Shiga Toxin Effects on Gene Regulation

Shiga toxin-producing E. coli (STEC) have emerged as a major health problem in the developed world and are associated with diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Our understanding of the role of Shiga toxins in the pathogenesis of STEC-associated disease is incomplete. Classical thinking about Shiga toxins' role in disease pathogenesis is that Shiga toxins harm the host by causing toxic effects on sensitive cells by inhibiting protein synthesis of critical proteins needed for cell survival. We have found that Shiga toxins are capable of inducing and superinducing IL-8, in intestinal epithelial cells. Paradoxically, IL-8 is secreted by these cells despite overall blockade of mRNA translation. Our data suggest that this may occur through alterations in host signal transduction. These data support a new model of how Stxs may contribute to disease, namely that Stxs are involved in altering the regulation of one or more host cell processes, resulting in synthesis of proteins by the host that contribute to pathogenesis. The goals of the proposed work are: 1) to characterize how Shiga toxins affect IL-8 gene regulation in intestinal epithelial cells 2) to determine how Shiga toxins are able to increase IL-8 mRNA and protein while inhibiting translation, and 3) to assess the effects of Shiga toxins on related genes in the intestine and the kidney. Shiga toxin-induced alterations in IL-8 gene regulation will be assessed at the level of transcription. The role of Shiga toxin glycohydrolase activity in IL-8 mRNA induction will be assessed. Effects of Shiga toxins on host signal transduction will be assessed. Intracellular trafficking pathway inhibitors will be employed to determine if specific Shiga toxin trafficking pathways are necessary for IL-8 synthesis. Using confocal microscopy, co-localization of IL-8 mRNA and intracellular Shiga toxin will determine if the site of IL-8 mRNA translation is distinct from where Shiga toxin traffics. Finally, the effects of Shiga toxins on gene regulation in epithelial cells will be assessed on a genomic level using cDNA microarray hybridization techniques. Understanding how this toxin may augment a host inflammatory response through affecting host gene regulation may allow us to better understand the pathogenesis of a disease for which there is no vaccine and only supportive therapies. Through coursework, seminars, and supervised research, Dr. Thorpe will gain knowledge and expertise in techniques pertinent to the proposed research.