The objectives are to examine the role of V. cholerae motility and associated structures in V. cholerae pathogenesis. And evaluate the contribution of flagellar structures to immunity against infection. The flagellar structure of V. cholerae is unique among enteric pathogens in a number of ways, most conspiciously in that the flagellum is surrounded by a sheath. Studies suggest that mot-motility and/or flagellar components may involved in colonization and immunity. We propose to isolate using transposon mutagenesis a series of V. cholerae mutants deficient in motility and synthesis of the flagellar apparatus. The mutants will be characterized genetically to observe whether the motility genes are arranged in clusters on the bacterial chromosome and also determine whether mutant formation is due to a single insertional event. The location of the transposon insertions will be determined by probing Southern blots with labeled transposon DNA. Motility genes will be mapped relative to other chromosomal genes by classical recombination techniques. The ability of selected mutants to colonize and cause disease will be evaluated in animal models. The composition of the flagellar cores and sheaths of parental and mutant strains will be determined. The flagella will then be examined with human immune sera and jejunal fluids to identify immunogenic components. Flagella from in vivo grown V. cholerae will also be analyzed to identify proteins uniquely expressed in vivo and if they are immunogenic. Immunogenic flagellar components will be evaluated for their potential as protective antigens in animal models. These protective antigens could provide a rationale for vaccine construction. Cloned V. cholerae chromosomal DNA will be probed to obtain flagellar structural genes and other genes associated with motility for use in future studies. The proposed studies will help elucidate the structure, function and genetics of V. cholerae flagella, as well as the role of motility and flagella in pathogenesis.
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