Trypan blue uptake by chinese hamster ovary cultured epithelial cells: a cellular model to study Escherichia coli STb enterotoxin

Abstract

The thermostable enterotoxin b (STb) produced by enterotoxigenic Escherichia coli strains is responsible for diarrheal diseases mainly in weaning piglets. For now, the only available assay for biological activity of STb toxin was in the animal host (i.e. piglet) or in an animal model (i.e. rat, mouse). In this study, we developed a cellular model for the study of the biological activity of STb enterotoxin. Using a trypan blue vital stain method, we showed that STb-treated cells of three out of the five cell lines tested absorbed more vital stain than their controls. Of all the cell lines tested, the chinese hamster's ovary derivated cells (CHO) were the most sensitive, absorbing 50% more trypan blue than their control. Maximal stain uptake was observed after 2 h. We then evaluated the trypan blue uptake for 16 STb mutants, produced in a previous work, on the CHO cell lines in order to compare it with the in vivo rat loop assay data. Interestingly, we observed a good correlation between the two bioassays. In fact, the biological activity observed in the rat could be correlated with the trypan blue uptake by the CHO cells ( R 2=0.78) for STb toxin and the 16 mutants. Using the variance analysis statistical test, we determined that the correlation between the two bioassays is significant ( F c≥ F 0.005). These results suggest that the trypan blue uptake bioassay could represent a new method to evaluate the biological activity and facilitate the elucidation of the mechanism of action of E. coli STb enterotoxin.