Open AccessCompositional and stoichiometric analysis of Clostridium perfringens enterotoxin complexes in Caco‐2 cells and claudin 4 fibroblast transfectants
Author(s) -
Robertson Susan L.,
Smedley James G.,
Singh Usha,
Chakrabarti Ganes,
Van Itallie Christina M.,
Anderson James M.,
McClane Bruce A.
Publication year - 2007
Publication title -
cellular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.542
H-Index - 138
eISSN - 1462-5822
pISSN - 1462-5814
DOI - 10.1111/j.1462-5822.2007.00994.x
Subject(s) - claudin , biology , enterotoxin , clostridium perfringens , occludin , tight junction , immunoprecipitation , microbiology and biotechnology , receptor , fibroblast , transfection , molecular mass , biochemistry , escherichia coli , in vitro , bacteria , gene , enzyme , genetics
Summary 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 Ca 2+ 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.