Phages and Genomic Variation in V. Cholerae Evolution

描述（由申请人提供）：本项目的目标是描述在自然流行环境中具有流行潜力的霍乱弧菌菌株进化的机制。重点将放在弧菌噬菌体（生长在弧菌物种上的细菌病毒）在毒性相关基因的水平转移中的作用的分子遗传学分析上，以及在调节环境中的霍乱弧菌种群动态中的作用。将从孟加拉国的10个环境监测点分离弧菌噬菌体，并对其宿主范围、环境流行率以及包装和转移编码霍乱弧菌毒力相关基因的DNA的能力进行表征。将用特异性PCR或核酸探针更密切地监测与霍乱弧菌的特定亚组的存在负相关或正相关的任何噬菌体。通过这种方式，我们希望评估调节特定霍乱弧菌菌株流行的能力，从而影响这些菌株引起霍乱暴发的能力。还将收集环境和临床霍乱弧菌菌株，通过微阵列、DNA测序和遗传指纹分析方法进行分析，以建立菌株之间的系统发育关系。将确定收集的菌株的毒力基因含量，并测试某些基因的变体等位基因介导毒力相关功能的能力（例如，肠道定植）。将对不具有已知定植因子基因的临床分离株进行遗传分析，以鉴定这些菌株携带的新决定簇。据报道，霍乱粪便中存在的霍乱弧菌在幼鼠中显示出增加的感染性。我们将使用各种方法来研究这种现象，包括生理学测定、用霍乱弧菌微阵列进行表达谱分析和定量趋化性测定。该项目的结果不仅将使人们充分了解致病性霍乱弧菌菌株出现、进化和传播的过程，而且还将指导制定可用于在霍乱流行地区防治霍乱的监测和预防措施。

DESCRIPTION (provided by applicant): The goal of this project is to characterize the mechanisms governing the evolution of Vibrio cholerae strains with epidemic potential within the context of a natural endemic environment. Emphasis will be placed on molecular genetic analysis of the role of vibriophages (bacterial viruses that grow on Vibrio species) in horizontal transfer of virulence-related genes, and in modulating V. cholerae population dynamics in the environment. Vibriophages will be isolated from 10 environmental surveillance sites in Bangladesh and characterized with respect to their host range, environmental prevalence, and ability to package and transfer V. cholerae DNA encoding virulence-related genes. Any phage that correlates negatively or positively with the presence of specific subgroups of V. cholerae will be monitored more closely with specific PCR or nucleic acid probes. In this way we hope to assess the phages ability to modulate the prevalence of specific V. cholerae strains, thereby affecting the ability of these strains to cause outbreaks of cholera. Environmental and clinical V. cholerae strains will also be collected, analyzed by microarray, DNA sequencing, and genetic fingerprinting methods to establish the phylogenetic relationships among the strains. The virulence gene content of the collected strains will be determined, and variant alleles of certain genes will be tested for their ability to mediate virulence-related functions (e.g., intestinal colonization). Clinical isolates that do not possess known colonization factor genes will be subjected to genetic analysis in order to identify the novel determinants carried by these strains. V. cholerae present in cholera stools have been reported to show increased infectivity in infant mice. We will examine this phenomenon using a variety of approaches including physiology assays, expression profiling with V. cholerae microarrays and quantitative chemotaxis assays. The results of this project will not only provide a full understanding of the processes by which pathogenic V. cholerae strains emerge, evolve, and transmit, but will also guide the development of surveillance and preventive measures that can be used to combat cholera in a cholera endemic area.