The research described is aimed at understanding the genetics ofpathogenesis of V. choleare and the generation of genetically engeneered v. cholerae cell as a vaccine carrier.Cholera is a major cause of illness and death in many developing countries. Currently available vaccines provide only short lived modest protection. Vibrio cholerae produces and secretes a potent toxin that is effector of the diarrhea produced by this organism, as well as other proteins that are viruence accessory proteins. Using transposon mutagenesis secretory deficient mutants were generated in V. cholerae. In this reseach period, DNase secretion deficient mutants were generated, that are deficient in secretion of the XDS protein but not in secretion of cholera toxin. Previous work indicated that mutants defective in secretion of cholera toxin are not deficient in secretion of the DNase. Thus, two different mechanism appear to be involved in scretion of these two protein by V. cholerae. The structural gene, xds, coding for a DNase from V. cholerae, was sequenced. Sequence data generated were used to modify the gene by PCR, in order to anlyze the structure function relationship of the xds gene.Cholera is a major cause of illness and death in many developing countries. Currently available vaccines provide only short lived modest protection. Vibrio cholerae produces and secretes a potent toxin that is effector of the diarrhea produced by this organism. In addition to cholera toxin, the organism produces and secrets a variety of other proteins. Understanding the secretory apparatus involved in secretion of proteins from V. cholerae will enable the construction of V. cholerae as a bacterial vaccine vector. Using transposon mutagenesis secretory deficient mutants will be generated in V. cholerae. colonies will be tested for defect in secretion of cholera toxin, protease, and DNase. A chromosomal DNA library will be prepared from the parent strain to complement the excretory deficient phenotype. The gene(s) coding for the secretory apparatus will be characterized. In addition, a gene coding for a DNase gene from V. cholerae was cloned. A chromosomal library from V. cholerae will be transformed into E. coli containing the cloned DNase gene. Gene allowing for the DNAse to be secreted from E. coli will be analyzed.
