Infectious diarrhea remains an important cause of human morbidity and mortality. Detailed understanding of the mechanisms of pathogenesis for specific agents of diarrhea should allow the rational design of safe and effective vaccines. Shiga toxin from Shigella dysenteriae 1 and Shiga-like toxin (SLT) from Escherichia coli are cytotoxic proteins important in diarrhea and other illnesses in humans. These toxins contain an enzymatically active A subunit and multiple copies of a B subunit that bind toxin to specific receptors on the cell surface. Glutamic acid-167 is critical to the enzymatic activity of the A subunit of SLT-I (Slt-IA) from E. coli. This proposal describes methods for site-directed mutagenesis of this residue (and other residues important in enzymatic activity) to identify a mutant Slt-IA that is stable and properly folded but devoid of enzymatic activity. Recombination of the gene for this mutant Slt-IA into an expression vector for wild-type Slt-IB will allow efficient production of mutant SLT-I holotoxin. This mutant holotoxin or purified Slt-IB may be useful as immunogens against S. dysenteriae 1 infection, either by themselves or covalently coupled to suitable polysaccharide antigens. Other bacteria, including Vibrio cholerae, also produce SLT, but the role of this toxin in pathogenesis of infection due to these bacteria is unknown. This proposal describes methods to identify the SLT genes of V. cholerae, using DNA probes derived from the cloned SLT-I genes of E. coli and from mixed oligonucleotides designed to detect amino acids conserved at the active site of Slt-IA. After cloning and sequencing the genes for SLT from V. cholerae, in vivo marker exchange will be used to introduce defined deletions of SLT into the chromosome and the virulence of these deleted strains will be tested in animals. The production of Shiga toxin and SLT- I are regulated by iron. TnphoA will be used to construct a gene fusion between a cloned copy of the promoter-proximal gene of SLT in V. cholerae and phoA. Regulation of SLT production in V. cholerae by iron will be examined using alkaline phosphatase activity as a marker. TnphoA will also be used to mutagenize the chromosome of V. cholerae to generate a series of insertion mutations in genes that are iron-regulated. These mutants will be screened for loss of virulence in animals and iron-regulated virulence determinants characterized. The availability of cloned copies of iron- regulated genes from V. cholerae will allow comparison of the mechanism of iron regulation in this pathogen to that established previously in E. coli.
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