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 approximately 135, approximately 155, and approximately 200 kDa. However, confluent Transwell cultures of either cell line CPE-treated for 20 min formed only the approximately 155-kDa complex. Since those Transwell cultures also exhibited significant (86)Rb release, approximately 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 (86)Rb into both Transwell chambers, whereas CaCo-2 cells released (86)Rb 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 approximately 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 approximately 155-kDa complex. Resultant damage then provides CPE access to TJs, leading to approximately 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.
Highlights
The Gram-positive bacterium Clostridium perfringens uses its potent arsenal of 14 toxins to cause enteric and histotoxic infections in humans and domestic animals [1, 2]
Much lower amounts of the ϳ200-kDa complex were detected in equivalent numbers of isolated Vero cells versus isolated CaCo-2 cells subjected to similar C. perfringens enterotoxin (CPE) treatment (Fig. 1, left panel)
By comparing the CPE responsiveness of CaCo-2 cells and Vero cells under several experimental conditions, the current study provides a number of new insights into CPE action and CPE-mediated GI disease
Summary
CPE was purified to homogeneity from C. perfringens strain NCTC 8239. The biological activity of that purified toxin was assayed, as described previously [29]. Aliquots (2 mg) of that purified CPE were radiolabeled by a published protocol [11, 30] using lactoperoxidaseglucose oxidase (Bio-Rad) and 2 mCi of Na125I (17 mci/mg; ICN Radiochemicals). The biological activity of the 125I-CPE was confirmed using published protocols [11, 30]
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