Staphylococcal enterotoxins (SEs), the etiological agents of staphylococcal food poisoning syndrome, in addition to causing emesis and diarrhea are pyrogenic, enhance susceptibility to endotoxic shock, and induce T-cell mitosis, interleukin-1 production and interferon production. Recent experimental and epidemiological data suggests that the SEs are virulence factors in some cases of toxic shock syndrome.
The specific aim of this proposal is to better understand the relationship between structure and function for type A staphylococcal enterotoxin (SEA). Protein domains required for SEA's functions will be localized. SEA gene (entA) mutants containing deletions will be constructed using recombinant DNA and molecular biological techniques. The protein products of these deletion mutants will be assayed for emetic, mitogenic, interleukin-1 and interferon inducing activities. Amino acid residues critical for these functions will be identified by determining the biological properties of altered-SEA's encoded for by entA gene missense mutants. Missense mutants will be constructed by oligonucleotide directed mutagenesis. Codons targeted for mutagenesis will be selected based on the deletion analysis as well as published biochemical information. Conformation of some interesting altered-SEA's will be compared to unaltered-SEA by serological, UV spectrum, fluorescence spectrum and circular dichroism analysis. The structure-function information as well as the altered-SEAs obtained from this proposed work will provide the next step for my long term goal of understanding the SE's (1) mode of action (e.g. specific cellular targets, receptors and lesions) and (2) role in pathogenicity of S. aureus infections. In addition, perhaps altered-SEAs that are nontoxic may prove to be of therapeutic value as immunopotentiating agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI025574-02
Application #
3139039
Study Section
Bacteriology and Mycology Subcommittee 1 (BM)
Project Start
1988-02-01
Project End
1991-01-31
Budget Start
1989-02-01
Budget End
1990-01-31
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Earth Sciences/Resources
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Harris, T O; Betley, M J (1995) Biological activities of staphylococcal enterotoxin type A mutants with N-terminal substitutions. Infect Immun 63:2133-40
Rao, L; Karls, R K; Betley, M J (1995) In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus. J Bacteriol 177:2609-14
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Tremaine, M T; Brockman, D K; Betley, M J (1993) Staphylococcal enterotoxin A gene (sea) expression is not affected by the accessory gene regulator (agr). Infect Immun 61:356-9
Betley, M J; Borst, D W; Regassa, L B (1992) Staphylococcal enterotoxins, toxic shock syndrome toxin and streptococcal pyrogenic exotoxins: a comparative study of their molecular biology. Chem Immunol 55:1-35
Grossman, D; Lamphear, J G; Mollick, J A et al. (1992) Dual roles for class II major histocompatibility complex molecules in staphylococcal enterotoxin-induced cytokine production and in vivo toxicity. Infect Immun 60:5190-6
Hufnagle, W O; Tremaine, M T; Betley, M J (1991) The carboxyl-terminal region of staphylococcal enterotoxin type A is required for a fully active molecule. Infect Immun 59:2126-34
Soltis, M T; Mekalanos, J J; Betley, M J (1990) Identification of a bacteriophage containing a silent staphylococcal variant enterotoxin gene (sezA+). Infect Immun 58:1614-9

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