Membrane translocation of binary actin-ADP-ribosylating toxins from Clostridium difficile

26 Some hypervirulent strains of Clostridium difficile produce the binary actin-ADP-ribosylating 27 toxin CDT in addition to the Rho-glucosylating toxins A and B. It has been suggested that the 28 presence of CDT increases the severity of the C. difficile-associated diseases including 29 pseudomembranous colitis. CDT contains a binding and translocation component CDTb, which 30 mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, 31 where CDTa modifies actin. Here, we have investigated the cellular uptake mechanism of CDT 32 and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, 33 implying that CDTa translocates from acidified endosomal vesicles into the cytosol. Consistently, 34 CDTa translocates across the cytoplasmic membranes into the cytosol when cell-bound CDT is 35 exposed to acidic medium. Radicicol and cyclosporin A, inhibitors of the heat shock protein 36 Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not with 37 toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of 38 CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the 39 inhibitors did not interfere with ADP-ribosyltransferase activity, receptor-binding and 40 endocytosis of the toxin. We obtained comparable results for the closely related iota toxin from 41 Clostridium perfringens. Moreover, CDTa as well as Ia, the enzyme component of iota toxin 42 specifically bound to immobilized Hsp90 and cyclophilin A in vitro. In combination with our 43 recently obtained knowledge on the C2 toxin from C. botulinum, the results imply a common 44 Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP45 ribosylating toxins. 46 47 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom INTRODUCTION 48 Clostridium difficile (C. difficile) infection causes human diseases ranging from mild diarrhea to 49 severe and potentially live-threatening pseudomembranous colitis. The C. difficile-associated 50 diseases occur in patients treated with broad-spectrum antibiotics. Under these conditions the 51 disturbed gut flora allows germination of C. difficile spores and colonization of the gut by this 52 pathogen. C. difficile produces two exotoxins, toxin A (308 kDa) and toxin B (270 kDa), which 53 are the causative agents of pseudomembranous colitis. The toxin-catalyzed glucosylation of Rho, 54 Rac and Cdc42 results in the inhibition of GTPase-mediated cell-signalling, destruction of the 55 actin cytoskeleton, and cell rounding that is the reason for loss of integrity of the intestinal wall 56 (for review see (20,21)). 57 During the last 10 years, hypervirulent C. difficile strains were identified, which produce in 58 addition to toxins A and B a third exotoxin, the binary C. difficile transferase (CDT). Up to 35% 59 of the strains produce CDT and it has been suggested that the presence of CDT correlates with 60 severity of the C. difficile-associated diseases (12,14,25,27,52). CDT belongs to the family of 61 binary actin-ADP-ribosylating toxins and consists of two non-linked proteins: the 62 binding/translocation component CDTb and the separate enzyme component CDTa, which 63 harbors the ADP-ribosyltransferase activity. Like the other members of this toxin family, CDT 64 exerts its toxic effects on mammalian cells by mono-ADP-ribosylation of G-actin at arginine-177 65 (1) thereby inhibiting actin polymerization (54). This results in cell-rounding. Recently, it was 66 reported that CDT interferes with the organization of microtubule structures, too, resulting in 67 formation of long microtubule-based protrusions around the cell body. Importantly, C. difficile 68 can bind to these microtubule-based protrusions resulting in increased adherence and colonization 69 of bacteria in infection models (46). 70 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom The family of binary actin-ADP-ribosylating toxins comprises the C2 toxin from C. botulinum 71 and the iota-like toxins. The latter includes iota toxin, which is produced by C. perfringens type E 72 strains and causes sporadic diarrheic outbreaks in farm animals (47,50,51), as well as CDT from 73 C. difficile (37) and C. spiroforme transferase (CST) (36). Iota, CDT and CST are distinguished 74 from the C2 toxin (30) because they are closer related than to C2 toxin regarding sequence 75 homology, functional and immunological aspects (34) and the different modification of 76 individual actin isoforms (31,43). 77 The sophisticated mechanism by which the binding/translocation component mediates the 78 transport of the enzyme components into the cytosol of mammalian target cells was discovered 79 for C2 toxin and iota toxin (3,5). The binding/translocation component of iota toxin, Ib (98 kDa) 80 becomes proteolytically activated and then binds to an unknown protein receptor and forms 81 heptamers, which act as a docking platform for the enzyme component Ia (47 kDa) (5,16,49). 82 After receptor-mediated endocytosis of the Ib/Ia complex, Ib mediates the translocation of Ia 83 from acidified endosomal vesicles into the cytosol (29,48). Under acidic conditions Ib heptamers 84 convert into a pore-conformation and form pores in endosomal membranes (5), which serve as 85 translocation channels for the enzyme component. A widely comparable uptake mechanism was 86 reported for C2 toxin. For both toxins, pore formation by the binding/translocation components is 87 an essential prerequisite for translocation of the enzyme components C2I and Ia, respectively into 88 the cytosol (6,23). It was shown earlier that C2I unfolds to translocate cross endosomal 89 membranes (19). Most likely, the enzyme components translocate through the pore lumen driven 90 by the pH gradient between the endosomal lumen and cytosol (23). Although both toxins share an 91 overall comparable uptake mechanism via acidified endosomes, translocation of the enzyme 92 components from endosomal vesicles to the cytosol are different. While Ia appears to escape 93 from endocytotic carrier vesicles, which are in a state between early and late endosomes (13), C2I 94 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom translocates from early endosomes to the cytosol (3), suggesting that Ia requires more acidic 95 conditions to cross membranes. Moreover, translocation of Ia seems to require a membrane 96 potential gradient in addition to the pH gradient (13). We have reported earlier that the membrane 97 translocation of C2 as well as iota toxin is facilitated by the chaperone heat shock protein 90 98 (Hsp90) (17,18) and more recently, that cyclophilin A (CypA), a peptidyl-prolyl cis/trans 99 isomerase (PPIase) (22) is crucial for translocation of C2 toxin. PPIases catalyze cis-trans 100 isomerization of proline-peptide bonds, often a rate-limiting step during protein refolding 101 (2,9,44,45). So far it is not clear whether PPIases are also involved in membrane translocation of 102 iota toxin. 103 In contrast to C2 toxin and iota toxin, the cellular uptake mechanism of CDT is not known. 104 Therefore, we have investigated the uptake of CDT into cultured African green monkey kidney 105 epithelial cells (Vero cells) and in particular studied the membrane translocation of the toxin. We 106 focused on the role of the host cell factors Hsp90 and cyclophilin A in membrane translocation of 107 CDT in comparison to iota toxin. The specific pharmacological inhibition of Hsp90 by radicicol 108 (Rad), as well as the inhibition of cyclophilin by cyclosporin A (CsA), protected Vero cells from 109 intoxication by CDT and iota toxin and inhibited the pH-dependent membrane translocation of 110 both toxins. 111 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom MATERIAL AND METHODS 112 Materials Cell culture medium (MEM) and fetal calf serum were purchased from Invitrogen 113 (Karlsruhe, Germany) and cell culture materials were from TPP (Trasadingen, Switzerland). 114 Complete protease inhibitor and streptavidin-peroxidase were from Roche (Mannheim, 115 Germany). The protein molecular weight markers Page Ruler prestained Protein ladder and Page 116 Ruler stained Protein ladder were obtained from Fermentas (St. Leon-Rot, Germany). 117 Biotinylated NAD was supplied by R&D Systems GmbH (Wiesbaden-Nordenstadt, Germany). 118 Bafilomycin A1 (BafA1) was obtained from Calbiochem (Bad Soden, Germany), CsA from 119 Fluka (Munich, Germany) and Rad from Sigma Aldrich (Munich, Germany). The enhanced 120 chemiluminescence (ECL) system was purchased from Millipore (Schwalbach, Germany). 121 Alexa568-maleimide was from Invitrogen (Karlsruhe, Germany). 122 123 Protein expression, purification and biotinylation – Ia and Ib were purified as described earlier 124 (33). Recombinant CDTa and CDTb (from C. difficile strain 196) were produced and purified as 125 His-tagged proteins in the B. megaterium expression system as it was described by others for the 126 large clostridial glycosylating toxins (32,55). Labelling of CDTa with Alexa568-maleimide was 127 performed according to the manufacturer ́s protocol (Invitrogen, Karlsruhe, Germany). The 128 purified proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis 129 (SDS-PAGE), stained with Coomassie blue and the protein concentration was determined via 130 densitometry by using Photoshop 7.0 software (Adobe Systems Inc.). CypA was purified as 131 described earlier (8) and human Hsp90 was purified as described (41). The biotinylation of C2I, 132 Ia and CDTa was performed with sulfo-NHS-biotin (Pierce, Rockford, Illinois, USA) according 133 to the manufacturer ́s instructions. 134 135 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom Cell culture and cytotoxicity assays African green monkey kidney (Vero) cells and the human 136 intestinal Caco-2 cells were cultivated at 37 °C and 5% CO2 in MEM containing 10% heat137 inactivated fetal calf serum, 1.5 g/L sodium bicarbonate, 1 mM sodium-pyruvate, 2 mM 138 L glutamine, 0.1 mM non-essential amino acids and 10 mg/mL Penicillin/Streptomycin. Cells 139 were trypsinized and reseeded for at most 15-20 times. For cytotoxicity experiments, cells were 140 seeded in culture dishes and incubated in serum-free medium with CDT or iota toxin. To inhibit 141 the PPIase activity of Cyps or the activity of Hsp90, the cells were incubated for 30 min with the 142 indicated concentrations of CsA or Rad, respectively. Subsequently, toxin was added and cells 143 were further incubated at 37 °C with toxin plus inhibitor. After the given incubation periods, the 144 cells were visualized by using a Zeiss Axiovert 40CFl microscope (Oberkochen, Germany) with 145 a Jenoptik progress C10 CCD camera (Carl Zeiss GmbH, Jena, Germany). The cytopathic effects 146 caused by the toxins were analyzed in terms of morphological changes. 147 148 Fluorescence microscopy to detect internalized CDTa Caco-2 cells were preincubated with 10 149 μM CsA, 10 μM Rad for 30 min at 37 °C. Subsequently cells were cooled to 4°C and 1 μg/mL 150 CDTa labelled with Alexa568 plus 2 μg/mL CDTb was added. Cells were incubated at 4°C for 151 30 min to allow toxin binding. Cells were transferred to 37°C for 20 min to induce endocytosis 152 and fixed. Actin was stained by FITC-phalloidin. Fixed samples were analyzed with an inverted 153 Axiovert 200M microscope (Carl Zeiss GmbH, Jena, Germany) equipped with plan-apochromat 154 objectives, driven by Metamorph imaging software (Universal Imaging, Downingtown, PA). 155 Confocal images were collected with a Yokogawa CSU-X1 spinning disc confocal head (Tokyo, 156 Japan) with an emission filter wheel, a Coolsnap HQ II digital (Roper Scientific, Tucson, AZ) 157 camera and 488 nm, 561 nm laser lines. 158 159 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom Preparation of cell extracts, SDS-PAGE and immunoblot analysis Following incubation with 160 the toxin, cells were washed twice with ice-cold PBS and lysed in 20 mM Tris-HCl (pH 7.5) 161 containing 1 mM EDTA, 1 mM DTT, 5 mM MgCl2 and complete protease inhibitor. Following 162 lysis of the cells and centrifugation (20,800 ×g, 7 min, 4 °C), the supernatant was stored at −20 163 °C. For immunoblot analysis, equal amounts of lysate protein were subjected to SDS-PAGE 164 according to the method of Laemmli (24). Subsequently, the proteins were transferred to a 165 nitrocellulose membrane (Whatman, Dassel, Germany). The membrane was blocked for 30 min 166 with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBS-T). For the detection of actin, 167 the samples were probed with a mouse monoclonal anti-β-actin antibody (clone AC-15; Sigma168 Aldrich, Seelze, Germany). After washing with PBS-T, the membrane was incubated for 1 h with 169 an anti-mouse antibody coupled to horseradish-peroxidase (Santa Cruz Biotechnology, 170 Heidelberg, Germany). The membrane was washed and the proteins visualized using an enhanced 171 chemiluminescence (ECL) system according to the manufacturer’s instructions. 172 173 Sequential ADP-ribosylation of actin in lysates from toxin-treated cells For ADP-ribosylation of 174 actin in a cell-free system, 20 μg of whole-cell lysate protein were incubated for 30 min at 37 °C 175 in a buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1mM DTT, 5 mM MgCl2, 176 complete protease inhibitor, together with biotin-labelled NAD (10 μM) and 300 ng of C2I 177 protein. The reaction was stopped with 5 x SDS-sample buffer (625 mM Tris/HCl pH 6.8, 20% 178 SDS, 8.5% glycerol, 0.2% bromphenol blue, 100 mM DTT) and heating of the samples for 5 min 179 at 95 °C. The samples were subjected to SDS-PAGE, transferred to a nitrocellulose membrane 180 and the biotin-labelled ADP-ribosylated actin was detected with peroxidase-coupled streptavidin 181 and a subsequent chemiluminescence reaction. 182 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom ADP-ribosylation of actin by CDTa in a cell free system Vero cell lysate (50 μg of protein) was 183 incubated for 2, 5 and 15 min at 37 °C together with 50 ng/mL of CDTa, 10 μM biotin-labelled 184 NAD, and 10 μM CsA. Samples were subjected to SDS-PAGE, blotted onto a nitrocellulose 185 membrane and the ADP-ribosylated actin was detected with streptavidin-peroxidase. Intensity of 186 the biotin-labelled actin was determined by densitometry using the Adobe Photoshop 7.0 187 software. 188 189 Toxin-translocation assay with intact cells The pH-dependent translocation of CDTa and Ia 190 through their corresponding pores across endosomal membranes was experimentally mimicked 191 on the surface of intact cells as described for iota toxin earlier (5). In brief, Vero cells were 192 exposed to an acidic pulse (pH 4.0) after binding of either CDTb/CDTa or Ib/Ia to the cell 193 surface. Under acidic conditions CDTa and Ia translocate across the cytoplasmic membrane into 194 the cytosol. Cell rounding was monitored and documented by photography. 195 196 Dot Blot analysis of the interaction between immobilized CypA and Hsp90 with CDTa, Ia and 197 C2I – Different amounts of CypA and Hsp90 were vacuum aspirated onto a nitrocellulose 198 membrane using a dot-blot system (Bio-Rad, Munich, Germany) according to the manufacturer ́s 199 instructions. Subsequently the membrane was blocked for 1 h with PBS-T containing 5 % non fat 200 dry milk and incubated with either biotin-labelled C2I, Ia or CDTa (200 ng/mL) for 1 h. The 201 membrane was washed three times with PBS-T and the bound biotinylated proteins were detected 202 with streptavidin-peroxidase using the ECL system. 203 204 Reproducibility of the experiments and statistics All experiments were performed independently 205 at least 2 times. Results from representative experiments are shown in the figures. In each 206 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom individual immunoblot panel shown in the figures, the protein bands were originally detected on 207 the same membrane and only cut out and recombined for presentation in the figures. Values (n ≥ 208 3) are calculated as mean ± standard deviation (S.D.) using the Prism4 Software (GraphPad 209 Software, Inc.). 210 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom RESULTS 211 CDT translocates from acidified endosomal vesicles into the host cell cytosol – The binary actin212 ADP-ribosylating C2 and iota toxins deliver their A-components from endosomal vesicles into 213 the cytosol. Importantly, acidification of the endosomal lumen is an essential prerequisite for this 214 process. Therefore, we used BafA1, which inhibits the vesicular ATPase and thereby prevents 215 acidification of the endosomes to test whether CDT translocates from acidified endosomes to the 216 cytosol. Pre-treatment of Vero cells with BafA1 protected Vero cells from intoxication with CDT 217 very efficiently. While there was a time-dependent increase in the amount of round cells after 218 application of CDT, the toxin-induced cell-rounding was inhibited in the presence of 100 nM 219 BafA1 (Fig. 1A). This concentration of BafA1 exhibited its protective effect even when the cells 220 were incubated with CDT for 24 h in the presence of BafA1 but had no effect on the morphology 221 of cells (not shown). Moreover, 0.1% final concentration of DMSO as solvent control had no 222 inhibitory effect on the CDT-induced cell rounding (not shown). This result suggests that the A223 component CDTa translocates from acidified endosomal vesicle to the cytosol. 224 We performed an alternative assay to verify that the membrane translocation of CDTa essentially 225 requires acidic conditions. Therefore, we experimentally mimicked the acidic conditions of the 226 endosomal lumen on the surface of intact cells. Vero cells were pre-treated with BafA1 to block 227 the “normal” uptake of CDTa into the cytosol and then the cells were incubated at 4 °C with 228 CDTb plus CDTa to enable toxin binding to the cell surface receptors. Subsequently, the pH of 229 the culture medium was adjusted to pH 4.5 (for control pH 7.5) and cells were incubated at 37 °C 230 to trigger membrane translocation of CDTa. Cell rounding was monitored to detect that CDTa 231 was delivered into the cytosol and modified actin. Cell rounding was observed only when CDT232 treated cells were exposed to low pH (Fig. 1B), indicating that a pH-gradient is essential for 233 translocation of cell-bound CDTa across the cytoplasmic membrane into the cytosol. 234 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom Taken together, the results imply that CDTa translocates from acidified endosomal vesicles to the 235 host cell cytosol which is in agreement with other members of this toxin family (3,5). 236 237 Pharmacological inhibition of host cell cyclophilin and Hsp90 protects Vero cells from 238 intoxication with CDT – To investigate whether the cellular uptake of CDT depends on the host 239 cell cyclophilin and/or Hsp90, we first tested whether pharmacological inhibition of these factors 240 by CsA and Rad, respectively, has any effect on intoxication of cells with CDT. Pre-treatment of 241 Vero cells with each inhibitor alone and with the combination of CsA and Rad protected Vero 242 cells from CDT-induced cell-rounding within 4 h after toxin application (Fig. 2A). The inhibitors 243 alone had no effects on cell morphology under these conditions (not shown). A quantitative 244 analysis revealed that CsA and Rad caused a significant and time-dependent delay in intoxication 245 rather than a complete inhibition (Fig. 2B). The combination of CsA and Rad had a slightly 246 stronger protective effect compared to the individual inhibitors however this effect was not 247 statistically significant (Fig. 2B). Because higher concentrations of CsA induced some 248 morphological effects on Vero cells, the concentration of 10 μM was used in this study. 249 The morphology-based analysis of the protective effect was confirmed by analyzing the ADP250 ribosylation status of actin from cells. Cells were treated with CDT in the absence and presence 251 of the inhibitors. After 1.5 h, cells were lysed and lysates were incubated with fresh C2I and 252 biotin-NAD as a co-substrate to enable ADP-ribosylation of actin in vitro and thereby its biotin253 labelling. The biotin-labelled, i.e. ADP-ribosylated actin was detected in a Western blot analysis 254 with streptavidin-peroxidase (Fig. 2C, upper panel) and the intensity of the bands was quantified 255 (Fig. 2C, the bars correspond to the bands of ADP-ribosylated actin in the upper panel). In this 256 assay, actin from control cells gives a strong signal, because it was not ADP-ribosylated in the 257 intact cells before lysis. In contrast, actin from CDT-treated cells gives a weaker signal, because 258 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom most of the actin was already modified in the intact cells by the toxin and is, therefore, no more 259 substrate in vitro. Most importantly, cells, incubated with CDT in the presence of CsA, Rad or 260 the combination of both inhibitors, gave a stronger signal of biotin-labelled actin compared to 261 cells, which have been treated with CDT alone. This result indicates that less actin was modified 262 by the toxin in intact cells when CsA or Rad was present. An anti-β-actin immunoblot of the 263 identical lysates confirmed comparable protein loading (Fig. 2C). 264 Taken together, the results clearly indicate that there was less CDTa activity in the cytosol of 265 cells in the presence of CsA or Rad, strongly suggesting that cyclophilin and Hsp90 are crucial 266 for intoxication of cells with CDT. However, from this result it is not clear whether the inhibitors 267 interfere with the enzyme activity of CDTa and/or the uptake of CDTa into the host cell cytosol. 268 Therefore, we first excluded that CsA and Rad inhibit the CDTa-catalyzed ADP-ribosylation of 269 actin in vitro (data not shown). This finding implies that the inhibitors interfere with the uptake of 270 CDTa into the cytosol and, therefore, we investigated which step during toxin uptake into the 271 cytosol is affected. First, we investigated whether the inhibitors interfere with binding of 272 CDTa/CDTb to the cell surface and the subsequent internalization of the toxin into endosomal 273 vesicles. Caco-2 cells, which have been pre-treated with either CsA or Rad, were incubated for 274 30 min at 4°C with CDTb and Alexa568-labelled CDTa to allow binding and for 20 min at 37 °C 275 for internalization of the toxin complex. The internalized CDTa-Al568 protein was visualized by 276 fluorescence microscopy. As shown in Fig. 3, there was a comparable amount of CDTa 277 detectable in the cells, independent whether the cells have been treated with inhibitors. Thus, 278 neither CsA nor Rad inhibited binding of the toxin to the receptor or internalization by receptor279 mediated endocytosis. CsA and Rad did not inhibit uptake of the C. difficile toxins A and B into 280 Vero cells under comparable experimental conditions (data not shown). These toxins are 281 internalized via receptor-mediated endocytosis and translocate from acidified endosomal vesicles 282 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom into the cytosol, likewise, where they modify Rho proteins leading to cell rounding. In our 283 experiments, the combination of CsA and Rad did not cause a significant delay in toxin A284 induced rounding-up of Vero cells (data not shown), indicating endocytosis in the presence of 285 these inhibitors. 286 287 CsA and Rad inhibit the pH-dependent membrane translocation of CDTa – Having excluded that 288 CsA and Rad interfere with the early steps of toxin uptake we focused on the membrane 289 translocation of CDTa. To test an effect of the inhibitors on this process, we performed a well290 established assay, which mimics endosomal conditions on the surface of intact cells. In brief, 291 Vero cells were pre-treated with BafA1 to block the “normal” uptake of CDT. Then, cells were 292 incubated at 4 °C with CDTb plus CDTa and, thereafter, cells were exposed to warm acidified 293 medium (37 °C, pH 4.5) as described before to trigger translocation of cell-bound CDTa across 294 the cytoplasmic membrane into the cytosol. During this step, CsA, Rad or the combination of 295 both inhibitors was present in the culture medium. The successful translocation of CDTa into the 296 cytosol was determined by the amount of round cells (Fig. 4). In the presence of CsA or Rad 297 there was a significant decrease in the amount of round cells after 1, 1.5 and 2 h, indicating that 298 both CsA and Rad inhibit membrane translocation of CDTa. The combination of CsA and Rad, 299 however, exhibited a synergistic inhibitory effect which caused a prolonged delay in intoxication 300 of cells compared to the single substances. In conclusion, these results imply that cyclophilin as 301 well as Hsp90 are crucial for the pH-dependent membrane translocation of CDTa and suggest 302 that both factors might act in a synergistic manner during this process. 303 304 CsA and Rad inhibit membrane translocation of the C. perfringens iota toxin and thereby protect 305 cells from intoxication – We have observed earlier that pharmacological inhibition of Hsp90 306 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom protected Vero cells from intoxication with iota toxin; however, the underlying molecular 307 mechanism was not investigated so far. Prompted by the results obtained for CDT, we finally 308 tested whether Hsp90 is crucial for membrane translocation of the enzyme component Ia and 309 whether cyclophilin is also involved in this process. To determine whether cyclophilins are 310 involved in the uptake of iota toxin, Vero cells were incubated with iota toxin in the presence or 311 absence of CsA. Toxin-induced cell rounding was analyzed after 4 h (Fig. 5A). Most of the toxin312 treated cells were round while the presence of CsA prevented cell rounding. The observed iota 313 toxin-induced cell rounding correlated with the ADP-ribosylation status of actin in these cells 314 (not shown). CsA inhibited the iota toxin-induced cell rounding in a timeand concentration315 dependent manner (Fig. 5B) and as observed before for CDT, the combination of CsA and Rad 316 showed a synergistic protective effect compared to the single inhibitors (Fig. 5C). Most 317 important, CsA as well as Rad inhibited the pH-dependent translocation of cell-bound iota toxin 318 across the cytoplasmic membrane into the cytosol. This becomes evident in a significantly 319 decreased amount of round cells in the presence of the inhibitors (Fig. 5D). In conclusion, the 320 data imply that Hsp90 as well as cyclophilin are crucial for membrane translocation of iota toxin 321 which is consistent to the results obtained for the closely related binary toxin CDT. Moreover, in 322 this aspect the iota-like toxins behave comparable to the binary actin-ADP-ribosylating C2 toxin 323 from C. botulinum. 324 325 The enzyme components of CDT and iota toxin directly interact with Hsp90 and CypA in vitro – 326 From these results we were not able to conclude which particular cyclophilin is involved in 327 uptake of CDT and iota toxins. We hypothesized, however, that cyclophilin A might interact with 328 the enzyme components of both toxins. This hypothesis is plausible because cyclophilin A is the 329 prominent cyclophilin in the cytosol of mammalian cells and the major molecular target of CsA. 330 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom Moreover, we have reported earlier that it interacts with C2I, the enzyme component of the C2 331 toxin. Therefore, we have finally investigated whether purified cyclophilin A and Hsp90 proteins 332 interact with CDTa and Ia in a dot blot analysis in vitro and we included C2I as a positive control 333 (Fig. 6). Starting with 1 μg of protein, decreasing amounts of Hsp90 and CypA proteins were 334 spotted onto a nitrocellulose membrane and the membranes (for control PBS, indicated as con) 335 were incubated in an overlay assay with either biotin-labelled CDTa, Ia or C2I protein in solution 336 (200 ng/ml final concentration). After extensive washing, the membrane-bound enzyme 337 components of the toxins were detected. Most importantly, CDTa, Ia and C2I bound to Hsp90 as 338 well as CypA and this binding was specific because there was no toxin bound to the membrane in 339 the absence of Hsp90 or CypA. Moreover, there was no signal when the immobilized Hsp90 and 340 CypA proteins were mock-incubated with PBS instead of toxin or incubated with the non-binding 341 lethal factor from Bacillus anthracis, as demonstrated recently (7). 342 In conclusion, this result indicates that the enzyme components of the binary CDT and iota toxins 343 directly interact with Hsp90 and CypA in vitro. This is in line with our recently obtained results 344 for the C2 toxin, implying that this interaction with Hsp90 and CypA might be a common feature 345 of the family members of binary actin-ADP-ribosylating toxins. 346 347 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom DISCUSSION 348 In the present paper, we have performed a series of experiments to analyze the cellular uptake of 349 the binary actin-ADP-ribosylating toxin CDT from C. difficile, in particular the membrane 350 translocation of its enzyme component CDTa. We demonstrate that CDTa translocates from 351 acidified endosomal vesicles into the cytosol and that the translocation depends on a pH-gradient 352 across the membrane. This is in agreement to earlier findings on the translocation of the binary 353 iota and C2 toxins (3,5). Recently, we reported that membrane translocation of some binary 354 toxins but not of others are facilitated by host cell chaperones and PPIases (7,17,18,22). 355 Therefore, we tested here whether CDT and the closely related iota toxin the iota-like toxins 356 require such factors for translocation. Indeed, membrane translocation of the enzyme components 357 CDTa and Ia was blocked by the pharmacological inhibitors Rad and CsA, implying that Hsp90 358 as well as cyclophilin facilitate this step. Consequently, both inhibitors protected cultured cells 359 from intoxication by CDT and iota toxins and the relative effects of CsA and Rad on CDT and 360 iota actions were overall comparable. The inhibitory effect on the intoxication of Vero cells with 361 iota toxin was significantly stronger when CsA and Rad were combined compared to CsA or Rad 362 alone while this synergistic inhibitory effect was less pronounced and not statistically significant 363 for the intoxication of Vero cells with CDT. Importantly, we ruled out that the inhibitors did 364 influence the enzyme activities of CDTa and Ia or other steps in toxin internalization, such as 365 binding of the toxin complex to the cell surface or endocytosis. Thus, we conclude that the 366 inhibitors exclusively interfere with toxin translocation and thereby inhibit uptake of CDTa and 367 Ia into the cytosol. To investigate membrane translocation of CDTa and Ia, we mimicked the 368 endosomal conditions on the surface of intact Vero cells. Only when cells were exposed to an 369 acidic pulse, membrane translocation of the cell-bound toxin was triggered. CsA and Rad, 370 however, blocked pH-driven translocation of CDTa and Ia. This assay was originally established 371 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom to investigate pH-dependent membrane translocation of diphtheria toxin (42) and successfully 372 used for a variety of toxins, which translocate from acidified endosomes into the cytosol 373 (10,15,28), including the binary actin-ADP-ribosylating C2 and iota toxins (3,5,6). Interestingly, 374 the inhibitory effect of CsA and Rad on the CDT-induced cell rounding appeared less efficient 375 when CDTa was introduced into the cytosol by acidic shift in comparison to the “normal” uptake 376 of the toxin via receptor-mediated endocytosis and subsequent translocation from acidified 377 endosomes. One possible explanation for this observation might be the synchronous translocation 378 of a comparatively large amount of CDTa across the cytoplasmic membrane under these artificial 379 conditions while less CDTa might translocate into the cytosol when the toxin is taken up via 380 acidified endosomes. In agreement with this hypothesis, cell rounding was faster when CDT was 381 introduced into cells by acidic pulse compared to “normal” uptake. On the other hand, there 382 might be differences regarding the recruitment of chaperones/PPIases which are crucial for 383 translocation of CDT to the endosomal membrane compared to the cytoplasmic membrane. From 384 our results obtained by this method, we conclude that Hsp90 and cyclophilins facilitate 385 translocation of CDTa and Ia across the membranes of acidified endosomes during uptake of the 386 toxin into mammalian cells. Moreover, this is most likely the explanation for an earlier finding 387 that Rad prevents intoxication of cells with iota toxin although it did not inhibit ADP388 ribosyltransferase activity of Ia (17). 389 The results corroborate our recent finding that cyclophilins and Hsp90 facilitate membrane 390 translocation of C2I, the enzyme component of the binary actin-ADP-ribosylating C2 toxin (22). 391 Immunoprecipitation experiments revealed that CypA, the most abundant cyclophilin in the 392 cytosol of mammalian cells and the major target for CsA, interacts with C2I (22). In the present 393 study we found that CDTa as well as Ia bound to immobilized Hsp90 and CypA proteins in vitro, 394 a hint that CypA might be the relevant cyclophilin that interacts with CDT and iota toxins during 395 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom cellular uptake, too. As observed for CDT and iota toxin in the present study, the inhibitors Rad 396 or CsA, respectively, prevented membrane translocation of C2I. Thus, in the presence of Rad or 397 CsA less, if any, C2I reached the cytosol and therefore cells were protected from intoxication by 398 C2 toxin (22). The finding that membrane translocation of CDTa, Ia, and C2I is facilitated by 399 Hsp90 and cyclophilins is interesting, because differences have been reported between C2 and 400 iota toxins during uptake of their enzyme components into the target cell cytosol. First, C2I 401 translocates from early endosomes into the cytosol while Ia is released at a later stage in vesicle 402 transport between early and late endosomes, implying that translocation of Ia is triggered by more 403 acidic conditions (13). Second, translocation of Ia but not of C2I requires a membrane potential 404 gradient in addition to the pH-gradient (13). Finally, there are different regions within the Ia and 405 C2I proteins that respectively mediate their interaction with Ib and C2IIa, as well as their 406 membrane translocation (4,26). 407 The observation that membrane translocation of C2I but also of CDTa and Ia is facilitated by the 408 same host cell factors is a strong hint for a common role of Hsp90 and cyclophilins during 409 translocation of binary actin-ADP-ribosylating toxins. Interestingly, the intoxication of cells with 410 the binary lethal toxin from Bacillus anthracis was not influenced by Rad and CsA (7,18,57), 411 although lethal toxin shares significant sequence and structural homology and an overall common 412 cellular uptake mechanism with binary actin-ADP-ribosylating toxins (for review see (38,56). 413 Just like Ib and C2IIa, the activated binding-/translocation component, protective antigen (PA63), 414 forms heptameric pores in membranes of acidified endosomes, which facilitate pH-dependent 415 membrane translocation of the enzyme component lethal factor (56). However, when the enzyme 416 domain of lethal factor (LF), a protease, was replaced by the enzyme domain of diphtheria toxin 417 (DTA), an ADP-ribosyltransferase, the PA63-dependent uptake of the LFn-DTA fusion toxin was 418 inhibited by Rad and CsA (7). Moreover, we demonstrated that the inhibitors blocked the pH419 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom dependent membrane translocation of this fusion toxin across endosomal membranes, as found 420 for the binary actin-ADP-ribosylating toxins (7). This unexpected finding strongly suggests that 421 the interaction with Hsp90 and cyclophilin during membrane translocation might be specific for 422 bacterial ADP-ribosyltransferases. This hypothesis is confirmed by earlier reports that Hsp90 is 423 crucial for the membrane translocation of the enzyme moieties of diphtheria toxin (40) and 424 cholera toxin (53). 425 The findings are in agreement with a an earlier report by Ratts and co-workers that translocation 426 of diphtheria toxin from early acidified endosomes is facilitated by a multi-protein translocation 427 complex containing Hsp90 and thioredoxin reductase (40). The composition of such complexes 428 and contribution of individual PPIases might differ depending on the type of toxin. However, 429 PPIases such as Cyp-40, FKBP51 and FKBP52 have been identified as functional co-chaperones 430 in Hsp90-containing protein complexes (35,39). Therefore, we can not exclude at present that 431 other cyclophilins besides CypA are involved in translocation of CDTa and/or Ia because CsA 432 inhibits the PPIase activity of most human cyclophilins. Moreover, it will be important to 433 investigate whether further PPIases besides the cyclophilins, for instance FK506 binding proteins 434 (11), are also involved in translocation of the binary actin-ADP-ribosylating toxins. 435 In conclusion, our study provides new information on the interaction of binary clostridial toxins 436 with target cell cyclophilins and Hsp90. However, further investigation is required to unravel the 437 precise molecular mechanisms how these host cell factors facilitate membrane translocation of 438 the toxins in mammalian cells. 439 440 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom ACKNOWLEDGEMENTS 441 HB was financially supported by the Deutsche Forschungsgemeinschaft DFG (grant BA 2087/2-1 442 and to K.A. AK6/20-1). 443 We thank Ulrike Binder for excellent technical assistance. 444