Study of transmissible forms of Vibrio cholerae

描述(申请人提供)：霍乱弧菌与浮游生物一起生活在世界各地的咸水和温带水域，是地方性和流行性霍乱的病原体。这种疾病的特征包括由分泌的霍乱毒素(CT)作用引起的巨大水样腹泻，以及罕见但致命的爆炸性流行病。暴发性流行病与人类拥挤以及未经处理的饮用水之间的强烈联系表明，粪便-口腔传播是一种非常有效的方式。我们发现，霍乱弧菌的粪便传播性增强(简称“高度传染性”)，即使在排入水库后仍会持续存在。了解这种表型的分子基础，以及对这种可传播形式的霍乱弧菌的一般特征，将有助于设计疫苗，以在感染的初始阶段预防霍乱。这项提案的目标1将使用转录图谱和蛋白质组学来帮助定义这种可传播的形式。斑点DNA微阵列将用于检测在池塘水中孵化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。粪便中霍乱弧菌的显微镜和转录组数据预测了在缺乏趋化信号的情况下细菌的运动状态。这种违反直觉的状态被认为至少在一定程度上导致了高度感染的表型。在这项建议的目标2中，将使用Paralog特异性抗血清进行定量免疫检测，以测试所有三种咀嚼连接蛋白和所有三种Cher甲基转移酶在淡水和池塘水孵化的霍乱弧菌粪便中的表达减少，正如当前转录组数据所预测的那样。此外，毛细管趋化试验将直接对这些样本中的霍乱弧菌进行，以证实这一假设。Aim 2还将测试第二个假设，即ToxR调节因子在高感染状态中不起作用，而ToxR调节因子在发病中是必不可少的。最后，目标3将使用突变和传染性分析来确定细菌的其他代谢、生理或表型特性是否有助于高度感染的表型，或者，替代地，有助于环境浮游宿主多变鱼腥藻的定居。这些研究将为了解淡水和池塘水孵化的粪便霍乱弧菌表现出的高度感染表型和一般特性奠定基础。反过来，这一知识将加强我们对这种或其他水传播病原体传播的了解，它将有助于开发针对“传入”弧菌抗原的新霍乱疫苗，并可能为防止这种致命微生物的传播提供新的方法。

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.