Nonmuscle actin ADP-ribosylated by botulinum C2 toxin caps actin filaments

The effect of nonmuscle actin ADP-ribosylated by botulinum C2 toxin on the polymerization of nonmuscle actin was investigated in order to clarify whether nonmuscle actin is converted into a capping protein by ADP-ribosylation. ADP-ribosylated actin was found to decrease the rate of polymerization of actin filaments which are free at both ends. ADP-ribosylated actin turned out to have no effect on the rate or extent of polymerization at the pointed ends of actin filaments the barbed ends of which were capped by gelsolin. The monomer concentration reached at the final stage of polymerization was similar to the critical concentration of the pointed ends of actin filaments. The results suggest that nonmuscle actin ADP-ribosylated by botulinum C2 toxin acts as a capping protein which binds to the barbed ends to inhibit polymerization.     

Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells

Botulinum C2 toxin ADP-ribosylates actin in [32P]orthophosphate-labelled intact chick embryo cells (CEC). The toxin-induced rounding up of CEC is correlated with ADP-ribosylation of actin in intact cells in a time and concentration-dependent manner. Both, rounding up of cells and actin ADP-ribosylation, depend on the presence of both components of botulinum C2 toxin (components I and II) and are independent of the ability of CEC to divide. Treatment of CEC with botulinum C2 toxin induced a time-dependent disorganization of the typical architecture of the microfilament network as shown by fluorescein-phalloidin staining. Botulinum C2 toxin decreased the amount of Triton X-100 insoluble actin, while the fraction of Triton soluble actin was increased. Actin, which was 32P-labelled by botulinum C2 toxin in intact CEC, was recovered in the Triton soluble but not in the Triton insoluble actin fraction. It is suggested that in intact CEC botulinum C2 toxin causes ADP-ribosylation of G-actin but not of F-actin thereby leading to an accumulation in the pool of monomeric actin.     

ADP-ribosylation of platelet actin by botulinum C2 toxin

Botulinum C2 toxin is a microbial toxin which possesses ADP-ribosyltransferase activity. In human platelet cytosol a 43-kDa protein was ADP-ribosylated by botulinum C2 toxin. Labelling of the 43-kDa protein using [32P]NAD as substrate was reduced by unlabelled NAD and nicotinamide. The label was removed by treatment with snake venom phosphodiesterase. Half-maximal and maximal ADP-ribosylation occurred at 0.1 microgram/ml and 3 micrograms/ml botulinum C2 toxin, respectively. The Km value of the ADP-ribosylation reaction for NAD was about 1 microM. The peptide map of the ADP-ribosylated 43-kDa protein was almost identical with platelet actin. The ADP-ribosylated 43-kDa substrate protein bound to and was eluted from immobilized DNase I in a manner similar to G-actin. Trypsin treatment of platelet cytosol decreased subsequent ADP-ribosylation of the 43-kDa protein without occurrence of smaller labelled polypeptides. Purified platelet actin was also ADP-ribosylated by botulinum C2 toxin with similar characteristics found with actin in platelet cytosol. Phalloidin decreased the ADP-ribosylation of actin in platelet cytosol and of isolated platelet actin. Half-maximal and maximal, about 90%, reduction of actin ADP-ribosylation was observed at 0.4 microM and 10 microM phalloidin, respectively. ADP-ribosylation of purified actin, induced by botulinum C2I toxin, abolished the formation of the typical microfilament network. The data indicate that platelet G-actin but not F-actin is a substrate of botulinum C2 toxin and that this covalent modification largely affects the functional properties of actin.     

ADP-ribosylation of gelsolin-actin complexes by clostridial toxins.

ADP-ribosylation of the 1:1 (G-A) and 1:2 (G-A-A) gelsolin-actin complexes by Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin was studied. Iota toxin ADP-ribosylated actin in the G-A complex from human platelets as effectively as skeletal muscle actin. The Km for NAD (4 microM) was identical for both substrates. C2 toxin ADP-ribosylated actin in the G-A complex with lower efficacy than nonmuscle actin from platelet cytosol. In the G-A-A complex both actin molecules were ADP-ribosylated by iota toxin. The G-A complex bound ADP-ribosylated actin (Ar) to form the G-A-Ar complex in which the weakly bound actin is ADP-ribosylated. Vice versa, ADP-ribosylated 1:1 gelsolin-actin complex (G-Ar) was able to bind unmodified actin to yield the G-Ar-A complex. ADP-ribosylation did not change the nucleation activity of either the G-Ar complex or the G-Ar-A complex. When monomeric actin was added to the G-A-Ar complex, polymerization of actin was delayed by about 10 min. According to a quantitative kinetic analysis, the delay of polymerization corresponded to the rate of dissociation of ADP-ribosylated actin from the G-A-Ar complex. This suggests that the nucleation activity of the G-A-A complex is inhibited by ADP-ribosylation of the weakly bound actin and that the inhibition can be removed by dissociation of ADP-ribosylated actin from the G-A-Ar complex.     

ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin

The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.     

ADP-ribosylation in Clostridium difficile toxin-treated cells is not related to cytopathogenicity of toxin B

ADP-ribosylation of a protein in human fibroblasts treated with partially purified Clostridium difficile toxin B was previously reported. Here we show that the same protein was ADP-ribosylated also in human fibroblasts exposed to supernatant from a C. difficile strain producing neither toxin A nor toxin B. Furthermore, in Chinese hamster ovary and in Vero cells, showing toxin B-induced cytopathogenic effect, the protein was not significantly ADP-ribosylated. The results indicate that the ADP-ribosylation is unrelated to the cytopathogenic effect of toxin B. It appears to be caused by another unidentified factor from C. difficile, and the substrate may correspond to a protein modified endogenously in cells exposed to stressful situations. Cellular actin was not ADP-ribosylated by toxin B.     

Characterization of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin

Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin belong to a novel family of actin ADP-ribosylating toxins. ADP-ribosylation of actin inhibits actin polymerization and G-actin-associated ATPase activity. The ADP-form of actin is ADP-ribosylated at a higher rate than actin with bound ATP. ADP-ribosylation of actin is reversible, a reaction, which is accompanied by reconstitution of actin ATPase activity.     

NAD-glycohydrolase activity of botulinum C2 toxin: a possible role of component I in the mode of action of the toxin

C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two separate protein components, designated components I and II, which individually have little activity, but, when mixed and treated with trypsin, exert the potent activity. The present study provides the evidence that component I of the toxin catalyzes the hydrolysis of NAD into nicotinamide and ADP-ribose, whereas component II does not, indicating that component I of C2T has NAD-glycohydrolase activity, which ability is shared with cholera and diphtheria toxins. However, C2T affected neither glycerol production of fat cells nor protein synthesis in cell-free system. Component I of C2T in the presence of [alpha-32P]NAD radiolabeled a protein of Mr 46,000 in the supernatant fractions of mouse tissue homogenates; the protein was abundant in brain, lung and intestine, whereas there was little or none of the protein in muscle. These results indicate that component I can catalyze the covalent attachment of the ADP-ribose moiety of NAD to intracellular protein, which differs from those modified with cholera and diphtheria toxins. The present data, together with previous findings, suggest that the biological activity of C2T is elicited by ADP-ribosylation activity of component I, which is internalized into the cells after binding to the receptor site introduced with the binding of component II to the cell surface membrane.     

Inhibition of cytochalasin D-stimulated G-actin ATPase by ADP-ribosylation with Clostridium perfringens iota toxin.

Clostridium perfringens iota toxin belongs to a novel family of actin-ADP-ribosylating toxins. The effects of ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin on cytochalasin D-stimulated actin ATPase activity was studied. Cytochalasin D stimulated actin-catalysed ATP hydrolysis maximally by about 30-fold. ADP-ribosylation of actin completely inhibited cytochalasin D-stimulated ATP hydrolysis. Inhibition of ATPase activity occurred at actin concentrations below the critical concentration (0.1 microM), at low concentrations of Mg2+ (50 microM) and even in the actin-DNAase I complex, indicating that ADP-ribosylation of actin blocks the ATPase activity of monomeric actin and that the inhibitory effect is not due to inhibition of the polymerization of actin.     

Salmonella enterica SpvB ADP-ribosylates actin at position arginine-177-characterization of the catalytic domain within the SpvB protein and a comparison to binary clostridial actin-ADP-ribosylating toxins

The SpvB protein from Salmonella enterica was recently discovered as an actin-ADP-ribosylating toxin. SpvB is most likely delivered via a type-III secretion system into eukaryotic cells and does not have a binding/translocation component. This is in contrast to the family of binary actin-ADP-ribosylating toxins from various Bacillus and Clostridium species. However, there are homologies in amino acid sequences between the C-terminal domain of SpvB and the catalytic domains of the actin-ADP-ribosylating toxins such as C2 toxin from Clostridium botulinum and iota toxin from Clostridium perfringens. We compared the biochemical properties of the catalytic C-terminal domain of SpvB (C/SpvB) with the enzyme components of C2 toxin and iota toxin. The specificity of C/SpvB concerning the modification of G- or F-actin was comparable to the C2 and iota toxins, although there were distinct differences regarding the recognition of actin isoforms. C/SpvB and iota toxin modify both muscle alpha-actin and nonmuscle beta/gamma-actin, whereas C2 toxin only modifies beta/gamma-actin. In contrast to the iota and C2 toxins, C/SpvB possessed no detectable glycohydrolase activity in the absence of a protein substrate. The maximal reaction rates were comparable for all toxins, whereas variable K(m) values for NAD were evident. We identified arginine-177 as the modification site for C/SpvB with the actin homologue protein Act88F from Drosophila.     

New insights into the mode of action of the actin ADP-ribosylating virulence factors Salmonella enterica SpvB and Clostridium botulinum C2 toxin

The C2 toxin from Clostridium botulinum represents the prototype of the family of binary actin-ADP-ribosylating toxins. These toxins covalently transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD(+)) onto arginine-177 of actin in the cytosol of eukaryotic cells resulting in depolymerization of actin filaments and cell rounding. The C2 toxin consists of two non-linked proteins, the enzyme component C2I and the binding and translocation component C2II, which delivers C2I into host cells. The ADP-ribosyltransferase SpvB from Salmonella enterica also modifies actin, but is delivered into the host cell cytosol from intracellular growing Salmonella, most likely via type-III-secretion. We characterized the mode of action of SpvB in comparison to C2 toxin in vitro and in intact cells. We identified arginine-177 as the target for SpvB-catalyzed mono-ADP-ribosylation of actin. To compare the cellular responses following modification of actin by SpvB or by the binary toxins without the influence of other Salmonella virulence factors, we constructed a cell-permeable fusion toxin to deliver the catalytic domain of SpvB (C/SpvB) into the cytosol of target cells. This review summarizes recent findings of research on the actin ADP-ribosylating toxins regarding their cellular uptake, molecular mode of action and the cellular consequences following ADP-ribosylation of actin.     

The host cell chaperone Hsp90 is essential for translocation of the binary Clostridium botulinum C2 toxin into the cytosol

Clostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I. The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I. This toxin complex is taken up by receptor-mediated endocytosis. In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol. We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin. ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors. Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I. At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I. Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa. The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro. The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors. In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells. The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II.     

ADP-ribosylation of actin causes increase in the rate of ATP exchange and inhibition of ATP hydrolysis

ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin increased the rate of exchange of actin-bound [gamma-32P]ATP by unlabelled ATP about twofold. Increased exchange rates were observed with ATP and ATP[gamma S], much less with ADP but not with AMP or NAD. ADP-ribosylation of skeletal muscle actin reduced "basal" and Mg2+ (1 mM)-induced ATP hydrolysis by about 80%. Similar inhibition of ATP hydrolysis was observed with liver actin ADP-ribosylated by Clostridium botulinum C2 toxin. The data indicate that ADP-ribosylation of actin at Arg-177 largely affects the ATP-binding and ATPase activity.     

De-ADP-ribosylation actin by Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin

The reverse reaction of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota-toxin was studied. In the presence of nicotinamide (30-50 mM) C2 toxin and iota-toxin decreased the radioactive labeling of [32P]ADP-ribosylated actin and catalyzed the formation of [32P]NAD. The pH optima for both reactions were 5.5-6.0. Concomitant with the removal of ADP-ribose, the ability of actin to polymerize was restored and actin ATPase activity increased. Neither ADP-ribosylation nor removal of ADP-ribose was observed after treatment of actin with EDTA, indicating that the native structure of actin is required for both reactions. ADP-ribosylation of platelet actin by C2 toxin was reversed by iota-toxin, confirming recent reports that both toxins modify the same amino acid in actin. However, C. botulinum C2 toxin was not able to cleave ADP-ribose from skeletal muscle actin which had been incorporated by iota-toxin, corroborating the different substrate specificities of both toxins.     

A cell-permeable fusion toxin as a tool to study the consequences of actin-ADP-ribosylation caused by the salmonella enterica virulence factor SpvB in intact cells

The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N- and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells.     

G-protein-stimulated phospholipase D activity is inhibited by lethal toxin from Clostridium sordellii in HL-60 cells.

Lethal toxin (LT) from Clostridium sordellii has been shown in HeLa cells to glucosylate and inactivate Ras and Rac and, hence, to disorganize the actin cytoskeleton. In the present work, we demonstrate that LT treatment provokes the same effects in HL-60 cells. We show that guanosine 5'-O-(3-thiotriphosphate)-stimulated phospholipase D (PLD) activity is inhibited in a time- and dose-dependent manner after an overnight treatment with LT. A similar dose response to the toxin was found when PLD activity was stimulated by phorbol 12-myristate 13-acetate via the protein kinase C pathway. The toxin effect on actin organization seemed unlikely to account directly for PLD inhibition as cytochalasin D and iota toxin from Clostridium perfringens E disorganize the actin cytoskeleton without modifying PLD activity. However, the enzyme inhibition and actin cytoskeleton disorganization could both be related to a major decrease observed in phosphatidylinositol 4,5-bisphosphate (PtdIns(4, 5)P2). Likely in a relationship with this decrease, recombinant ADP-ribosylation factor, RhoA, Rac, and RalA were not able to reconstitute PLD activity in LT-treated cells permeabilized and depleted of cytosol. Studies of phosphoinositide kinase activities did not allow us to attribute the decrease in PtdIns(4,5)P2 to inactivation of PtdIns4P 5-kinase. LT was also found to provoke a major inhibition in phosphatidylinositol 3-kinase that could not account for the inhibition of PLD activity because wortmannin, at doses that fully inhibit phosphatidylinositol 3-kinase, had no effect on the phospholipase activity. Among the three small G-proteins, Ras, Rac, and RalA, inactivated by LT and involved in PLD regulation, inactivation of Ral proteins appeared to be responsible for PLD inhibition as LT toxin (strain 9048) unable to glucosylate Ral proteins did not modify PLD activity. In HL-60 cells, LT treatment appeared also to modify cytosol components in relationship with PLD inhibition as a cytosol prepared from LT-treated cells was less efficient than one from control HL-60 cells in stimulating PLD activity. Phosphatidylinositol transfer proteins involved in the regulation of polyphosphoinositides and ADP-ribosylation factor, a major cytosolic PLD activator in HL-60 cells, were unchanged, whereas the level of cytosolic protein kinase Calpha was decreased after LT treatment. We conclude that in HL-60 cells, lethal toxin from C. sordellii, in inactivating small G-proteins involved in PLD regulation, provokes major modifications at the membrane and the cytosol levels that participate in the inhibition of PLD activity. Although Ral appeared to play an essential role in PLD activity, we discuss the role of other small G-proteins inactivated by LT in the different modifications observed in HL-60 cells.     

Alteration of the cytoskeleton of mammalian cells cultured in vitro by Clostridium botulinum C2 toxin and C3 ADP-ribosyltransferase

The effects of Clostridium botulinum C3 ADP-ribosyltransferase and of Clostridium botulinum C2 toxin were studied on the cytoskeleton of rat hepatoma FAO and human glioma U333 cells. After treatment of these cells for 24 to 48 h with C3 (3-30 micrograms/ml), the actin microfilaments disappeared, and the intermediate filament network was found to collapse, while microtubules remained intact. Similar alterations of the cytoskeletal filaments without affecting microtubules were induced by the actin-ADP-ribosylating C2 toxin. In FAO cells, C3 caused the rounding up of cells. Concomitantly, cytosolic 22 to 24 kDa proteins were ADP-ribosylated in a guanine nucleotide-dependent manner. Rounding up of cells and ADP-ribosylation of proteins in intact cells were observed at similar concentration of the transferase. These data suggest a role of the protein substrates of C3 in the regulation of the cytoskeletal integrity.     

Cellular uptake of Clostridium botulinum C2 toxin: membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain

The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP-ribosylating toxins. C2 toxin is composed of two separated nonlinked proteins. The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells. The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol. After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol. For this step, the cellular heat shock protein Hsp90 is essential. We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I. Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component. The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa. Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain. Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin. In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited. The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes. Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells. The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited.     

Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin

The culture medium of certain strains of Clostridium botulinum type C contains two separable ADP-ribosyltransferases. Besides the ADP-ribosylation of actin due to botulinum C2 I toxin, a second microbial enzyme causes the mono-ADP-ribosylation of a eukaryotic protein with a molecular mass of about 20 kDa found in platelets, neuroblastoma X glioma hybrid cells, S49 lymphoma cells, chick embryo fibroblasts and sperm. The eukaryotic substrate is inactivated by heating and trypsin treatment. In contrast, the novel ADP-ribosyltransferase, which can be separated by DEAE-Sephadex chromatography, is largely resistant in the short term to trypsin digestion.     

FK506-binding protein 51 interacts with Clostridium botulinum C2 toxin and FK506 inhibits membrane translocation of the toxin in mammalian cells

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I. C2IIa delivers C2I into the cytosol of eukaryotic target cells where C2I ADP-ribosylates actin. After receptor-mediated endocytosis of the C2IIa/C2I complex, C2IIa forms pores in membranes of acidified early endosomes and unfolded C2I translocates through the pores into the cytosol. Membrane translocation of C2I is facilitated by the activities of host cell chaperone Hsp90 and the peptidyl-prolyl cis/trans isomerase (PPIase) cyclophilin A. Here, we demonstrated that Hsp90 co-precipitates with C2I from lysates of C2 toxin-treated cells and identified the FK506-binding protein (FKBP) 51 as a novel interaction partner of C2I in vitro and in intact mammalian cells. Prompted by this finding, we used the specific pharmacological inhibitor FK506 to investigate whether the PPIase activity of FKBPs plays a role during membrane translocation of C2 toxin. Treatment of cells with FK506 protected cultured cells from intoxication with C2 toxin. Moreover, FK506 inhibited the pH-dependent translocation of C2I across membranes into the cytosol but did not interfere with the enzyme activity of C2I or binding of C2 toxin to cells. Furthermore, FK506 treatment delayed intoxication with the related binary actin ADP-ribosylating toxins from Clostridium perfringens (iota toxin) and Clostridium difficile (CDT) but not with the Rho-glucosylating Clostridium difficile toxin A (TcdA). In conclusion, our results support the hypothesis that clostridial binary actin-ADP-ribosylating toxins share a specific FKBP-dependent translocation mechanism during their uptake into mammalian cells.