Effect of Escherichia coli STb toxin on NIH-3T3 cells.

Previous studies have shown that STb causes microscopic histological alterations in animal intestinal models. Disrupted intestinal epithelium at the villous tips could be the result of an altered physiological cell state induced by the toxin. As a cellular model we used NIH-3T3 cells, a mouse fibroblast cell line, previously shown to be capable of internalizing the STb toxin. Using various probes specific for the cellular physiological state or cell organelles, we investigated STb activity using flow cytometry and confocal microscopy. In NIH-3T3 cells, labelled with propidium iodide and carboxyfluorescein diacetate, STb permeabilized the plasma membrane but the cellular esterases remained active. Confocal microscopy showed that fluorescein isothiocyanate (FITC)-labelled STb toxin molecules were internalized and were found scattered in the cytoplasm. Moreover, important clusters of FITC-STb were observed inside the cells after 6 h and these clusters matched with mitochondria labelling. After cell treatment with STb, using a fluorescent mitochondrial potential sensor, we observed mitochondria hyperpolarization, as an early event of intoxication. This phenomenon increased linearly with the dose of STb. The cell population treated with STb showed histological alterations such as membrane budding, granular cytoplasm and enlarged nucleus. Altogether, these results provide new information, at the cellular level, on the effect of the STb toxin.