Comparative biochemical and immunocytochemical studies reveal differences in the effects of Clostridium perfringens enterotoxin on polarized CaCo-2 cells versus Vero cells

Since most in vitro studies exploring the action of Clostridium perfringens enterotoxin (CPE) utilize either Vero or CaCo-2 cells, the current study directly compared the CPE responsiveness of those two cell lines. When OPE-treated in suspension, both CaCo-2 and Vero cells formed SDS-resistant, CPE-containing complexes of similar to 135, similar to 155, and similar to 200 kDa. However, confluent Transwell (R) cultures of either cell line CPE-treated for 20 min formed only the similar to 155-kDa complex. Since those Transwell (R) cultures also exhibited significant Rb-86 release, similar to 155-kDa complex formation is sufficient for CPE-induced cytotoxicity. Several differences in CPE responsiveness between the two cell lines were also detected. i) CaCo-2 cells were more sensitive when CPE-treated on their basal surface, whereas Vero cells were more sensitive when CPE-treated on their apical surface; those sensitivity differences correlated with CPE binding the apical versus basolateral surfaces of these two cell lines. (ii) CPE-treated Vero cells released Rb-86 into both Transwell (R) chambers, whereas CaCo-2 cells released Rb-86 only into the CPE-containing Transwell (R) chamber. (iii) Vero cells express the tight junction (TJ) protein occludin but (unlike CaCo-2 cells) cannot form Ws. The ability of TJs to affect CPE responsiveness is supported by the similar effects of CPE on Transwell (R) cultures of CaCo-2 cells and Madin-Darby canine kidney cells, another polarized cell forming Ws. Confluent CaCo-2 Transwell (R) cultures CPE-treated for >I h formed the similar to 200-kDa CPE complex (which also contains occludin), exhibited morphologic damage, and had occludin removed from their Ws. Collectively, these results identify CPE as a bifunctional toxin that : in confluent polarized cells, first exerts a cytotoxic effect mediated by the similar to 155-kDa complex. Resultant damage then provides CPE access to Ws, leading to similar to 200-kDa complex formation, internalization of some TJ proteins, and TJ damage that may increase paracellular permeability and thereby contribute to the diarrhea of CPE induced gastrointestinal disease.