Toxic shock syndrome toxin-1 (TSST-1) has been strongly implicated in the pathogenesis of toxic shock syndrome, an acute onset multisystem disorder. this staphylococcal exotoxin triggers the activation of T- lymphocytes and macrophages resulting in the induction of cytokines. The potent immunomodulatory effects of this toxin is likely to be a key factor in the pathogenesis of toxic shock syndrome. The overall goal of this proposal is to define structure-function correlations for the various biologic effects of the toxin using mutational analysis. Mutant TSST-1 toxins will be generated using site-directed mutagenesis on the cloned gene. Mutant TSST-1 will be tested for the ability to induce cytokine secretion by macrophages and to activate T-cells. This approach can be used to determine whether functionally distinct sites exist on the toxin molecule. The mutant toxins will also be used to study the interaction between TSST-1, the T-cell receptor (TCR) and the MHC class II molecules, a critical event in the initiation of T-cell mitogenesis by TSST-1. The TSST-1 mutants will be tested for the ability to bind to class II MHC molecules. Conversely, cell lines expressing mutant class II MHC antigens will be used to define the sites on the class II molecule that interact with native TSST-1. T-cell receptor interactions with TSST-1 will be studied by comparing the TCR-Vbeta genes expressed on T- cells activated by mutant TSST-1 with those selected by the wild-type TSST-1. This selective activation of T-cells bearing particular subsets of Vbeta elements is characteristic of the TSST-1 """"""""superantigen"""""""". In summary, the proposed work will define sites on TSST-1 molecule which are critical for toxin-mediated effects on T-cells and macrophages. Molecular interactions between the cellular receptors and the TSST-1 toxin will also be characterized. This information can ultimately be used to understand the relative contribution of TSST-1 functions in the pathogenesis of toxin shock syndrome.
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