|Droleskey, Robert - Bob|
Submitted to: Poisonous Plants Symposium Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 8/7/2001
Publication Date: N/A
Technical Abstract: Escherichia coli O157:H7 produce Shiga toxins, Stx 1 and Stx 2, which are responsible for much of the diarrhea and enterocolitis associated with disease resulting from infection. The primary receptor on the epithelial cell surface for Stx is the glycolipid receptor globotriaosylceramide, GB3. Recent work has indicated that tunicamycin, an inhibitor of N-linked glycoprotein synthesis, inhibits expression of GB3 on the surface of HeLa cells in culture which results in reduced cell cytotoxicity after exposure to shiga toxin from Shigella dysenteriae. We explored the possibility that the known alpha-D-mannosidase II inhibitor swainsonine could also regulate the expression of GB3 and/or a second low affinity Stx binding site. The supernatant from an overnight culture of Stx 1 and 2 producing E. coli O157:H7 was tested for toxin activity against Vero cells using a LDH cytotoxicity assay procedure. Vero cells were seeded into the wells of a 96 6well microtiter plate at 10e4 cells/well. For wells in which the protectiv effects of swainsonine were evaluated, the cells were co-incubated with swainsonine during the 24 h establishment period. After establishment, the Vero cells were incubated overnight with toxin, toxin plus swainsonine, or the appropriate control. LDH activity was assayed 24h after the addition of Stx. Toxic effects were not observed in swainsonine control wells co-incubated with as much as 5 ug/well. When cells were incubated with 1/16 or 1/8 dilutions of Stx toxin and 2.5 ug of swainsonine, LDH release levels were reduced by 50% and 22% compared to controls. These results suggest a protective effect from co-incubation of Vero cells with swainsonine. swainsonine.