Our hypothesis is that a cell free protein toxin plays a critical role in pathogenesis of Shigella infections. The toxin may be responsible for both clinical manifestations of the disease, watery diarrhea and dysentery. A basic understanding of the mechanisms by which the toxin mediates its biological effects will aid in the development of improved therapeutic treatment programs and of safe and effective vaccine strains. To study the mechanism of action of Shigella toxin we will investigate toxin mediated inhibition of protein synthesis. Using a cell free system we will determine specifically what biochemical step in protein synthesis is inhibited by the toxin. We will employ various methods for protein and RNA separation and characterization to investigate the actual toxin catalyzed ribosomal alteration which leads to the protein synthesis inhibition. Using both in vivo and in vitro systems we will determine whether toxin inhibits protein synthesis in the lumen of the small intestine. By using cell separation techniques, the effects of toxin on the various cell components of the intestinal lumen will be determined. Finally by separating A and B chains and by using monoclonal antibodies directed against the toxin chains we will investigate the role of these chains in the various biological activities of the toxin. In addition to clarifying the role of toxin in pathogenesis, these studies should also provide some basic information concerning the structure and function of the eukaryotic ribosome.
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