CARDIOTOXICITY OF STREPTOCOCCAL PYROGENIC EXOTOXINS

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DESCRIPTION (Adapted from the applicant's abstract): The long term goals of this project are two fold: a) to evaluate the role of pyrogenic toxin superantigens, notably streptococcal pyrogenic exotoxins (SPEs, scarlet fever toxins), in causing both acute toxic shock syndrome and vascular illnesses and chronic autoimmune and allergic diseases, and b) to analyze the structure-function relationships among the SPEs and between the SPEs and staphylococcal enterotoxins and toxic shock syndrome toxin-1, with the intent to clarify the molecular mechanism(s) of action of the toxins and develop toxoid vaccines against the toxins. Specific aims of the present application include: a) determination of the three dimensional structure of SPE C and SPEA/staphylococcal enterotoxin C (SEC/SEB complexed. with the T cell receptor beta chain. The investigator's role in these studies is to provide sufficient toxins for structural analyses by ethanol precipitation from culture fluids, resolubilization in acetate buffered saline at pH 4.0 or water, and preparative isoelectric focusing. Crystallization and three dimensional structure analysis of SPE C will be done in collaboration with Dr. Douglas H. Ohlendorf, Department of Biochemistry, University of Minnesota and that of SPE A/SEB complexed to the beta chain of the T cell receptor by Dr. Roy A.. Mariuzza, Center for Advanced Research in Biotechnology, University of Maryland; b) Domains and amino acid residues on SPE C and the SPE A/SEC/SEB subgroup of pyrogenic toxin superantigens required for biological activity (pyrogenicity, enhancement of lethal endotoxin shock and cardiotoxicity, ability to induce TSS when administered subcutaneously in miniosmotic pumps, superantigenicity, and lipopolysaccharide binding) will be localized through use of PCR mutagenesis. Nucleotide sequencing will be done to verify changed amino acids and structural analysis where possible to assess alterations of three dimensional structure of mutants. It is hoped in addition to localizing domains required for toxicity, that these studies will clarify important mechanisms of T cell activation and lead to useful toxoid vaccines.