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.Clostridium difficile infection causes human diseases ranging from mild diarrhea to severe and potentially life-threatening pseudomembranous colitis. C. difficile-associated diseases occur in patients treated with broad-spectrum antibiotics. Under these conditions, the disturbed gut flora allows germination of C. difficile spores and colonization of the gut by this pathogen. C. difficile produces two exotoxins, toxin A (308 kDa) and toxin B (270 kDa), which are the causative agents of pseudomembranous colitis. The toxin-catalyzed glucosylation of Rho, Rac, and Cdc42 results in the inhibition of GTPase-mediated cell signaling, destruction of the actin cytoskeleton, and cell rounding that are the reasons for loss of integrity of the intestinal wall (for a review, see references 20 and 21).During the last 10 years, hypervirulent C. difficile strains were identified that produce, in addition to toxins A and B, a third exotoxin, the binary C. difficile transferase (CDT). Up to 35% of the strains tested produce CDT, and it has been suggested that the presence of CDT correlates with the severity of C. difficile-associated diseases (12,14,25,27,52). CDT belongs to the family of binary actin-ADP-ribosylating toxins and consists of two nonlink...

BEC, a Novel Enterotoxin of Clostridium perfringens Found in Human Clinical Isolates from Acute Gastroenteritis Outbreaks

“…The ADP ribosylation assay was performed as described previously (29,30). Briefly, two g of non-muscle actin (Cytoskeleton, Inc.) was incubated with 300 ng of each recombinant toxin component in the presence of 10 M biotinylated NAD ϩ (Trevigen) or nonlabeled NAD ϩ in the reaction buffer (20 mM Tris-HCl at pH 7.5, 1 M dithiothreitol, 40 M ATP, 40 M CaCl 2 , and 5 M MgCl 2 ) at 37°C for 60 min, followed by the addition of 3ϫ SDS sample buffer to terminate reactions.…”

Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

“…Ia probably passes through the channel formed by oligomeric Ib following a pH gradient, and in addition, a membrane potential gradient is necessary for translocating Ia (8). Furthermore, it has been reported that heat shock protein 90 (Hsp-90) and cyclophilin A are crucial for translocation of Ia across the endosomal membrane (14). In the cytosol, Ia ADP-ribosylates G-actin, resulting in actin depolymerization and cell rounding.…”

Diphtheria Toxin and Cytosolic Translocation Factors

“…A potential role for proyl isomerases in DT T domain membrane insertion and channel formation has been proposed (See Pore Formation above). Cyclophilin does facilitate the cytosolic entry of the C. botulinum C2 toxin, C. perfingens toxin, and the C difficile actin-ADP ribosylating CDT toxin (Kaiser et al, 2011). Dmochewitz et al (2011) demonstrated that in vitro translocation of the DT C domain across endosomal membranes using the anthrax pore was inhibited by cyclosporin, a specific inhibitor of cyclophilin.…”