The objective is to study the genetic determinants of virulence in Campylobacter jejuni, a common cause of diarrhea of children and adults thoughout the world. We propose to begin by developing a library of Campylobacter genes in a foreign genetic background by cosmid or bacteriophage vector cloning. Clones will be screened to detect expression of Campylobacter-specific sequences, including those which restore physiological deficiencies of the recipient E. coli K12 cells and sequences directing the synthesis of outer membrane polypeptides, employing in vivo and in vitro protein synthesis systems. Once it has been determined that Campylobacter genes are functional in E. coli, we will search for clones expressing three determinants which appear to contribute to the pathogenesis of gastrointestinal tract infection, including enterotoxin, cytotoxin, and the ability to invade epithelial cells. These will be detected using bioassays and immune sera directed against specific Campylobacter proteins. Cloned genetic determinants of virulence will then be characterized by subcloning, mapping and nucleotide sequencing. DNA probes containing these sequences will be used to examine their distribution in clinical isolates and to assess their contribution to different clinical syndromes. Ultimately, we wish to produce isogenic mutants in Campylobacter jejuni, each deficient in a single, potential virulence factor, which can be studied in relevant animals models of gastrointestinal tract infection. This could be accomplished by exchanging cloned, inactivated sequences with active genes in Campylobacter via recombination or by developing a genetic exchange system permitting transposon-directed mutagenesis in vivo. Thus, we have also proposed an alternative means of manipulating Campylobacter genetic determinants of virulence, in the event that expression of Campylobacter DNA is not achieved in E. coli K12.
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