title: 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 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.
authors.affiliation: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: U
authors.initials: U
authors.lastname: Singh
authors.forename: L L
authors.initials: LL
authors.lastname: Mitic
authors.forename: E U
authors.initials: EU
authors.lastname: Wieckowski
authors.forename: J M
authors.initials: JM
authors.lastname: Anderson
authors.forename: B A
authors.initials: BA
authors.lastname: McClane
mesh_terms: D000199:Actins / Q000096:biosynthesis; D000818:Animals; D015153:Blotting, Western; D018938:Caco-2 Cells; D002450:Cell Communication; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D004285:Dogs; D004768:Enterotoxins / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000096:biosynthesis; D062793:Occludin; D010750:Phosphoproteins / Q000096:biosynthesis; D011485:Protein Binding; D012413:Rubidium / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D013997:Time Factors; D014709:Vero Cells; D062826:Zonula Occludens-1 Protein
chemical_list: D000199:Actins; D004768:Enterotoxins; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D013501:Surface-Active Agents; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C035598:enterotoxin, Clostridium; D012967:Sodium Dodecyl Sulfate; D012413:Rubidium