B-162. Effect of STb Toxin Produced by Escherichia coli on the Mitochondrial Membrane Potential and Cell Viability

C. Gonçalves, J. D. Dubreuil;
Univ. de Montreal, Saint-Hyacinthe, QC, CANADA.

Escherichia coli STb enterotoxin induces rapid fluid accumulation in the small intestine in various animal species but is principally associated with swine. Previous studies have shown that STb could cause histological microscopic alterations in animal intestinal models. The occurrence of disrupted intestinal epithelium at the villous tips could be the result of cell death induced by the toxin. We used, as a cellular model, NIH-3T3, a fibroblast cell line to study the effects of STb toxin. Using various probes specific for cell organelles/or the cellular physiological state, we tried to understand STb activity using flow cytometry and confocal microscopy. NIH-3T3 cells labeled with propidium iodine and carboxyfluorescein diacetate showed that STb permeabilizes the plasma membrane and that the cellular esterases were still active. Using JC-1 probe, a fluorescent mitochondrial potential sensor, we observed mitochondria hyperpolarization produced by STb. The cell population treated with STb showed histological alterations such as membrane bledding, granular cytoplasm and enlarged nucleus. Thus, some cell sub-populations treated with STb indicated an apoptotic or a necrotic state corroborating the inferred cellular death of previous studies. However, apoptosis was the main effect associated with STb toxin activity as the cell percentage increased with the amount of STb used. Confocal microscopy showed that FITC-labelled STb toxin molecules were internalized and found scattered in the cytoplasm. FITC-STb was also associated with the plasma membrane and observed inside vesicles. Moreover, important clusters of FITC-STb were observed after 6 hours in the cells and these clusters matched with mitochondria labelling using Mitotracker red. This colocalization could explain the mitochondria hyperpolarization, the histological alterations as well as the altered cell viability observed. Altogether, these results provide a cellular explanation of the role of STb toxin in the development of diarrhea.