This is a competitive renewal of a collaborative project between the Novick and Schlievert laboratories aimed toward understanding the molecular genetics and pathobiology of one prototypical superantigen, toxic shock syndrome (TSST 1). Their results and the results of others suggest that the interaction of TSST 1 with the TCR and MHC Class II receptors is responsible for their superantigen activity. However, these investigators have provided evidence that its interaction with different types of receptors on other cells is responsible for its lethality. They have recently determined the structure of TSST 1 and several derivatives by X ray crystallography and have constructed a series of mutants with properties that have led to one hypothesis of this proposal, namely that lethality and superantigenicity are separately determined. The first two aims will focus on a definitive test of this hypothesis through confirmation of their preliminary results which suggest separable functions. This part of the project will investigate the pathobiology of TSST 1 by identifying and characterizing tissue specific receptors and determining the mechanism of intracellular cytotoxicity with reference to activities of mutant forms of the toxin.
Aims 3 and 4 will be a continuing investigation into the molecular genetics of S. aureus toxin production.
Aim 3 will focus on extending their preliminary genetic data suggesting that TSST 1 is encoded by a transposon, Tn557. This part of the project will determine the structure and function of Tn557 and also of closely linked heterologous insertions containing determinants of SEB and certain resistances.
Aim 4 will attempt to develop an explanation for the mutual exclusion of TSST 1 positive and alpha-hemolysin positive phenotypes in S. aureus, despite the fact that many strains have both genes. A similar situation exists in group A streptococci with two superantigens (SPEA and SPEB). They have obtained preliminary results suggestive of an explicit regulatory antagonism between TSST 1 and alpha hemolysin. They propose to determine whether other exoproteins are involved and what is the basis of this antagonism. They will perform similar studies with SPEA and SPEB.
Aim 5 will attempt to elucidate the mechanism of action for glycerol monolaurate effects on eukaryotic cells and on S. aureus. In a series of unrelated experiments, they have observed that GML inhibits the synthesis of TSST 1 and other exoproteins and also of the induction of resistance to several antibiotics, probably by interfering with signal transduction. Furthermore, they showed that GML is mitogenic for T lymphocytes but not for myoblasts. Although the study of GML was initially focused on TSST 1 they now propose to continue this investigation on a broader basis. They propose to identify the putative signal signalling pathway(s) in bacteria that are sensitive to GML inhibition, to confirm that signal transduction is the actual target, and to determine the putative signalling pathway used by GML to stimulate lymphocytes. The PI's expect that this work will lead to a comprehensive understanding of the structure function, genetics, regulation and pathobiology of TSST 1 and will help develop certain diagnostic and therapeutic applications plus a general method of immunization against superantigen toxins.
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