Eva Kaiser

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.

Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells.

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I, which mono‐ADP‐ribosylates actin in eukaryotic cells. Pore formation of C2IIa in early endosomal membranes facilitates translocation of unfolded C2I into the cytosol. We discovered earlier that translocation of C2I depends on the activity of the host cell chaperone heat shock protein Hsp90. Here, we demonstrate that cyclosporin A, which inhibits the peptidyl‐prolyl cis/trans isomerase activity of cyclophilins, inhibited intoxication of cells with C2 toxin and prevented uptake of C2I into the cytosol. Cyclosporin A blocked the pH‐dependent translocation of C2I activity across membranes of intact cells and of partially purified early endosomes. In vitro, the addition of cytosol to C2 toxin‐loaded endosomes induced translocation of C2I activity into the cytosol, which was prevented by pretreatment of the cytosol with an antibody against cyclophilin A. Pull‐down experiments with lysates from C2 toxin‐treated cells revealed specific binding of cyclophilin A to the N‐terminal domain of C2I. In conclusion, our results suggest an essential role of cyclophilin A for translocation of C2I across endosomal membranes during the uptake of C2 toxin into mammalian cells.

Role of CypA and Hsp90 in membrane translocation mediated by anthrax Protective Antigen

Bacillus anthracis lethal toxin consists of the protective antigen (PA) and the metalloprotease lethal factor (LF). During cellular uptake PA forms pores in membranes of endosomes, and unfolded LF translocates through the pores into the cytosol. We have investigated whether host cell chaperones facilitate translocation of LF and the fusion protein LFNDTA. LFN mediates uptake of LFNDTA into the cytosol, where DTA, the catalytic domain of diphtheria toxin, ADP‐ribosylates elongation factor‐2, allowing for detection of small amounts of translocated LFNDTA. Cyclosporin A, which inhibits peptidyl‐prolyl cis/trans isomerase activity of cyclophilins, and radicicol, which inhibits Hsp90 activity, prevented uptake of LFNDTA into the cytosol of CHO‐K1 cells and protected cells from intoxication by LFNDTA/PA. Both inhibitors, as well as an antibody against cyclophilin A blocked the release of active LFNDTA from endosomal vesicles into the cytosol in vitro. In contrast, the inhibitors did not inhibit cellular uptake of LF. In vitro, cyclophilin A and Hsp90 bound to LFNDTA and DTA but not to LF, implying that DTA determines this interaction. In conclusion, cyclophilin A and Hsp90 facilitate translocation of LFNDTA, but not of LF, across endosomal membranes, and thus they function selectively in promoting translocation of certain proteins, but not of others.

Selective and specific internalization of clostridial C3 ADP‐ribosyltransferases into macrophages and monocytes

The C3 transferases from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) mono‐ADP‐ribosylate and thereby inactivate RhoA, ‐B and ‐C of eukaryotic cells. Due to their extremely poor cellular uptake, C3 transferases were supposed to be exoenzymes rather than exotoxins, challenging their role in pathogenesis. Here, we report for the first time that low concentrations of both C3lim and C3bot are selectively internalized into macrophages/monocytes in less than 3 h, inducing the reorganization of the actin cytoskeleton by ADP‐ribosylation of Rho. We demonstrate that C3 transferases are internalized into the cytosol of macrophages/monocytes via acidified early endosomes. Bafilomycin A1, an inhibitor of endosomal acidification, protected J774A.1 macrophages and human promyelotic leukaemia cells (HL‐60) from intoxication by C3. Moreover, confocal laser scanning microscopy revealed colocalization of C3 with early endosomes. An extracellular acidic pulse enabled direct translocation of cell surface‐bound C3 across the cytoplasmic membrane to the cytosol. In line with this finding, both C3 proteins exhibited membrane activity in lipid bilayer membranes only under acidic conditions (pH < 5.5). In conclusion, we identified macrophages/monocytes as target cells for clostridial C3 transferases and shed light on their selective uptake mechanism, which might contribute to understand the role of C3 transferases in pathogenesis.

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.

The long-lived nature of Clostridium perfringens iota toxin in mammalian cells induces delayed apoptosis

ABSTRACT Mono-ADP ribosylation of actin by bacterial toxins, such as Clostridium perfringens iota or Clostridium botulinum C2 toxins, results in rapid depolymerization of actin filaments and cell rounding. Here we report that treatment of African green monkey kidney (Vero) cells with iota toxin resulted in delayed caspase-dependent death. Unmodified actin did not reappear in toxin-treated cells, and enzyme-active toxin was detectable in the cytosol for at least 24 h. C2 toxin showed comparable, long-lived effects in cells, while a C2 toxin control lacking ADP-ribosyltransferase activity did not induce cell death. To address whether the remarkable stability of the iota and C2 toxins in cytosol was crucial for inducing cell death, we treated cells with C/SpvB, the catalytic domain of Salmonella enterica SpvB. Although C/SpvB also mono-ADP ribosylates actin as do the iota and C2 toxins, cells treated with a cell-permeating C/SpvB fusion toxin became rounded but recovered and remained viable. Moreover, unmodified actin reappeared in these cells, and ADP-ribosyltransferase activity due to C/SpvB was not detectable in the cytosol after 24 h, a result most likely due to degradation of C/SpvB. Repeated application of C/SpvB prevented recovery of cells and reappearance of unmodified actin. In conclusion, a complete but transient ADP ribosylation of actin was not sufficient to trigger apoptosis, implying that long-term stability of actin-ADP-ribosylating toxins, such as iota and C2, in the cytosol is crucial for inducing delayed, caspase-dependent cell death.

Cyclophilin-Facilitated Membrane Translocation as Pharmacological Target to Prevent Intoxication of Mammalian Cells by Binary Clostridial Actin ADP-Ribosylated Toxins ☆

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium difficile CDT belong to the family of binary actin ADP-ribosylating toxins and are composed of a binding/translocation component and a separate enzyme component. The enzyme components ADP-ribosylate G-actin in the cytosol of target cells resulting in depolymerization of F-actin, cell rounding and cell death. The binding/translocation components bind to their cell receptors and form complexes with the respective enzyme components. After receptor-mediated endocytosis, the binding/translocation components form pores in membranes of acidified endosomes and the enzyme components translocate through these pores into the cytosol. This step is facilitated by the host cell chaperone heat shock protein 90 and peptidyl-prolyl cis/trans isomerases including cyclophilin A. Here, we demonstrate that a large isoform of cyclophilin A, the multi-domain enzyme cyclophilin 40 (Cyp40), binds to the enzyme components C2I, Ia and CDTa in vitro. Isothermal titration calorimetry revealed a direct binding to C2I with a calculated affinity of 101 nM and to Ia with an affinity of 1.01 μM. Closer investigation for the prototypic C2I revealed that binding to Cyp40 did not depend on its ADP-ribosyltransferase activity but was stronger for unfolded C2I. The interaction of C2I with Cyp40 was also demonstrated in lysates from C2-treated cells by pull-down. Treatment of cells with a non-immunosuppressive cyclosporine A derivative, which still binds to and inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, protected cells from intoxication with C2, iota and CDT toxins, offering an attractive approach for development of novel therapeutic strategies against binary actin ADP-ribosylating 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.

Role of Host Cell Chaperones in Cellular Uptake of Clostridium Botulinum C2 Toxin

The binary C2 toxin from Clostridium botulinum consists of two separate proteins: the transport component C2IIa delivers the enzyme component C2I into the cytosol of eukaryotic host cells. In the cytosol, C2I mono-ADP- ribosylates actin, thereby inducing depolymerization of actin filaments resulting in delayed caspase-dependent cell death. The sophisticated cellular uptake mechanism of C2 toxin, in particular our new results regarding the role of host cell chaperones and protein-folding helper enzymes during intracellular membrane translocation of C2I, are focused upon in this minireview. We discovered earlier that translocation of C2I across endosomal membranes in mammalian cells depends on the chaperone activity of the heat shock protein Hsp90. Recently we have demonstrated that cyclosporin A (CsA), an inhibitor of peptidyl-prolyl cis/trans isomerase (PPIase) activity of cyclophilins, inhibited intoxication of various mammalian cell lines with C2 toxin. The underlying reason for this effect was the prevented uptake of C2I into the host cell cytosol. CsA, as well as a specific antibody against cyclophilin A, blocked the pH-dependent translocation of C2I-ADP- ribosyltransferase activity across membranes of intact cells and of partially-purified early endosomes in vitro. In conclusion, our results imply that the activities of Hsp90 and cyclophilin A are crucial for translocation of the C2I ADP- ribosyltransferase from early endosomes into the cytosol of mammalian cells. This is the first observation that a host cell PPIase, in concert with a heat shock protein, facilitates intracellular membrane translocation of a bacterial protein toxin.