Study of transmissible forms of Vibrio cholerae

描述（由申请人提供）：霍乱弧菌，与浮游生物一起生活在全世界微咸的温带沃茨，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）引起的巨大水样腹泻，以及罕见但致命的爆发性流行病。爆发性流行病与人群拥挤和未经处理的饮用水之间的密切联系表明，粪-口传播模式非常有效。我们已经发现粪便霍乱弧菌的传播性增强状态（简称为“高感染性”），即使在脱落到水库中后也会持续存在。了解这种表型的分子基础以及这种可传播形式霍乱弧菌的一般特征，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。将通过定量测定选定基因的稳态mRNA和蛋白质水平来验证结果。粪便霍乱弧菌的显微镜检查和转录组数据预测了在不存在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假设为是负责的，至少部分，高感染表型。在本提案的目标2中，使用旁系同源特异性抗血清的定量免疫检测将用于测试新鲜和池塘水孵育的粪便霍乱弧菌中所有三种CheW接头蛋白和所有三种CheR甲基转移酶的表达降低，如通过当前转录组数据所预测的。此外，将直接对这些样本中的霍乱弧菌进行毛细管趋化性试验，以证实这一假设。目的2还将测试第二个假设，即ToxR调节因子，这是必不可少的发病机制，没有发挥作用的高度感染状态。最后，目标3将使用突变和感染性分析，以确定细菌的其他代谢，生理或表型特性是否有助于高感染表型，或替代地，对环境寄生虫宿主鱼腥藻的定殖。这些研究将建立一个基础，了解高感染性表型，并在一般情况下，所表现出的新鲜和池塘水培养粪便霍乱弧菌的属性。反过来，这方面的知识将提高我们对这种和其他水传播病原体传播的理解，它将有助于开发针对“传入”弧菌抗原的新霍乱疫苗，并可能提出预防这种致命生物传播的新方法。

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.