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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI027329-02
Application #
3454949
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1988-12-01
Project End
1993-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
2
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
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Litwin, C M; Calderwood, S B (1994) Analysis of the complexity of gene regulation by fur in Vibrio cholerae. J Bacteriol 176:240-8
Butterton, J R; Calderwood, S B (1994) Identification, cloning, and sequencing of a gene required for ferric vibriobactin utilization by Vibrio cholerae. J Bacteriol 176:5631-8
Litwin, C M; Calderwood, S B (1993) Cloning and genetic analysis of the Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker exchange. J Bacteriol 175:706-15
Litwin, C M; Calderwood, S B (1993) Role of iron in regulation of virulence genes. Clin Microbiol Rev 6:137-49
Acheson, D W; Calderwood, S B; Boyko, S A et al. (1993) Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or iron-regulated promoter systems. Infect Immun 61:1098-104
Butterton, J R; Boyko, S A; Calderwood, S B (1993) Use of the Vibrio cholerae irgA gene as a locus for insertion and expression of heterologous antigens in cholera vaccine strains. Vaccine 11:1327-35
Butterton, J R; Stoebner, J A; Payne, S M et al. (1992) Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae. J Bacteriol 174:3729-38
Deresiewicz, R L; Calderwood, S B; Robertus, J D et al. (1992) Mutations affecting the activity of the Shiga-like toxin I A-chain. Biochemistry 31:3272-80
Stoebner, J A; Butterton, J R; Calderwood, S B et al. (1992) Identification of the vibriobactin receptor of Vibrio cholerae. J Bacteriol 174:3270-4

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