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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Specialized Center--Cooperative Agreements (U54)
Project #
Application #
Study Section
Special Emphasis Panel (ZAI1-DDS-M)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Maryland Baltimore
United States
Zip Code
Li, Huiguang; Hwang, Young; Perry, Kay et al. (2016) Structure and Metal Binding Properties of a Poxvirus Resolvase. J Biol Chem 291:11094-104
Ramachandran, Girish; Tennant, Sharon M; Boyd, Mary A et al. (2016) Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella. PLoS One 11:e0151875
Ray, Greeshma; Schmitt, Phuong Tieu; Schmitt, Anthony P (2016) C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles. J Virol 90:3650-60
Chou, Yi-ying; Cuevas, Christian; Carocci, Margot et al. (2016) Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor. J Virol 90:4494-510
Fraley, Stephanie I; Athamanolap, Pornpat; Masek, Billie J et al. (2016) Nested Machine Learning Facilitates Increased Sequence Content for Large-Scale Automated High Resolution Melt Genotyping. Sci Rep 6:19218
Levy, Revital; Rotfogel, Ziv; Hillman, Dalia et al. (2016) Superantigens hyperinduce inflammatory cytokines by enhancing the B7-2/CD28 costimulatory receptor interaction. Proc Natl Acad Sci U S A 113:E6437-E6446
Molleston, Jerome M; Sabin, Leah R; Moy, Ryan H et al. (2016) A conserved virus-induced cytoplasmic TRAMP-like complex recruits the exosome to target viral RNA for degradation. Genes Dev 30:1658-70
Riblett, Amber M; Blomen, Vincent A; Jae, Lucas T et al. (2016) A Haploid Genetic Screen Identifies Heparan Sulfate Proteoglycans Supporting Rift Valley Fever Virus Infection. J Virol 90:1414-23
Ramachandran, Girish; Boyd, Mary Adetinuke; MacSwords, Jennifer et al. (2016) Opsonophagocytic Assay To Evaluate Immunogenicity of Nontyphoidal Salmonella Vaccines. Clin Vaccine Immunol 23:520-3
Plaut, Roger D; Stibitz, Scott (2015) Improvements to a Markerless Allelic Exchange System for Bacillus anthracis. PLoS One 10:e0142758

Showing the most recent 10 out of 360 publications