Shiga toxin (Stx) of Shigella dysenteriae type 1, Stx1 and Stx2 of Escherichia coli O157:H7, and other Shiga toxin-producing E. coli (STEC), and ricin from the castor bean plant cause depurination of a critical residue in the 28S rRNA of 60S ribosomes and, hence, inhibition of protein synthesis, apoptosis, and cell death. Humans are usually at risk of intoxication by Stx or ricin through ingestion of Stx-expressing organisms or accidental intake of castor beans, respectively. Yet, these toxins are categorized as CDC Select Agents because of their potency and potential use as bioterrorist weapons. Deliberate delivery of the toxins may occur through intentional infection with STEC, or by ingestion, inhalation, or injection of ricin. When the toxins are introduced orally, they must first interact with enterocytes of the gastrointestinal tract by steps not yet defined, and, in some instances, breach the mucosa. Exactly how the association between Stxs and colonic epithelial cells occurs for the non-invasive E .coli O157:H7 and other STEC is unclear because these cells in humans do not express the Stx receptor globotriaosylceramide (Gb3). Nevertheless, we demonstrated dose-dependent binding of Stx1 and Stx2 to human colonic epithelial HCT-8 cells despite our inability to detect Gb3 on the surface of the cells. Moreover, Thorpe et al. reported that Stx1 is cytotoxic for HCT-8 cells at high doses (a finding that we confirmed but could not show for Stx2) and that Stxl, Stx2, and ricin cause varying degrees of inhibition of protein synthesis in HCT-8 cells. Thus, these ribotoxins appear to gain entry into and at some level intoxicate human intestinal cells in culture, and one long range goal of this project is to define the mechanisms by which Stxs and ricin cross the mucosal barrier. A second ultimate objective is to design therapeutic compounds that inactivate the toxins in intoxicated cells. The specific aims are to: 1. exploit non-Gb3-surface-expressing HCT-8 cells as a surrogate for normal human colonic cells to evaluate the nature of Stx binding and the basis for the finding that Stx1 but not Stx2 can, at high doses, kill HCT-8 cells; 2. monitor, by immunochemistry and biological activity, the translocation of Stxs produced by E. coli O157:H7 during infection of a 3-dimensional (3-D) organoid model of HCT-8 cells, and evaluate whether Stxs or ricin added to the surface of such multi-layered organoid tissue can transit from the apical surface to underlying cell layers and/or elicit tissue damage; and, 3.develop recombinant Stx or ricin cell-binding domains as platforms for conjugated therapeutics that selectively deliver into toxin-sensitive cells either toxin-neutralizing antibodies or toxin inhibitors and then test these chimeric molecules on cell lines and in mice for the capacity to ablate the lethal effects of Stx or ricin.