾病的特点包括由分泌型霍乱毒素 (CT) 作用引起的大量水样腹泻，以及罕见但致命的爆发性流行病。爆发性流行病与人口拥挤以及未经处理的饮用水之间的密切联系表明，粪口传播是一种非常有效的模式。我们发现粪便霍乱弧菌的传播能力处于增强状态（简称为“超感染性”），即使在流入水库后这种传播能力仍然持续存在。了解这种表型的分子基础以及霍乱弧菌这种传播形式的一般特征，将有助于设计在感染初期预防霍乱的疫苗。该提案的目标 1 将使用转录分析和蛋白质组学来帮助定义这种可传播的形式。点状 DNA 微阵列将用于确定在池塘水中孵育的粪便霍乱弧菌的转录组，并将其与新鲜粪便霍乱弧菌的转录组进行比较，以确定潜在的差异。结果将通过定量分析选定基因的稳态 mRNA 和蛋白质水平来验证。粪便霍乱弧菌的显微镜检查和转录组数据预测了在没有趋化信号传导的情况下细菌的运动状态。据推测，这种违反直觉的状态至少部分是造成过度感染表型的原因。在该提案的目标 2 中，将使用旁系同源特异性抗血清进行定量免疫检测，以测试新鲜和池塘水培养的霍乱弧菌粪便中所有三种 CheW 连接蛋白和所有三种 CheR 甲基转移酶表达的减少，正如当前转录组数据所预测的那样。此外，将直接对这些样本中的霍乱弧菌进行毛细管趋化性测定，以证实这一假设。目标 2 还将检验第二个假设，即对于发病机制至关重要的 ToxR 调节因子在过度感染状态中不起作用。最后，目标 3 将使用突变和感染性分析来确定细菌的其他代谢、生理或表型特性是否会导致过度感染表型，或者是否会导致环境浮游宿主多变鱼腥藻的定殖。这些研究将为了解新鲜霍乱弧菌粪便和池塘水培养粪便所表现出的过度感染表型和一般特性奠定基础。反过来，这些知识将增强我们对这种以及其他水生病原体传播的理解，它将有助于开发针对“传入”弧菌抗原的新型霍乱疫苗，并且可能会提出预防这种致命生物体传播的新方法。