Mode of Action of Shigella Toxin: Effects on Ribosome Structure and Function

The goal of this contract research project was to describe, in biochemical terms, the detailed mechanism of Shiga toxin inhibition of eukaryotic protein synthesis. It was previously determined that Shiga toxin preferentially inhibited peptide elongation, but the exact steps of peptide elongation representing the toxin target had not been described in detail. In the present study, purified components for protein synthesis were utilized to measure individual steps of peptide elongation. Our results clearly indicate that Shiga toxin is a primary inhibitor or eukaryotic elongation factor-1 eEF- 1 dependent binding of aminoacyl-tRNA to ribosomes. In addition, Shiga toxin was shown to be a less potent inhibitor of eEF-2-dependent translocation of aminoacyl-tRNA to ribosomes. Excess eEF-1 protein could not reverse the toxin- related effects on aminoacyl-tRNA binding whereas excess exogenous eEF-2 protein could easily overcome toxin-induced inhibitions of peptidyl-tRNA translocation. An additional discovery during this project was the observation that Shiga toxin exhibited a latent cytotoxic response towards human vascular endothelial cells, in vitro. The significance of these results is that Shiga toxin action described in this study may explain the nature of hemolytic uremic syndrome HUS, vascular disease of children. HUS occurs 3 to 4 days after a peak of bacillary dysentery. aw