Compositional and stoichiometric analysis of Clostridium perfringens enterotoxin complexes in Caco-2 cells and claudin 4 fibroblast transfectants

Abstract

Clostridium perfringens enterotoxin (CPE) binds to host cell receptors, forming a small complex precursor for two large complexes reportedly having molecular masses of ∼155 or ∼200 kDa. Formation of the ∼155 kDa complex causes a Ca2+ influx that leads to apoptosis or oncosis. CPE complex composition is currently poorly understood, although occludin was identified in the ∼200 kDa complex. The current study used heteromer gel shift analysis to show both CPE large complexes contain six CPE molecules. Ferguson plots and size exclusion chromatography re-sized the ∼155 and ∼200 kDa complexes as ∼425-500 kDa and ∼550-660 kDa respectively. Co-immunoprecipitation and electroelution studies demonstrated both CPE-binding and non-CPE-binding claudins are associated with all three CPE complexes in Caco-2 cells and with small complex and ∼425-500 kDa complex of claudin 4 transfectants. Fibroblast transfectants expressing claudin 4 or C-terminal truncated claudin 4 were CPE-sensitive and formed the ∼425 kDa complex, indicating claudin-induced cell signalling is not required for CPE action and that expression of a single receptor claudin suffices for ∼425-500 kDa CPE complex formation. These results identify CPE as a unique toxin that combines with tight junction proteins to form high-molecular-mass hexameric pores and alter membrane permeability. © 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd.