Genetic and Ecological Factors in Transmissibility and Epidemic Cycle of Cholera

说明(申请人提供)：属于O1和O139血清群的产毒霍乱弧菌可引起霍乱，这是一种急性水源性腹泻疾病，在孟加拉国等许多发展中国家引起季节性流行。虽然霍乱通常通过被病原体污染的水传播，但在孟加拉国，促进霍乱疫情传播和支持病原体在水中生存的自然机制尚不清楚。我们将应用我们开发的一种名为AST的新方法来监测和研究孟加拉国地表水中存在的致病性霍乱弧菌的“条件存活环境细胞(CVEC)”的性质。由于CVEC可能来自霍乱弧菌在人类霍乱粪便中的生物膜状聚集体，我们提出了一个广泛的计划来了解霍乱弧菌在霍乱粪便中的感染形式及其在水环境中的生存。我们将从环境水中分离出CVEC，并在动物模型中从代谢、形态、免疫化学以及它们的先天感染性等方面对它们进行表征。由于我们已经证明噬菌体捕食明显结束了霍乱流行，我们将确定霍乱弧菌的CVEC是否由于其可能的低代谢活动或物理特性而免受噬菌体捕食。我们提议的分析将包括生化和遗传学研究，旨在了解粪便来源弧菌的增强传播性、它们作为CVEC在环境中的生存以及CVEC对致病活生物体的复苏。我们建议从霍乱粪便(CVEC-H)、实验感染兔的细胞(CVEC-R)或完全实验室条件下(CVEC-L)培养出类似CVEC的霍乱弧菌细胞。为了了解不同细胞类型之间转换的遗传基础，我们将进行基于微阵列的基因组转录分析。标准的遗传方法将被用来鉴定参与CVEC-R和CVEC-L形成的基因。霍乱弧菌2623个明确的转座子插入突变体的文库将有助于系统地鉴定与低钠渗透胁迫相关的基因。结合来自环境监测的数据，这些研究应能更好地了解霍乱的感染循环，从而启发新的有效干预措施，以中断发展中国家的这种疾病的自然历史。

DESCRIPTION (provided by applicant): Toxigenic Vibrio cholerae belonging to the O1 and O139 serogroups cause cholera, an acute, waterborne diarrheal disease that causes seasonal epidemics in many developing countries such as Bangladesh. Although transmission of cholera typically occurs through water contaminated with the pathogenic organisms, the natural mechanisms that facilitate the epidemic spread of cholera and support the survival of the pathogen in water between seasonal epidemics in Bangladesh are not clear. We will apply a new method we developed called AST to monitor and study the nature of "conditionally viable environmental cells (CVEC)" of pathogenic V. cholerae that exist in surface waters of Bangladesh. Because CVEC are likely derived from biofilm-like aggregates of V. cholerae shed in human cholera stools, we propose a broad program to understand the infectious form of V. cholerae in cholera stools, and their survival in the aquatic environment. We will isolate CVEC from environmental waters and characterize them metabolically, morphologically, immunochemically, and for their innate infectivity in animal models. Because we have shown phage predation apparently ends cholera epidemics, we will determine whether CVEC of V. cholerae are protected from phage predation due to their possible low metabolic activity or physical properties. Our proposed analysis will include biochemical and genetic studies designed to understand enhanced transmissibility of stool-derived vibrios, their environmental survival as CVEC and resuscitation of CVEC to pathogenic viable organisms. We propose to produce CVEC-like V. cholerae cells from cholera stools (CVEC-H), or from cells shed by experimentally infected rabbits (CVEC-R) or under fully laboratory conditions (CVEC-L). To understand the genetic basis for the transition between various cell types, we will conduct microarray-based, genomic transcriptional analysis. Standard genetic methods will be utilized to identify genes involved in formation of CVEC-R and CVEC-L. A library of 2623 defined transposon insertion mutants of V. cholerae will aid in the systematic identification of genes involved in low sodium osmo-stress. Together with data from environmental monitoring, these studies should provide a better understanding of the infectious cycle of cholera and thus inspire new effective interventions for interrupting the natural history of this disease in developing countries.