title: Membrane translocation of binary actin-ADP-ribosylating toxins from Clostridium difficile and Clostridium perfringens is facilitated by cyclophilin A and Hsp90.
abstract: Some hypervirulent strains of Clostridium difficile produce the binary actin-ADP-ribosylating toxin C. difficile transferase (CDT) in addition to Rho-glucosylating toxins A and B. It has been suggested that the presence of CDT increases the severity of C. difficile-associated diseases, including pseudomembranous colitis. CDT contains a binding and translocation component, CDTb, that mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, where CDTa modifies actin. Here we investigated the mechanism of cellular CDT uptake and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, implying that CDTa is translocated from acidified endosomal vesicles into the cytosol. Consistently, CDTa is translocated across the cytoplasmic membranes into the cytosol when cell-bound CDT is exposed to acidic medium. Radicicol and cyclosporine A, inhibitors of the heat shock protein Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not from intoxication with toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the inhibitors did not interfere with the ADP-ribosyltransferase activity, receptor binding, or endocytosis of the toxin. We obtained comparable results with the closely related iota-toxin from Clostridium perfringens. Moreover, CDTa and Ia, the enzyme component of iota-toxin, specifically bound to immobilized Hsp90 and cyclophilin A in vitro. In combination with our recently obtained data on the C2 toxin from C. botulinum, these results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Kaiser
authors.forename: Claudia
authors.initials: C
authors.lastname: Kroll
authors.forename: Katharina
authors.initials: K
authors.lastname: Ernst
authors.forename: Carsten
authors.initials: C
authors.lastname: Schwan
authors.forename: Michel
authors.initials: M
authors.lastname: Popoff
authors.forename: Gunter
authors.initials: G
authors.lastname: Fischer
authors.forename: Johannes
authors.initials: J
authors.lastname: Buchner
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
authors.forename: Holger
authors.initials: H
authors.lastname: Barth
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000378:metabolism* / Q000472:pathogenicity; D003016:Clostridium perfringens / Q000378:metabolism* / Q000472:pathogenicity; D021984:Cyclophilin A / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D018942:Macrolides / Q000494:pharmacology; D021381:Protein Transport; D014709:Vero Cells
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D018841:HSP90 Heat-Shock Proteins; D018942:Macrolides; C057620:bafilomycin A; D036002:ADP Ribose Transferases; C410164:actin-specific ADP-ribosyltransferase, Clostridium; D021984:Cyclophilin A