Intoxication of mammalian cells with binary clostridial enterotoxins is inhibited by the combination of pharmacological chaperone inhibitors

Abstract

Binary enterotoxins Clostridioides difficile CDT toxin, Clostridium botulinum C2 toxin, and Clostridium perfringens iota toxin consist of two separate protein components. The B-components facilitate receptor-mediated uptake into mammalian cells and form pores into endosomal membranes through which the enzymatic active A-components translocate into the cytosol. Here, the A-components ADP-ribosylate G-actin which leads to F-actin depolymerization followed by rounding of cells which causes clinical symptoms. The protein folding helper enzymes Hsp90, Hsp70, and peptidyl-prolyl cis/trans isomerases of the cyclophilin (Cyp) and FK506 binding protein (FKBP) families are required for translocation of A-components of CDT, C2, and iota toxins from endosomes to the cytosol. Here, we demonstrated that simultaneous inhibition of these folding helpers by specific pharmacological inhibitors protects mammalian, including human, cells from intoxication with CDT, C2, and iota toxins, and that the inhibitor combination displayed an enhanced effect compared to application of the individual inhibitors. Moreover, combination of inhibitors allowed a concentration reduction of the individual compounds as well as decreasing of the incubation time with inhibitors to achieve a protective effect. These results potentially have implications for possible future therapeutic applications to relieve clinical symptoms caused by bacterial toxins that depend on Hsp90, Hsp70, Cyps, and FKBPs for their membrane translocation into the cytosol of target cells.