Comparative Biochemical and Immunocytochemical Studies Reveal Differences in the Effects of Clostridium perfringens Enterotoxin on Polarized CaCo-2 Cells Versus Vero Cells

Abstract

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 CPE-treated in suspension, both CaCo-2 and Vero cells formed SDS-resistant, CPE-containing complexes of ∼135, ∼155, and ∼200 kDa. However, confluent Transwell® cultures of either cell line CPE-treated for 20 min formed only the ∼155-kDa complex. Since those Transwell® cultures also exhibited significant 86Rb release, ∼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 86Rb into both Transwell® chambers, whereas CaCo-2 cells released 86Rb only into the CPE-containing Transwell® chamber. (iii) Vero cells express the tight junction (TJ) protein occludin but (unlike CaCo-2 cells) cannot form TJs. The ability of TJs to affect CPE responsiveness is supported by the similar effects of CPE on Transwell® cultures of CaCo-2 cells and Madin-Darby canine kidney cells, another polarized cell forming TJs. Confluent CaCo-2 Transwell® cultures CPE-treated for >1 h formed the ∼200-kDa CPE complex (which also contains occludin), exhibited morphologic damage, and had occludin removed from their TJs. Collectively, these results identify CPE as a bifunctional toxin that, in confluent polarized cells, first exerts a cytotoxic effect mediated by the ∼155-kDa complex. Resultant damage then provides CPE access to TJs, leading 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.