The mode of action of Shiga toxin in bacterial pathogensis is to be explored at the whole cell and subcellular levels. The mechanism of our newly discovered latent cytotoxicity effect exhibited by Shiga toxin on human vascular endothelial cells in culture is to be investigated in regard to the temporal relationship of toxin binding and entry into these cells. A role for this in vitro latent cytotoxicity phenomenon will be explored to help explain a similar latent effect of Shiga toxin in human vascular diseases such as hemorrhagic colitis and hemolytic uremic syndome. At the subcellular level, three different molecular approaches are described to reveal how Shiga toxin enzymatically inactivates mammalian ribosomes, the primary target of this toxin which is responsible for inhibition of protein synthesis and Shiga-related cytotoxicity. First, emphasis is placed on our efforts to define the ribonuclease activity which continues to appear to be an inherent property of purified Shiga toxin. Shiga RNase activity is to be studied using the intact ribosome, highly purified rRNA and RNA homopolymer substrates. Second, to more fully answer the question of how Shiga toxin recognizes and interacts with eukaryotic ribosomes, we present a research plan based on hybridzation of defined cloned rRNA gene fragments to intact ribosomes to protect the ribosome against inactivation by Shiga toxin. This experimental approach will help reveal which rRNA regions are required for Shiga toxin recognition of ribosomes. Third, (125I)Shiga toxin will be covalently crosslinked to its natural ribosomal binding site using a photoaffinity reagent. A research plan is described for identification of those ribosomal protein and rRNA species located within the toxin binding site to which Shiga toxin becomes crosslinked. Together, these molecular studies would provide a description of how Shiga toxin identifies and catalytically inactivates mammalian ribosomes. In addition, the endothelial cellular investigations should yield new information about Shiga toxin cytotoxic mechanisms in clinically- related vascular diseases.
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