GENETIC ANALYSIS OF STAPHYLOCOCCAL ENTEROTOXIN A

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The biochemical and genetic properties of staphylcoccal enterotoxin A (SEA) will be characterized. Knowledge gained from the nucleotide sequence of the SEA structural gene (entA) will be used to perform site-specific mutagenesis of the entA gene in regions likely to encode amino acid residues involved in formation of the SEA active site or receptor binding domain. The mutant entA gene products will be assayed for emetic activity, mitogenic activity and interferon induction. If these studies lead to the identification of regions involved in the formation of SEA's biological active site(s), then short synthetic peptides homologous to these regions will be tested for the induction or inhibition of biological activities. These studies could lead to the development of SEA toxoids, and possibly new pharmacological agents for the induction of interferon or mitogenesis. The true incidence of SEA production in clinical isolates of S. aureus will be determined using an entA gene probe. In addition, the active site studies discussed above may also result in the development of an oligonuleotide probe (directed at homologies present in the three enterotoxins SEA, SEB and SEC) that might identify all enterotoxin producing Staphylococcus aureus strains in a hybridization assay. To determine if SEA can contribute to the pathogenicity of S. aureus strains, the relative virulence of isogenic EntA+ and EntA- S. aureus strains will be tested in several animal models. Characterization of the entA-converting phages and related non-entA converting phages will continue with respect to genetic properties and genome structure. These studies may lead to a better understanding of how entA-converting phages arise in nature and the mechanism by which antigenic heterogeneity is generated in the staphylococcal enterotoxins. The possibility that entA gene expression is regulated will be examined by assessing the effects of strain background, media composition, and gene dosage on a chloramphenicol-entA-promoter fusion.