Shiga toxin (Stx) producing E. coli (STEC) are foodborne pathogens that can cause severemorbidity and mortality, including hemorrhagic colitis (HC) and hemolytic uremic syndrome(HUS) and remain a major challenge for food safety and public health. There are no specifictherapeutics or vaccines against infection by bacteria producing these toxins. Shiga toxins are afamily of AB5 toxins consisting of an A1 subunit, which depurinates a universally conservedadenine in the ribosome, and a pentamer of identical B subunits, which bind to a glycolipidglobotriaosylceramide receptor. E. coli strains producing Shiga toxin 2 (Stx2) are associatedwith progression to more severe disease than strains producing Shiga toxin 1 (Stx1). Themechanism that accounts for the differences in toxicity of Stx1 and Stx2 is not known. Althoughseveral studies suggested a role for the B subunit in the increased toxicity of Stx2, the role ofthe A1 subunit in the higher toxicity of Stx2 has not been investigated. The proposed studiesseek to fill a critical gap in our understanding of Stx toxicity by examining the role of the A1subunit. We propose that the A1 subunit in combination with the B subunit contributes to toxicity.We recently demonstrated that the A1 subunit of Stx2 has higher affinity for mammalianribosomes and is more active than the A1 subunit of Stx1 and that differences in surface chargeare critical for the interaction of the A1 subunits with ribosomes. We will test the hypothesis thatribosome binding kinetics and catalytic activity, which determine the rate of depurination,contribute to the differential toxicity of Stx1 and Stx2. In Aim 1 we will identify the residues thatcontribute to differences in surface charge of the A1 subunits and determine if they contribute tothe differential activity and toxicity; in Aim 2, we will determine if depurination activity of the A1subunit contributes to the lethality of Stx2 holotoxin in an animal model; and in Aim 3 we willidentify peptides that can bind to ribosome docking surfaces and determine if they reducetoxicity of Stx2 by disrupting its interaction with the ribosome. This approach has alreadyculminated in peptides that inhibit depurination activity of Stx2. We expect that our uniqueapproach, the powerful set of tools we developed, ability to dissect the toxin activity in vitro andin vivo will reveal the factors critical for STEC pathogenesis and will provide a path to newtherapy.