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Originally published In Press as doi:10.1074/jbc.M000596200 on April 3, 2000

J. Biol. Chem., Vol. 275, Issue 25, 18704-18711, June 23, 2000
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Cellular Uptake of Clostridium botulinum C2 Toxin Requires Oligomerization and Acidification*

Holger BarthDagger , Dagmar BlöckerDagger , Joachim Behlke, Wilma Bergsma-Schutter||, Alain Brisson||, Roland Benz**, and Klaus AktoriesDagger DaggerDagger

From the Dagger  Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany,  Max-Delbrück-Zentrum für Molekulare Medizin, Robert-Rössle-Str. 10, D-13125 Berlin, Germany, || Department of Biophysical Chemistry, University of Groningen, Nijenborgh 4, NL-9747 AG Groningen, The Netherlands, and ** Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany

Received for publication, January 27, 2000, and in revised form, March 28, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. Here we report that binding of C2II to the surface of target cells requires cleavage of C2II by trypsin. Trypsin cleavage causes oligomerization of the activated C2II (C2IIa) to give SDS-stable heptameric structures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme component C2I is blocked by bafilomycin but not by brefeldin A or nocodazole, indicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells involves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cleaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface and assembly with C2I, (iv) receptor-mediated endocytosis of both C2 components into endosomes, and finally (v) translocation and release of C2I into the cytosol after acidification of the endosomal compartment.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial exotoxins that enzymatically modify substrates in eukaryotic target cells have developed delivery systems to transport their active components across lipid membranes into the cytoplasm to mediate their cytotoxic effects. In most cases, this process requires four steps: receptor-binding on the surface of target cells, receptor-mediated endocytosis, translocation of the enzyme domain into the cytosol, and modification of the specific target (1). Bacterial protein toxins of the AB type consist of a binding domain (B domain), which binds to a specific receptor on target cells and mediates translocation of the enzymatic A domain into the cytosol. In some toxins (e.g. diphtheria toxin), the A and B domains are located on the same polypeptide chain (2). In binary bacterial protein toxins, the enzyme component and the binding component are individual and nonlinked proteins that assemble on the target cell (3, 4). Members of this binary toxin family are iota -toxin from Clostridium perfringens (5), the ADP-ribosyltransferase from Clostridium difficile (6), Clostridium spiroforme toxin (7), the vegetative insecticidal proteins from Bacillus cereus (8), the anthrax toxin from Bacillus anthracis (9), and C. botulinum C2 toxin (10).

C2 toxin consists of the enzyme component C2I, an ADP-ribosyltransferase that modifies G-actin (11, 12) and the binding component C2II, which mediates cell entry of the toxin (13, 14). A prerequisite of the toxin action is the assembly of both components at the surface of the target cells. Recently, we reported that C2II binds to asparagine-linked glycans on the cell surface (15). Subsequent to binding, C2II and C2I are taken up by receptor-mediated endocytosis (16). C2I is translocated into the cytosol, and finally, it ADP-ribosylates G-actin at arginine 177 (17). This modification of G-actin causes inhibition of actin polymerization (11). Moreover, ADP-ribosylated G-actin acts like a capping protein to block polymerization of unmodified actin at the barbed ends of F-actin (18). Finally, these effects cause depolymerization of actin filaments, breakdown of the actin cytoskeleton, and rounding up of target cells (19, 20). While the enzyme component C2I (21, 22) and its action (20) was characterized in detail, much less is known about the binding component C2II and the cellular uptake mechanism for C2 toxin.

Here the activation and oligomerization of C2II and the requirements for toxin entry into mammalian cells were studied. We show that proteolytic activation of the C2II binding component by trypsin (23) is a prerequisite for cellular C2 toxin uptake. Thereby, a 20-kDa peptide is cleaved off from the N terminus of native C2II, resulting in active C2IIa. C2IIa but not native C2II oligomerizes to heptamers, which form ion-permeable channels in artificial lipid bilayer membranes (24). Moreover, we present evidence for the cell entry of C2 toxin from an acidic compartment.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Cell culture medium was from Biochrom (Berlin, Germany), fetal calf serum was from PAN Systems (Aidenbach, Germany), and cell culture materials were from Falcon (Heidelberg, Germany). The C2I enzyme component was purified as recombinant glutathione S-transferase fusion proteins with the glutathione S-transferase Gene Fusion System from Amersham Pharmacia Biotech as described (21). Anti-HB4 antiserum was raised from rabbits against the 15 N-terminal amino acids from trypsin-activated C2IIa coupled to keyhole lympet hemocyanine (synthesized by H. R. Rackwitz, Deutsches Krebsforschungszentrum Heidelberg, Germany). Donkey anti-rabbit antibody coupled to peroxidase and the enhanced chemiluminescence detection kit as well as the x-ray films were purchased from Amersham Pharmacia Biotech. Thrombin, nocodazole, brefeldin A, and cytochalasin D were purchased from Sigma. [32P]NAD (30 Ci/mmol) was from NEN Life Science Products (Bad Homburg, Germany). Trypsin and trypsin inhibitor were from Roche Molecular Biochemicals. Bafilomycin A1 was from Calbiochem (Bad Soden, Germany). Vivaspin 4 ML Concentrator devices were from Vivascience Ltd. (Binbrook Hill, United Kingdom). Hanks' balanced salt solution (HBSS)1 contained 0.185 g/liter CaCl2,, 0.098 g/liter MgSO4, 0.4 g/liter KCl, 0.06 g/liter KH2PO4, 8 g/liter NaCl, 0.048 g/liter Na2HPO4, 1 g/liter glucose, to which 10 mM HEPES (pH 7.4) was added.

Purification, Activation, and Oligomerization of C2II-- The C2II binding component from C. botulinum C2 toxin was purified and activated with trypsin as described (10, 23). Recombinant C2II was purified as recombinant glutathione S-transferase fusion protein with the glutathione S-transferase Gene Fusion System from Pharmacia Biotech and cleaved with thrombin as described.2 For activation, C2II was incubated for 20 min at 37 °C with 0.2 µg of trypsin/µg of C2II and subsequently with 2 µg of trypsin inhibitor for 1 h at 4 °C to block trypsin effects. C2IIa oligomers were isolated by centrifugation at 4 °C with a Vivaspin 4 ML Concentrator device with a cut-off mass of 100 kDa. Activation and oligomerization of C2IIa were analyzed by 3-12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with or without prior boiling of the samples, respectively.

Analytical Ultracentrifugation-- Molecular mass studies on recombinant C2IIa oligomers were performed using an An-60 Ti rotor in an XL-A type analytical ultracentrifuge (Beckman Instruments, Palo Alto, CA) equipped with UV absorbance optics. Sedimentation equilibrium technique was employed to directly determine the molecular mass. Sedimentation equilibrium was analyzed using externally loaded six-channel centerpieces of 12-mm optical path filled with about 70 µl of liquid. This cell type allows the analysis of three solvent-solution pairs. Three of these cells were used to simultaneously analyze different samples in one run. Sedimentation equilibrium was reached after 2 h of overspeed at 14,000 rpm followed by an equilibrium speed of 10,000 rpm (7266 × g) at 20 °C for about 26 h. The radial absorbancies of each compartment were recorded at three different wavelengths (275, 280, and 285 nm) using the molar absorbance coefficients. Molecular mass calculations were carried out by simultaneously fitting the sets of three radial absorbance distribution curves described by the equation Ar = Ar,mexp[MK(r2 - rm2)] with K = [(1 - rho <A><AC>&ngr;</AC><AC>&cjs1171;</AC></A>)omega 2]/2RT, using our computer program Polymole (25). In these equations, rho  is the solvent density, <A><AC>&ngr;</AC><AC>&cjs1171;</AC></A> is the partial specific volume, omega  is the angular velocity, R is the gas constant, and T is the absolute temperature. Ar is the radial absorbance, and Ar,m is the corresponding value at the meniscus position. The partial specific volume was calculated from the amino acid composition and the density increments of the individual amino acids.

Black Lipid Membrane Experiments-- Channel formation experiments of C2IIa oligomer in black lipid bilayer membranes were performed as described previously (24, 26). In brief, membranes were formed by painting onto a 1% solution of diphytanoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) in n-decane across a 0.5-mm2 circular hole, which connects two aqueous compartments of a Teflon chamber. The single channel recordings were performed by the use of calomel electrodes (with salt bridges) connected in series to a voltage source and a current amplifier. The amplified signal was monitored on a storage oscilloscope (Tektronix 7633) and recorded on a strip chart or tape recorder.

Electronic Microscopy of C2IIa Oligomers-- The stock protein solution (0.15 mg/ml) was diluted 20-fold in a buffer containing 10 mM HEPES, 150 mM NaCl, pH 7.4. Samples (5 µl) were deposited on carbon-coated electron microscopy grids rendered hydrophilic by glow discharge in air and negatively stained with 1% uranyl acetate. Electron microscopy was performed with a CM120 Philips microscope operating at 120 kV. Images were recorded under low dose conditions at × 60,000 magnification.

Cell Culture and Cytotoxicity Assays-- CHO-K1, HeLa, and NIH3T3 cells, respectively, were cultivated in tissue culture flasks at 37 °C and 5% CO2. CHO cells were grown in Ham's F-12/Dulbecco's modified Eagle's medium (1:1), and HeLa and NIH3T3 cells were grown in Dulbecco's modified Eagle's medium. All media contained 5% heat-inactivated (30 min, 56 °C) fetal calf serum, 2 mM L-glutamate, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were routinely trypsinized and reseeded twice a week. For cytotoxicity experiments, cells were seeded in small dishes and incubated with the respective drug or toxin in either complete medium or in serum-free Dulbecco's modified Eagle's medium or Hanks' balanced salt solution, respectively.

ADP-ribosylation Assay-- Cells were washed with cold PBS, scraped into 300 µl of cold lysis buffer (2 mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 80 µg/ml benzamidine in 50 mM HEPES, pH 7.4) and sonicated on ice with 10 strokes each for 5 s with 50% of maximal power (Bandelin Sonopuls HD60). Protein concentration was determined by the method of Bradford (27). ADP-ribosylation was performed as described (21). In brief, 50 µg of lysate proteins were incubated with [32P]NAD (0.5 µM) and 50 ng of C2I for 15 min at 37 °C. The reaction was stopped by the addition of Laemmli buffer, and the samples were heated for 3 min at 95 °C and subsequently subjected to SDS-PAGE. [32P]ADP-ribosylated proteins were detected by autoradiography with a PhosphorImager from Molecular Dynamics (Krefeld, Germany).

SDS-PAGE and Western Blotting-- SDS-PAGE was performed according to the methods of Laemmli (28). For immunoblot analysis, the proteins were transferred from the gel onto a nitrocellulose membrane using the semidry system. The membrane was blocked for 30 min with 5% nonfat dry milk in PBS containing 0.05% Tween 20 (PBS-T). The proteins were probed for 1 h with anti-HB4 antiserum (rabbit, 1:10,000 in PBS-T), washed with PBS-T, and incubated for a further hour with donkey anti-rabbit antibody coupled to horseradish peroxidase (1:4000 in PBS-T). The membrane was washed again, and proteins were visualized using the ECL system according to the manufacturer's instructions.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Trypsin Cleavage of C2II Is Required for Binding and Cytotoxic Effects-- The importance of trypsin activation of C2II for the action of C2 toxin was reported earlier (23). So far, it was not clear which step of the intoxication process depends on trypsin activation of the binding component C2II. As shown in Fig. 1A, the activated C2IIa but not the native C2II mediated cytotoxic effects when delivered together with C2I into CHO cells. The morphological observation that only C2IIa plus C2I act cytotoxic was confirmed by subsequent in vitro ADP-ribosylation of the respective cell lysates with [32P]NAD and C2I. In Fig. 1B, the autoradiogram of radiolabeled actin is shown. [32P]ADP-ribosylated actin was observed in lysates from control cells (lane 1) and after treatment of intact cells with nonactivated C2II/C2I (lane 3) but not after treatment of cells with activated C2IIa plus C2I (lane 2). This means that in cells treated with C2IIa plus C2I, all G-actin had been ADP-ribosylated and was no more substrate for subsequent in vitro ADP-ribosylation because all sites had been saturated by C2I in intact cells.


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  		Fig. 1.   Cytotoxic effects of native C2II and C2IIa on cells. A, CHO cells were incubated at 37 °C in HBSS with trypsin-activated C2IIa (200 ng/ml) plus C2I (100 ng/ml) or with native C2II (200 ng/ml) plus C2I (100 ng/ml), respectively. For control, cells were incubated without C2 toxin. Phase-contrast pictures of the cells were taken after a 3-h incubation. B, autoradiography of [32P]ADP-ribosylated actin. Cells were treated as described above and lysed after the 3-h incubation period, and 50 µg of each lysate protein was subjected to in vitro ADP-ribosylation assay with C2I. Lane 1, control cells; lane 2, cells treated with C2IIa plus C2I; lane 3, cells treated with C2II plus C2I. C, immunoblot analysis of cell-bound C2II. Subconfluently growing NIH3T3, HeLa, CHO-wild type (WT), and CHO-C2RK14 monolayer cells were incubated at 4 °C in HBSS with C2IIa (200 ng/ml) or C2II (200 ng/ml), respectively. After 3 h, cells were washed and lysed, and 100 µg of cell protein was heated for 3 min at 95 °C and subjected to 12.5% SDS-PAGE. C2II was identified by immunoblot analysis with anti-HB4 antiserum.

Next, we compared the binding of C2II and C2IIa to cells. Therefore, NIH3T3, HeLa, and CHO cells were incubated on ice with C2II or C2IIa, respectively, and cell-bound C2II was detected by immunoblot analysis with anti-C2II antiserum HB-4, which recognizes C2II as well as C2IIa (Fig. 1C). Exclusively trypsin-activated C2IIa but not full-length C2II bound significantly to cells. To test the specificity of C2IIa binding, we used the C2 toxin-resistant CHO-C2RK14 mutant cell line, which lacks the C2 toxin receptor (29). As expected, these cells showed no significant binding of C2IIa, indicating the specificity of C2IIa binding to C2-sensitive cells. The same results were obtained with C2II/C2IIa preparations of recombinant C2II and C2I.

Activation of C2II by Trypsin Cleavage Leads to Oligomerization of C2IIa-- When recombinant full-length C2II (80 kDa; Fig. 2A, lane 1) was cleaved by trypsin and the resulting C2IIa was heated for 3 min at 95 °C, the cleaved protein migrated as a 60-kDa protein on SDS-PAGE (Fig. 2A, lane 2). Additionally, a faint band of slowly migrating protein was detected almost at the start of the separation gel. When C2IIa was subjected to 3-12.5% gradient SDS-PAGE without prior heating for 3 min at 95 °C, no monomeric C2IIa but a protein band of a high molecular weight was detected after Coomassie Blue staining (Fig. 2A, lane 3). By contrast, noncleaved C2II showed no oligomerization, even without heating. Immunoblot analysis with anti-C2II antiserum confirmed this high migrating band as oligomeric C2II (Fig. 2B, lane 3). Fig. 2C gives a time course of the activation and oligomerization of C2II by trypsin treatment. C2II was incubated with trypsin, and after the indicated times, aliquots were removed. Laemmli sample buffer was added, and the proteins were heated for 3 min at 95 °C. After a 1-min incubation with trypsin, a significant amount of C2IIa was detected, and at 4 min after trypsin addition, oligomers appeared. Oligomerization of C2IIa without heating of the samples followed a comparable time course, but the amount of oligomeric protein was much higher than with heating and no monomers were detectable. These findings indicate that after trypsin cleavage, C2IIa immediately forms SDS-stable but heat-sensitive oligomers in solution. Oligomerization of C2IIa was also observed when C2II purified from C. botulinum was used for the experiments instead of recombinant C2II.


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  		Fig. 2.   In vitro oligomerization of trypsin-activated C2IIa. Recombinant C2II (100 µg/ml) was incubated with 0.2 µg of trypsin/µg of C2II for 20 min at 37 °C. Native C2II and trypsin-activated C2IIa were analyzed on 3-12.5% SDS gels with or without prior heating at 95 °C. Proteins (2 µg each) were stained with Coomassie Blue (A), or proteins (200 ng each) were detected by anti-C2II antiserum HB-4 in an immunoblot analysis (B). Lane 1, native C2II (not heated); lane 2, C2IIa (3 min at 95 °C); lane 3, C2IIa (not heated). C, time course of C2II activation by trypsin. Trypsin was added to C2II, and immediately and after the indicated incubation times at 37 °C, aliquots were taken, heated for 3 min at 95 °C (alternatively not heated), and analyzed by 3-12.5% SDS-PAGE and Coomassie Blue staining.

The C2IIa Oligomers Form Ring-shaped Structures in Solution-- In an attempt to visualize the oligomeric structure of C2IIa, the protein solutions were characterized by electron microscopy. Negatively stained solutions showed many particles exhibiting a characteristic annular (Fig. 3, two upper rows) or horseshoe (Fig. 3, bottom row) shape. The particle outer diameter was 11-13 nm, with a 2-4-nm central stained filled hole. It is yet unclear whether the horseshoe-shaped structures result from a staining artifact or correspond to an incomplete oligomerization state of the C2 toxin. Several particles presented 2-4-nm protrusions at their periphery; further work is needed for determining their number and providing a detailed structure of these protrusions.


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  		Fig. 3.   Gallery of negatively stained C2IIa oligomers. The two upper rows show annular-shaped particles, and the bottom row shows horseshoe-shaped particles. Scale bar, 10 nm.

C2IIa Forms Heptamers in Solution-- To analyze the stoichiometry of the C2IIa oligomers, the molecular mass of the complex was determined by analytical ultracentrifugation. Therefore, the C2IIa protein solution was analyzed at different concentrations between 14 and 70 µg/ml. Since the solutions contained some low molecular weight material besides the oligomers, an equilibrium speed of 10,000 rpm was used. This allowed us to consider the low molecular weight material to be buried in the base line with negligible increase of the radial absorbance at sedimentation equilibrium. Using the Polymole program, the data (Fig. 4) were fitted simultaneously. A mass average molecular mass of 422 ± 78 kDa was determined. Similar results were obtained from solutions with loading concentrations of 50-70 mg/ml. In the more diluted samples, an average molecular mass of nearly 60 kDa was determined. The high molecular mass component of 420 kDa exceeds the monomer molecular mass about 7 times, indicating that the oligomers should form on the average heptamers. Because the calculated molecular mass of trypsin-activated C2IIa monomer is 59.8 kDa, the deviation from average molecular mass (78 kDa) allows us to speculate that hexamers and octamers may be present, too.


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  		Fig. 4.   Determination of C2IIa heptamers by analytical ultracentrifugation. Radial absorbance distribution of recombinant C2IIa (loading concentration 0.57 mg/ml) at sedimentation equilibrium was recorded at 275 nm (open circle ), 280 nm (), and 285 nm (). T = 20 °C. From the simultaneous curve fit, an average molecular mass of 422 ± 78 kDa was determined.

C2IIa Oligomers Form Channels in Artificial Lipid Bilayer Membranes-- As described previously, trypsin-activated C2IIa but not full-length C2II forms ion-permeable channels in artificial lipid bilayer membranes (24). Therefore, we tested the channel forming activity of purified C2IIa oligomers. A typical experiment is given in Fig. 5. Ten min after membrane formation, 100 ng/ml C2IIa oligomers were added to the aqueous 1 M KCl solution (pH 6) on one side of the membrane. Shortly after the addition, the membrane conductance started to increase in a stepwise fashion. Most of the conductance steps had a single channel conductance of about 150 pS in 1 M KCl.


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  		Fig. 5.   Single-channel recording of a diphytanoyl phosphatidylcholine/n-decane membrane in the presence of recombinant C2IIa oligomers expressed in Escherichia coli. 10 min after formation of the membrane, 100 ng/ml oligomers were added to the aqueous phase on one side of the membrane. The aqueous phase contained 1 M KCl (pH 6). The applied membrane potential was 20 mV; T = 20 °C.

C2IIa Oligomers Bind to Cells and Mediate Cell Entry of C2I-- As shown above, activated C2IIa but not native C2II binds to cells and mediates C2 toxin effects when delivered together with C2I. Because C2IIa forms SDS-stable but heat-sensitive heptamers, which are able to insert into lipid bilayers, we analyzed which species of C2IIa binds to cells. Therefore, CHO cells were incubated with C2II and C2IIa, respectively, and analyzed in an immunoblot without prior heating of the samples. CHO-WT and the C2 receptor-defective CHO-C2RK14 cells were incubated for 1 h at 4 °C in HBSS with native C2II or trypsin-activated C2IIa to allow binding of the proteins. For controls, cells were incubated without C2II. Cells were washed, lysed, and immediately subjected to SDS-PAGE (3-12.5%) without heating. Proteins were blotted onto a nitrocellulose membrane and analyzed with an antiserum against C2II. The immunoblot shows that only the oligomeric C2IIa was significantly bound to CHO-WT cells (Fig. 6). Only a weak signal was detected when CHO-C2RK14 cells were incubated with C2IIa oligomers, which may be due to spontaneously reverted cells. This indicated that the oligomeric C2IIa pre-pores bound specifically to the cellular receptor. Moreover, when C2IIa was bound at 4 °C to cells, which were subsequently washed and then shifted to 37 °C, only oligomeric C2IIa was detected after 30 min, indicating that C2II oligomers do not dissociate during endocytosis. Finally, we incubated CHO-WT cells at 4 °C with both C2IIa and C2I. Cells were washed and further incubated at 37 °C. After 2 h, the cells were completely rounded up. Cells were lysed and subjected to immunoblot analysis. Even after 2 h, C2IIa oligomers remained stable, and no C2IIa monomer was detected. This demonstrates that the C2IIa oligomer represents the species of C2II that binds to the carbohydrate receptor for C2II on the surface of target cells and mediates the uptake of C2I.


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  		Fig. 6.   Binding of C2IIa oligomers to CHO cells. CHO-WT as well as CHO-C2RK14 cells were incubated for 1 h at 4 °C in HBSS with C2II (200 ng/ml) or C2IIa (200 ng/ml), respectively. Control cells were without C2II or C2IIa. Cells were washed and lysed, and C2II binding was tested by 3-12.5% SDS-PAGE followed by anti-C2II immunoblot analysis. Alternatively, after incubation with C2IIa at 4 °C, CHO-WT cells were incubated for an additional 30 min at 37 °C prior to lysis. In parallel, CHO-WT cells were incubated with C2IIa plus C2I (100 ng/ml) at 4 °C, washed, exposed for 2 h to 37 °C, and lysed.

Cellular Uptake of C2 Toxin Requires an Acidic Compartment-- Previously, it was reported that C2 toxin enters the cell via receptor-mediated endocytosis (16). Next, we studied the intracellular route of C2 toxin to reach its final destination in the cytosol. To answer the question of whether C2 toxin uptake requires an acidic cellular compartment, bafilomycin A1 was applied (30). Bafilomycin is a specific inhibitor of the vacuolar-type H+-ATPase (31) and is frequently used to detect toxin uptake from acidic compartments. As shown in Fig. 7A, cells that were incubated with C2 toxin without bafilomycin A1 (C2) were round. In contrast, cells that were treated with C2 toxin in the presence of bafilomycin (Baf + C2) showed control morphology. Bafilomycin itself did not induce any morphological effects within the incubation times indicated. In line with morphological changes, actin was ADP-ribosylated. The cell lysates were subjected to an in vitro ADP-ribosylation assay with [32P]NAD and C2I. The autoradiography of [32P]ADP-ribosylated actin in Fig. 7B shows that only the actin from the cells treated with C2 toxin without bafilomycin was not radiolabeled, indicating entry and action of C2 toxin. By contrast, radiolabeling of actin was observed when cells were previously treated with C2 toxin in the presence of bafilomycin. These results indicate that bafilomycin A1 prevents C2 toxin uptake and suggest that an acidic compartment is required for the translocation of C2 toxin into the cytosol.


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  		Fig. 7.   Influence of bafilomycin A1 on C2 toxin effects. A, NIH3T3 cells were preincubated for 15 min with 100 nM bafilomycin A1 followed by a 3-h incubation with C2 toxin (C2IIa (200 ng/ml) plus C2I (100 ng/ml)) in complete medium in the presence of bafilomycin (Baf + C2). Control cells were incubated with bafilomycin A1 (Baf) or with C2 toxin (C2) or without both (con). B, autoradiography of [32P]ADP-ribosylated actin from 50 µg of lysate protein from the cells after in vitro ADP-ribosylation with [32P]NAD and C2I.

C2 Toxin Can Be Delivered into Cells by Extracellular Acidification in the Presence of Bafilomycin A1-- Because C2IIa mediates translocation of C2I across the endosomal membrane, we wondered whether cell surface-bound C2 toxin could be delivered directly into the cytosol after extracellular acidification. To block endosomal toxin uptake, experiments were performed in the presence of bafilomycin A1. NIH3T3 cells were preincubated for 15 min at 37 °C with bafilomycin A1, prechilled, and subsequently incubated for 1 h with C2IIa and C2I at 4 °C to allow binding of the toxin. Cells were washed and incubated for 3 min at 37 °C with prewarmed medium containing bafilomycin at pH 7.5 or 5.0, respectively. Thereafter, fresh prewarmed medium (37 °C, pH 7.5) containing bafilomycin A1 was added, and cells were incubated for further 1.5 h at 37 °C. As shown by phase-contrast microscopy in Fig. 8A, only those cells exhibited C2 morphology that were allowed to bind C2 toxin and were subsequently exposed to pH 5.0 medium. Cells that bound C2 toxin but were not shifted to acidic pH did not round up. The observation that C2 toxin was taken up into the cells after an acidic shift was confirmed by subsequent in vitro ADP-ribosylation of the lysates from these cells (Fig. 8B). Only actin from cells that showed C2 morphology was not radioactively labeled. In these cells, all G-actin was ADP-ribosylated by C2I, which entered the cells after the extracellular acidic shift. For a more detailed characterization of the pH dependence of C2 toxin uptake, the effects of pH values between 4.0 and 7.0 were studied. Cells that were exposed to pH 4.0 and 5.0 after C2 toxin binding were completely round. By contrast, cells showed control morphology after exposure to pH 6.0 and 7.0, respectively (data not shown). Testing small pH steps, we observed the typical C2 morphology (rounding up of cells) at pH <= 5.4. By contrast, cells that were exposed to pH >= 5.6 showed control morphology. Even after 3 h, no C2 toxin effects were observed on cells that were exposed to pH >= 5.6. The percentage of rounded cells per field after 1.5- and 3-h exposure is shown in Fig. 9A, again indicating the sharp pH step allowing entry of toxin at pH 5.4. 


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  		Fig. 8.   Influence of extracellular pH on C2 toxin uptake into bafilomycin A1-treated cells. NIH3T3 cells were incubated for 1 h at 4 °C in HBSS containing 100 nM bafilomycin A1 and 200 ng/ml C2IIa plus 100 ng/mlC2I (C2). Control cells were incubated without C2 toxin (con). Cells were washed and incubated for 3 min in serum-free medium (37 °C with bafilomycin) at pH 7.5 or 5.0, respectively. Cells were further incubated in complete medium at 37 °C, pH 7.5 with bafilomycin. After 1.5 h, phase-contrast pictures were taken (A), cells were lysed, and in vitro ADP-ribosylation with [32P]NAD and C2I was performed (B).


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  		Fig. 9.   C2 toxin is delivered across the cytoplasmic membrane when extracellular pH is lowered to 5.4. NIH3T3 cells were incubated for 1 h at 4 °C with C2 toxin in HBSS plus bafilomycin. Cells were treated for 3 min with serum-free medium (37 °C with bafilomycin, pH 5.0, 5.2, 5.4, 5.6, 5.8, or 6.0, respectively). Cells were further incubated in complete medium at 37 °C, and after 1.5 and 3 h, pictures were taken. The percentage of round cells per field is given (A). B, model for the mechanism of how external acidification of whole cells mimics the endosomal environment. ec, extracellular; ic, intracellular; cm, cytoplasm membrane. C2IIa, heptameric C2IIa pre-pore. Bafilomycin A1 blocks the vacuolar-type H+-ATPase and thereby acidification of the endosome. C, influence of cytochalasin D, nocodazole, and brefeldin A on C2 toxin effects of CHO cells. CHO cells were incubated for 6 h with cytochalasin D (Cyt, 2 µg/ml) or with nocodazole (Noc, 20 µg/ml), respectively. Control cells were incubated without any drug (con). C2 toxin (200 ng/ml C2IIa plus 100 ng/ml C2I) was added, and cells were incubated for a further 12 h. Alternatively, CHO cells were treated for 30 min with brefeldin A (Bref, 10 µM final concentration), C2 toxin was added, and cells were incubated for a further 3 h. Cells were lysed, and in vitro ADP-ribosylation with [32P]NAD and C2I was performed.

Cells also rounded up when the time of external acidification was decreased from 3 min to 30 s (not shown). Notably, cells did not round up when they were incubated at 4 °C with C2II (in the absence of C2I), were shifted to acidic external pH (3 min, pH 4.5), and were subsequently incubated with C2I in complete medium (pH 7.5 plus bafilomycin A1) (data not shown). This indicates that C2I must be bound to C2IIa prior to acidification. Thus, we propose that the C2IIa pre-pore binds C2I and assembles with the receptor on the cell surface (Fig. 9B). After exposure of the extracellular site to acidic pH, the C2IIa pre-pore inserts into the cytoplasmic membrane and forms a pore similar to that in the endosomal membrane upon acidification. Subsequently, C2I translocates to the intracellular site.

Actin Filaments but Not Microtubules Are Involved in the Uptake of C2 Toxin-- Microtubules are involved in the transport of vesicles from the early to the late endosomal compartment (32, 33). To test any influence of the cytoskeletal components on uptake of C2 toxin, microtubules and actin filaments were disassembled by nocodazole and cytochalasin D, respectively. Therefore, CHO cells were incubated for 6 h with nocodazole or cytochalasin D, respectively. C2IIa plus C2I was added, and cells were incubated for further 12 h. Because nocodazole or cytochalasin D changed cell morphology in their own right, the cellular effects of C2 toxin were determined by subsequent in vitro actin ADP-ribosylation (Fig. 9C). Actin from nocodazole-pretreated cells was not radioactively labeled when cells were subsequently treated with C2 toxin. In contrast, a significant amount of [32P]ADP-ribosylated actin was detected when cells were pretreated with cytochalasin D and subsequently incubated with C2 toxin. These data indicate that the microtubule system is not essential for C2 toxin and suggest that C2I is released from the early endosomes into the cytosol. As expected, brefeldin A, which prevents retrograde transport of proteins to the endoplasmatic reticulum, also had no influence on C2 toxin uptake. When CHO cells were preincubated for 30 min with brefeldin A and subsequently C2IIa plus C2I was added, cells were completely rounded up after a further 3 h (not shown). Cells were lysed, and in vitro ADP-ribosylation was performed. As shown in Fig. 9C, no radiolabeled actin was detected in C2 toxin-treated cells and in cells that were preincubated with brefeldin A prior to the C2 toxin addition.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we have studied the binding/translocation component C2II of C2 toxin and describe its properties, cell binding, and carrier function to deliver C2I into the target cells. The binding component C2II exhibits significant sequence similarity with the protective antigen (PA), the binding component of anthrax toxin (9, 34). Like PA, C2II has to be activated by partial proteolysis (23, 35). C2II is highly trypsin-sensitive; however, at present it is not known whether trypsin is the physiological activator. As shown in this communication, trypsin activation was a prerequisite for C2II cell binding. The related PA (83 kDa) of anthrax toxin is cleaved near its N terminus and thereby activated by cellular proteases including furin (9, 36) but reportedly binds to cells even as a full-length protein (37). Similar to activated PA (PA63), activated C2IIa but not full-length C2II formed oligomers (9). The oligomerization of C2II occurred immediately after trypsin cleavage, and the oligomers were SDS-stable but heat-sensitive.

Using electron microscopy, we show that C2IIa oligomers form ring-shaped and horseshoe-shaped structures with outer diameters of 11-13 nm. In the middle, the oligomeric complexes are characterized by a "dark" spot, which might reflect a hole-like structure of 2-4 nm. More insights into the stoichiometry of the oligomeric structure were obtained by analytical ultracentrifugation of the complexes. These studies revealed that the oligomers are predominantly heptamers, a finding that is in correspondence with the electron microscopic data. However, the formation of some hexamers and octamers could not be excluded. Again the similarity to PA63 is evident. Also, PA63 forms heptamers (38).

Recently we have shown that activated C2IIa forms ion-permeable channels in artificial lipid bilayer membranes (24). Using isolated oligomers, we observed a more efficient channel formation than previously reported. Channel formation is also known for the protective antigen (38-40) and for several other toxins including diphtheria toxin (41, 42). Similar to channels formed by anthrax PA63 and diphtheria toxin, the C2IIa channel is cation-selective. This is based on point charges at the channel mouth (24).

In previous studies by Simpson (16), C2 toxin was shown to enter cells by receptor-mediated endocytosis. Recently, using a receptor-deficient mutant CHO cell line (RK14-CHO cells) (29), we identified asparagine-bound complex carbohydrates as toxin receptors or at least part of the receptor (15). After receptor binding, C2 toxin is endocytosed. Endocytosis appears to occur with C2IIa alone or with C2I attached to C2IIa (16). In general, two pathways of intracellular routing exist for bacterial protein toxins (43). One group of toxins is routed by a retrograde transport to the ER, where cellular uptake occurs. These toxins include cholera toxin (44), shiga toxin (45), and Pseudomonas exotoxin A (46). The other group of toxins, including diphtheria toxin (47) and anthrax toxin (48), translocate into the cytosol from an endosomal compartment. To get more insights into the cellular routing of C2 toxin, several inhibitors for cellular uptake and transport mechanisms were tested for their effects on C2 toxin-induced intoxication of cells. The drug brefeldin A, which blocks retrograde transport of cholera toxin and ricin to the ER (49, 50), did not decrease the C2 toxin effects. In contrast, the fungal metabolite bafilomycin A1 completely inhibited C2 toxin effects on cells. Bafilomycin A1 specifically blocks the vacuolar-type H+-ATPase in the endosomal membrane and thereby prevents acidification of endosomal vesicles (30, 51). This observation indicates that an acidic compartment is required for uptake of C2 toxin and that C2 toxin may be taken up in the same way as the anthrax toxins. Moreover, when C2 toxin was bound to whole cells at 4 °C in the presence of bafilomycin A1 and cells were subsequently exposed to low pH and incubated at 37 °C, the toxin was translocated directly from the cell surface into the cytosol. This system mimics the endosomal environment (52). Similar cell membrane translocation was reported for diphtheria toxin (53) and anthrax toxin (40).

We believe that after acidification of the early endosomal compartment, the oligomeric C2IIa pre-pores insert into the endosomal membrane and mediate subsequent C2I translocation into the cytosol. Fig. 10 shows our actual model for the uptake of C2 toxin into eukaryotic cells summarizing the results discussed above. Based on crystal structure analysis, it has been suggested that the central lumen of the PA heptameric channel has an average diameter of ~2 nm (54). In artificial membranes, a C2IIa channel diameter of about 1 nm has been proposed (24). Thus, in consideration of the sequence homologies between C2II and PA and the findings with black lipid membranes, a similar size of the channels of PA and C2II is likely. So far, the precise mechanism underlying the membrane insertion and translocation of the enzyme component into the cytosol is not clear. For the heptameric PA, it has been proposed that seven amphipathic hairpins each formed by the 2beta 2-2beta 3 loop of PA63 result in a 14-stranded beta -barrel structure that is basically involved in membrane insertion (54). This amphipathic 2beta 2-2beta 3 loop recognized in PA is highly conserved in C2II (amino acid residues 300-330), suggesting a similar function of these sequences. The translocation of C2I into the cytosol is even less clear. Two models are possible. In one case, the channel formed is capable of translocating several enzyme components into the cytosol. The other model implies that the process of membrane insertion and channel formation is also the driving force for the translocation of the enzyme component. Therefore, it is noteworthy that the intoxication of cells by C2 toxin in the presence of bafilomycin was not observed when the pH-dependent step was carried out with C2IIa alone and the enzyme component (C2I) was added after increasing the pH to 7.5 again. This may indicate that C2I must be present when the pH-dependent conformational change and/or the membrane insertion occurs, favoring a model that combines insertion and transport. On the other hand, it has been shown with anthrax toxin that the PA heptamer binds seven enzyme LF components (48), and the intoxication of cells is increased with increasing stoichiometry of PA/LF = 7:1 < 7:2 < 7:3 < 7:5, etc. It is difficult to believe that several enzyme components undergo the same conformational changes and subsequent translocation concomitantly with the membrane insertion of the binding heptamer. Thus, further studies are necessary to clarify the translocation mechanisms of this family of protein toxins in detail.


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  		Fig. 10.   Model of the uptake of C. botulinum C2 toxin into eukaryotic cells. After activation with trypsin, C2IIa forms heptamers and assembles with C2I. This complex binds to the carbohydrate receptor for C2IIa on the cell surface and is internalized via receptor-mediated endocytosis. After acidification of the early endosomes, C2II forms a channel in the membrane, and C2I escapes into the cytosol. This step can be blocked by bafilomycin A1 (Baf). Transport of vesicles from early to late endosomes is inhibited by nocodazole (Noc). Nocodazole has no influence on the uptake of C2 toxin.


    ACKNOWLEDGEMENTS

We thank Elke Maier, Ulrike Müller, and Otilia Wunderlich for expert technical assistance. Dr. H. R. Rackwitz is acknowledged for peptide synthesis. We thank Dr. Ingo Just for fruitful discussion of the results.

    FOOTNOTES

* This study was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 388) and by the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Institut für Pharmakologie und Toxikologie, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany. Tel.: 49-761-2035301; Fax: 49-761-2035311; E-mail: aktories@uni-freiburg.de.

Published, JBC Papers in Press, April 3, 2000, DOI 10.1074/jbc.M000596200

2 D. Blöcker, H. Barth, E. Maier, R. Bent, J. T. Barbieri, and K. Aktovies, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: HBSS, Hanks' balanced salt solution; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; PA, protective antigen; WT, wild type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Montecucco, C., Papini, E., and Schiavo, G. (1994) FEBS Lett. 346, 92-98
2. Collier, R. J. (1990) in ADP-ribosylating Toxins and G Proteins (Moss, J. , and Vaughan, M., eds) , pp. 3-19, American Society for Microbiology, Washington, D. C.
3. Considine, R. V., and Simpson, L. L. (1991) Toxicon 29, 913-936
4. Madshus, I. H., and Stenmark, H. (1992) Curr. Top. Microbiol. Immunol. 175, 2-26
5. Stiles, B. G., and Wilkens, T. D. (1986) Infect. Immun. 54, 683-688
6. Popoff, M. R., Rubin, E. J., Gill, D. M., and Boquet, P. (1988) Infect. Immun. 56, 2299-2306
7. Simpson, L. L., Stiles, B. G., Zepeda, H., and Wilkins, T. D. (1989) Infect. Immun. 57, 255-261
8. Han, S., Craig, J. A., Putnam, C. D., Carozzi, N. B., and Tainer, J. A. (1999) Nat. Struct. Biol. 6, 932-936
9. Leppla, S. H. (1995) in Bacterial Toxins and Virulence Factors in Disease (Moss, J. , Iglewski, B. , Vaughan, M. , and Tu, A. T., eds) , pp. 543-572, Marcel Dekker, New York
10. Ohishi, I., Iwasaki, M., and Sakaguchi, G. (1980) Infect. Immun. 30, 668-673
11. Aktories, K., Bärmann, M., Ohishi, I., Tsuyama, S., Jakobs, K. H., and Habermann, E. (1986) Nature 322, 390-392
12. Ohishi, I., Morikawa, Y., and Baba, T. (1990) J. Biochem. (Tokyo) 107, 420-425
13. Simpson, L. L. (1982) J. Pharmacol. Exp. Ther. 223, 695-701
14. Ohishi, I., and Miyake, M. (1985) Infect. Immun. 48, 769-775
15. Eckhardt, M., Barth, H., Blöcker, D., and Aktories, K. (2000) J. Biol. Chem. 275, 2328-2334
16. Simpson, L. L. (1989) J. Pharmacol. Exp. Ther. 251, 1223-1228
17. Vandekerckhove, J., Schering, B., Bärmann, M., and Aktories, K. (1988) J. Biol. Chem. 263, 696-700
18. Wegner, A., and Aktories, K. (1988) J. Biol. Chem. 263, 13739-13742
19. Reuner, K. H., Presek, P., Boschek, C. B., and Aktories, K. (1987) Eur. J. Cell Biol. 43, 134-140
20. Aktories, K., and Wegner, A. (1992) Mol. Microbiol. 6, 2905-2908
21. Barth, H., Preiss, J. C., Hofmann, F., and Aktories, K. (1998) J. Biol. Chem. 273, 29506-28511
22. Barth, H., Hofmann, F., Olenik, C., Just, I., and Aktories, K. (1998) Infect. Immun. 66, 1364-1369
23. Ohishi, I. (1987) Infect. Immun. 55, 1461-1465
24. Schmid, A., Benz, R., Just, I., and Aktories, K. (1994) J. Biol. Chem. 269, 16706-16711
25. Behlke, J., Ristau, O., and Schonfeld, H. J. (1997) Biochemistry 36, 149-156
26. Benz, R., Janko, K., and Lauger, K. (1979) Biochim. Biophys. Acta 551, 238-247
27. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254
28. Laemmli, U. K. (1970) Nature 227, 680-685
29. Fritz, G., Schroeder, P., and Aktories, K. (1995) Infect. Immun. 63, 2334-2340
30. Werner, G., Hagenmaier, H., Drautz, H., Baumgartner, A., and Zahner, H. (1984) J. Antibiotics 37, 110-117
31. Bowman, B. J., and Bowman, E. J. (1986) J. Membr. Biol. 94, 83-97
32. Gruenberg, J., Griffiths, G., and Howell, K. E. (1989) J. Cell Biol. 108, 1301-1316
33. Sakai, T., Yamashina, S., and Ohnishi, S. (1991) J. Biochem. (Tokyo) 109, 528-533
34. Kimura, K., Kubota, T., Ohishi, I., Isogai, E., Isogai, H., and Fujii, N. (1998) Vet. Microbiol. 62, 27-34
35. Eklund, M. W., and Poysky, F. T. (1972) Appl. Microbiol. 24, 108-113
36. Klimpel, K. R., Molloy, S. S., Thomas, G., and Leppla, S. H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10277-10281
37. Escuyer, V., and Collier, R. J. (1991) Infect. Immun. 59, 3381-3386
38. Milne, J. C., Furlong, D., Hanna, P. C., Wall, J. S., and Collier, R. J. (1994) J. Biol. Chem. 269, 20607-20612
39. Zhao, J., Milne, J. C., and Collier, R. J. (1995) J. Biol. Chem. 270, 18626-18630
40. Milne, J. C., and Collier, R. J. (1993) Mol. Microbiol. 10, 647-653
41. Donovan, J. J., Simon, M. I., Draper, R. K., and Montal, M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 172-176
42. Kagan, B. L., Finkelstein, A., and Colombini, M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4950-4954
43. Olsnes, S., Wesche, J., and Falnes, P. O. (2000) Handbook of Experimental Pharmacology , Springer, Heidelberg, in press
44. Majoul, I., Sohn, K., Wieland, F. T., Pepperkok, R., Pizza, M., Hillemann, J., and Söling, H.-D. (1998) J. Cell Biol. 143, 601-612
45. Sandvig, K., Garred, O., Prydz, K., Kozlov, J. V., Hansen, S. H., and Van Deurs, B. (1992) Nature 358, 510-512
46. Chaudhary, V. K., Jinno, Y., FitzGerald, D., and Pastan, I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 308-312
47. Madshus, I. H., Stenmark, H., Sandvig, K., and Olsnes, S. (1991) J. Biol. Chem. 266, 17446-17453
48. Singh, Y., Klimpel, K. R., Goel, S., Swain, P. K., and Leppla, S. H. (1999) Infect. Immun. 67, 1853-1859
49. Orlandi, P. A., Curran, P. K., and Fishman, P. H. (1993) J. Biol. Chem. 268, 12010-12016
50. Sandvig, K., Prydz, K., Hansen, S. H., and Van Deurs, B. (1991) J. Cell Biol. 115, 971-981
51. Umata, T., Moriyama, Y., Futai, M., and Mekada, E. (1990) J. Biol. Chem. 265, 21940-21945
52. Sandvig, K., and Olsnes, S. (1981) J. Biol. Chem. 256, 9068-9076
53. Moskaug, J. O., Stenmark, H., and Olsnes, S. (1991) J. Biol. Chem. 266, 2652-2659
54. Petosa, C., Collier, R. J., Klimpel, K. R., Leppla, S. H., and Liddingtom, R. C. (1997) Nature 385, 833-838

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