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J. Biol. Chem., Vol. 276, Issue 36, 33402-33412, September 7, 2001

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

Usha Singh, Laura L. Mitic^§, Eva U. Wieckowski^¶, James M. Anderson^§, and Bruce A. McClane^**

From the  Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 and the ^§ Department of Internal Medicine and the  Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06511

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.

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