Low pH-induced Formation of Ion Channels by Clostridium difficile Toxin B in Target Cells*

Holger BarthDagger , Gunther PfeiferDagger , Fred HofmannDagger , Elke Maier§, Roland Benz§, and Klaus AktoriesDagger

From the Dagger  Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany, and § Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany

Received for publication, October 17, 2000, and in revised form, December 20, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Clostridium difficile toxin B (269 kDa), which is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis, inactivates Rho GTPases by glucosylation. Here we studied the uptake and membrane interaction of the toxin with eukaryotic target cells. Bafilomycin A1, which prevents acidification of endosomal compartments, blocked the cellular uptake of toxin B in Chinese hamster ovary cells cells. Extracellular acidification (pH <=  5.2) induced uptake of toxin B into the cytosol even in the presence of bafilomycin A1. Toxin B increased 86Rb+ release when preloaded Chinese hamster ovary cells were exposed to low pH (pH <=  5.6) for 5 min. Release of 86Rb+ depended on the concentration of toxin B and on the pH of the extracellular medium. An antibody directed against the holotoxin prevented channel formation, whereas an antibody against the N-terminal enzyme domain was without effect. The N-terminally truncated toxin B fragment consisting of amino acids 547-2366 increased 86Rb+ efflux when cells were exposed to low pH. Toxin B also induced pH-dependent channel formation in artificial lipid bilayer membranes. Clostridium sordellii lethal toxin, another member of the family of large clostridial cytotoxins, also induced increased 86Rb+ release at low pH. The results suggest that large clostridial cytotoxins including C. difficile toxin B and C. sordellii lethal toxin undergo structural changes at low pH of endosomes that are accompanied by membrane insertion and channel formation.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The large clostridial cytotoxins from Clostridium difficile toxin A (308 kDa) and toxin B (269 kDa) are major virulence factors of antibiotic-associated diarrhea and the causative agents of pseudomembranous colitis (1-4). Both toxins are O-glucosyltransferases that modify the small GTPases of the Rho family (Rho, Rac, and Cdc42) by monoglucosylation at threonine 37 and threonine 35, respectively (5, 6). The glucosylation blocks the biological functions of Rho GTPases, which are molecular switches in a large array of signal processes including regulation of the actin cytoskeleton (7, 8). Accordingly, some of the best-documented effects of large clostridial cytotoxins are the toxin-induced redistribution of actin filaments, morphological changes, and rounding-up of cells.

Structure-function analyses of large clostridial cytotoxins suggest a tripartite organization of the toxins similar to that known for diphtheria toxin. According to this model, the catalytic domain is located at the N terminus (9), whereas the C-terminal part of the toxins is believed to bind to the cellular receptor (10-12). In the middle of the molecule is a rather small hydrophobic region possibly involved in the translocation of the toxin into the cytoplasm. Because the substrates of the toxins are intracellularly located, translocation of the toxins across the cell membrane is a prerequisite for their actions on Rho GTPases. However, little is known about the site and mechanism of translocation of the toxins. Two major traffic pathways for bacterial exotoxins have been described. One group of toxins including cholera toxin (13), shiga toxins (14), and Pseudomonas aeruginosa exotoxin A (15) appears to be internalized after binding to specific receptors and then follows a retrograde pathway back to the endoplasmic reticulum, where the membrane translocation occurs. Another group of toxins appears to enter the cytosol from the low pH compartment of endosomes. Prototypes of this group are diphtheria toxin (16), anthrax toxin (17), and Clostridium botulinum C2 toxin (18). It is believed that endosomal acidification leads to a conformational change of these toxins, thereby allowing insertion into the endosomal membrane and eventually allowing translocation across the endosomal membrane into the cytosol. Some previous reports suggested that toxin B also escapes from the endosome into the cytoplasm (19). Toxins that enter the cytosol from endosomes were shown to induce ion-permeable channels in artificial membranes and in cell membranes when exposed to acidic pH (20, 21). Therefore, we investigated whether acidification also triggers the formation of channels by large clostridial cytotoxins. Here we report that under acidic conditions, C. difficile toxin B and Clostridium sordellii lethal toxin induce membrane permeabilization in eukaryotic cells as well as channel formation in artificial lipid bilayer membranes.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Materials-- Cell culture medium was from Biochrom (Berlin, Germany), and fetal calf serum was from PAN Systems (Aidenbach, Germany). Cell culture materials were obtained from Falcon (Heidelberg, Germany). Thrombin was from Sigma (Deisenhofen, Germany). Rubidium-86 (specific activity, 2 mCi/mg) was from PerkinElmer Life Sciences (Boston, MA). Bafilomycin A1 was from Calbiochem (Bad Soden, Germany). Toxin B from C. difficile VPI 10463 and C. sordellii lethal toxin were purified as described elsewhere (22). A polyclonal antibody against toxin B (anti-Tox B) was produced in a rabbit. The toxin B fragment CDB1-546 (catalytic domain of C. diffcile toxin B (amino acids 1-546)) was cloned, expressed, and purified as described previously (22), and a monoclonal antibody (anti-CDB1-546) was produced against this fragment.

Cloning and Expression of Toxin B Fragment CDB547-2366-- CDB547-2366 (enzymatic inactive fragment of C. diffcile toxin B (amino acids 547-2366)) was amplified from genomic DNA from C. difficile VPI 10463 by polymerase chain reaction with Taq polymerase (Roche Diagnostics, Mannheim, Germany) and the oligonucleotide primers CDB547-BamHI (5'-GGGGATCCGATAATCTTGATTTTTCTCAAAAT-3') and CDB3N-EcoRI (5'-GAATTCCTATTCACTAATCACTAATTG-3'). Amplification was done by denaturing at 94 °C for 10 s, primer annealing at 55 °C for 30 s, and extension at 68 °C for 6 min and repeated for 25 cycles. The resulting polymerase chain reaction product (2 µl) was cloned into pCR2.1 vector (Invitrogen, NV Leek, The Netherlands) according to the manufacturer's instructions. For expression of CDB547-2366, the gene was excised with BamHI/EcoRI (NEB Biolabs, Schwalbach, Germany) and cloned in BamHI/EcoRI-digested pGEX2T plasmid (Amersham Pharmacia Biotech, Uppsala, Sweden) containing a double glycine linker. Proteins were expressed in Escherichia coli-TG1 as recombinant glutathione S-transferase-fusion proteins and purified by affinity chromatography with glutathione-Sepharose 4B according to the manufacturer's instructions. Glutathione S-transferase was cleaved off by thrombin, and proteins were analyzed by SDS-polyacrylamide gel electrophoresis according to the method of Laemmli (23).

Cell Culture and Toxin Translocation Assay-- CHO1-K1, HT-29, Caco-2, and Vero cells were cultivated in tissue culture flasks at 37 °C and 5% CO2 in Ham's F-12/Dulbecco's modified Eagle's medium (DMEM) (1:1) containing 5% heat-inactivated (30 min, 56 °C) fetal calf serum, 2 mM L-glutamate, 100 units/ml penicillin, and 100 µg/ml streptomycin. Rat basophilic leukemia (RBL) cells were cultivated in Eagle's minimal essential medium plus Earle's salts containing 15% (v/v) heat-inactivated fetal calf serum, 4 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. All cells were routinely trypsinized and reseeded twice a week. For the toxin B translocation assay, CHO cells were preincubated for 15 min at 37 °C with 100 nM bafilomycin A1 in serum-free medium (Ham's F-12/DMEM), toxin B (100 ng/ml) was subsequently added, and cells were incubated for an additional 2 h at 4 °C in serum-free medium. Cells were washed with cold medium and incubated for 5 min in serum-free medium (Ham's F-12/DMEM) at pH 7.5, pH 5.2, or pH 4.5, respectively. Subsequently, cells were incubated in complete medium, pH 7.5, at 37 °C in the presence of bafilomycin A1, and after 1.5 h, phase-contrast pictures were taken.

86Rb+ Efflux Measurements-- For 86Rb+ efflux experiments, cells were plated in complete medium (Ham's F-12/DMEM containing 5% fetal calf serum) at a density of ~2 × 105 cells/well in 24-well culture plates. At 8 h after plating, fresh medium containing 86Rb+ (1 µ Ci/ml) was added, and cells were incubated for an additional 14 h. Cells were chilled at 4 °C, and fresh medium (4 °C; Ham's F-12/DMEM without serum) containing toxin A or toxin B was added. Toxins were allowed to bind for 2 h at 4 °C and then washed two times with cold medium to remove unbound toxin. To initiate membrane insertion of the toxins, cells were treated with warm medium (37 °C; Ham's F-12/DMEM without serum, pH 4.0-7.5) for 5 min at 37 °C. Cells were further incubated at 4 °C, and after various incubation times, aliquots of the medium were removed, and 86Rb+ release was determined by liquid scintillation counting in a 1209 Rackbeta beta counter from LKB Wallac (Gräfeling, Germany).

Black Lipid Bilayer Experiments-- The methods used for black lipid bilayer experiments have been described previously in detail (24). Membranes were formed from a 1% solution of asolectin or diphytanoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) in n-decane.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Translocation of Cell-bound Toxin B into the Cytoplasm-- Treatment of CHO cells with bafilomycin A1, which blocks the vacuolar H+-ATPase pump, prevented the cytotoxic effect of toxin B. To test the effect of pH on toxin uptake, toxin B was allowed to bind to cells at 4 °C. After 2 h, cells were incubated for 5 min at 37 °C with bafilomycin-containing medium of pH 7.5, pH 5.2, or pH 4.5, respectively. Thereafter, the acidified media were replaced by neutral medium containing bafilomycin, and cells were incubated for an additional 1.5 h at 37 °C. The cells were then analyzed for cytopathic effects induced by toxin B by phase-contrast microscopy. As shown in Fig. 1, cells rounded up when the pH was shifted for 5 min to pH 5.2 or pH 4.5 but not at pH 7.5. The results indicate that at low pH, the toxin crosses the cell membrane directly even in the presence of bafilomycin. The data are in line with the recent report that toxin B escapes from an acidic endosomal compartment into the cytoplasm (19).



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Fig. 1.   Influence of extracellular pH on toxin B uptake into bafilomycin A1-treated CHO cells. CHO cells were preincubated for 15 min at 37 °C with 100 nM bafilomycin A1, followed by a 2-h incubation with toxin B (100 ng/ml) in serum-free medium. After washing, cells were incubated for 5 min in serum-free medium (37 °C + bafilomycin A1) at pH 7.5, pH 5.2, or pH 4.5, respectively. Cells were further incubated at 37 °C in complete neutral medium (pH 7.5) containing bafilomycin A1. After 1.5 h, phase-contrast pictures were taken. In parallel, cells were incubated with 100 ng/ml toxin B (Tox B) or without any drug (con).

Toxin B-induced 86Rb+ Release from CHO Cells-- The conditions allowing direct uptake of toxin B across the cytoplasmic membrane were used to test whether extracellular acidification leads to channel formation and membrane permeabilization by toxin B. To this end, the efflux of 86Rb+ from preloaded cells was studied. At first, 86Rb+-preloaded CHO cells were incubated without and with toxin B and heat-inactivated toxin B, respectively. An increase in 86Rb+ release was measured when toxin B-treated cells were incubated in an acidified medium of pH 5.2 (Fig. 2A). The increased 86Rb+ release depended not only on low pH but also required short-term (5 min) incubation at 37 °C. It is noteworthy that under these conditions, cells did not round up, indicating no intoxication by toxin B. Acidification of medium in the absence of toxin B and in the presence of heat-inactivated toxin or binding of toxin B to cells without subsequent acidification did not increase 86Rb+ release. This result indicates that toxin B was inserted into the membrane of eukaryotic cells and increased membrane permeability after exposure of cells to acidic pH. In Vero cells, toxin B caused a similar increase in efflux of 86Rb+ at low pH (Fig. 2B). This finding indicates that channel formation by toxin is not restricted to CHO cells. However, we were not able to detect an increase in 86Rb+ release in HT-29 and Caco-2 cells. Additional studies on toxin B-induced 86Rb+ release were performed with the CHO-K1 cell line.



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Fig. 2.   Toxin B-mediated release of 86Rb+ from CHO and Vero cells after acidification. A, CHO cells were preloaded with 86Rb+ (14 h, 1 µCi/ml) and subsequently incubated for 2 h at 4 °C with toxin B (100 ng/ml) or with heat-inactivated toxin B (100 ng/ml) in fresh medium without serum. For control, cells were incubated without toxin. Cells were washed to remove unbound toxin, and prewarmed medium (37 °C; pH 7.5 or pH 5.2, respectively) was added. Cells were incubated at 37 °C for 5 min and then at 4 °C for an additional 55 min. The complete medium (500 µl) was removed, and 86Rb+ release was determined by liquid scintillation counting. Values are given as mean ± S.D. (n = 3). B, Vero cells preloaded with 86Rb+ were incubated with toxin B (100 ng/ml) or, for control, without toxin B. Cells were shifted for 5 min to pH 5.2 and further incubated at 4 °C for 25 min. The medium was assayed for 86Rb+. Values are given as mean ± S.D. (n = 3).

Characterization of Toxin B-induced 86Rb+ Release from CHO Cells-- At first, the influence of pH on the kinetics of the 86Rb+ release from toxin B-treated CHO cells was studied. When the medium of toxin B-treated cells was shifted to pH 5.2 for 5 min at 37 °C and cells were subsequently incubated on ice at pH 5.2, the increase in 86Rb+ release occurred mainly within 20-30 min. A low additional increase in 86Rb+ release occurred in a second phase within 90 min (Fig. 3). A similar release of 86Rb+ was observed when toxin B-treated cells were kept for 5 min in a medium of pH 5.2 and then incubated at 4 °C at pH 7.5. This finding suggests that membrane permeabilization by toxin B is achieved by a brief acidic pulse. Once formation of channels has occurred, the channels remained open to release 86Rb+ even at physiological pH. To further characterize the formation of channels, we studied the pH dependence of the toxin effects in more detail. 86Rb+-preloaded CHO cells were incubated with toxin B and shifted to pH 7.5, pH 5.6, pH 5.2, or pH 5.0, respectively. At the lowest pH (5.0), the largest increase in release of 86Rb+ occurred. At pH 5.6, 86Rb+ efflux was much less pronounced than that at pH 5.2 or pH 5.0, and at pH 7.5, no increased release was measured (Fig. 4A). Next, 86Rb+-preloaded CHO cells were incubated with increasing concentrations of toxin B (25, 100, and 500 ng/ml, respectively). Cells were then shifted to pH 5.2, and released 86Rb+ was determined. 86Rb+ efflux depended on the concentration of the toxin. Treatment of cells with 100 and 500 ng/ml toxin B, respectively, did not result in significant differences in 86Rb+ release (Fig. 4B), most likely indicating saturation of the toxin binding capacity of CHO cells. This finding suggests that the specific binding of toxin B to its cell membrane receptor is a prerequisite for subsequent channel formation. Moreover, the data indicate that channel formation is not caused by an unspecific integration of toxin B into cell membranes.



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Fig. 3.   Effect of pH on toxin B-mediated 86Rb+ efflux. 86Rb+-preloaded CHO cells were incubated for 2 h at 4 °C with toxin B (100 ng/ml) or, for control, without toxin. Cells were washed to remove unbound toxin, and prewarmed medium (pH 5.2) was added. Cells were incubated at 37 °C for 5 min and subsequently at 4 °C at pH 5.2 (control, black-square; toxin B, black-triangle) or pH 7.5 (control, ; toxin B, black-down-triangle ), respectively. After an additional 5, 10, 15, 25, 55, and 85 min, 100 µl of the medium were removed, and 86Rb+ release was determined by scintillation counting.



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Fig. 4.   pH and concentration dependence of toxin B-mediated 86Rb+ efflux. 86Rb+-preloaded CHO cells were incubated for 2 h at 4 °C with toxin B (100 ng/ml) or, for control, without toxin. Cells were washed to remove unbound toxin, and prewarmed medium (pH 7.5, black-square; pH 5.6, ; pH 5.2, black-triangle; pH 5.0, black-down-triangle ) was added. Cells were incubated at 37 °C for 5 min and then incubated at 4 °C. After 10, 20, 30, and 60 min, aliquots (100 µl) of the medium were removed, and released 86Rb+ was determined (A). 86Rb+-preloaded CHO cells were incubated for 2 h at 4 °C with toxin B (25, 100, and 500 ng/ml, respectively) or, for control, without toxin B. Cells were washed, and prewarmed medium (pH 5.2) was added. Cells were incubated at 37 °C for 5 min and then incubated at 4 °C. After 10 (black-square), 20 (), 30 (black-triangle), and 60 min (black-down-triangle ), aliquots (100 µl) of the medium were removed, and 86Rb+ was determined (B).

Influence of Anti-toxin B Antibodies on Toxin B-induced 86Rb+ Release-- To confirm that receptor binding of toxin B is necessary for subsequent membrane permeabilization, toxin B was incubated with an antibody directed toward the complete toxin, which recognizes mainly the receptor binding domain of toxin B (25), or with an antibody against its N-terminal catalytic domain, respectively. CHO cells were then incubated with the respective toxin/antibody mixture. The antibody against the holotoxin inhibited binding of toxin B to CHO cells and prevented the cytotoxic effects (Fig. 5A). In contrast, the antibody against the catalytic domain did not inhibit toxin B-induced rounding of the cells (Fig. 5A). Accordingly, the latter antibody did not affect toxin B-induced 86Rb+ release from CHO cells (Fig. 5B). Incubation of toxin B with the antibody against the holotoxin prevented toxin-induced membrane permeabilization. However, this antibody was without effect when added to cells after binding of toxin B (Fig. 5B). This observation suggests that only toxin B that was already bound to the cell membrane receptor formed channels at low pH.



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Fig. 5.   Influence of anti-toxin B antibodies on cytotoxic toxin B effects and pH-induced release of 86Rb+ from CHO cells. Toxin B was preincubated with an antibody against the holotoxin (anti-Tox B) or with an antibody against its catalytic domain (anti-CDB1-546) for 30 min at 4 °C. For control, toxin B was preincubated with phosphate-buffered saline. A, CHO cells were incubated with the respective pretreated toxin B (100 ng/ml) at 37 °C. The morphology of the cells after 3 h is shown. B, 86Rb+-preloaded CHO cells were incubated for 2 h at 4 °C in serum-free medium with the respective pretreated toxin B (100 ng/ml), and pH shift assay (pH 7.5 versus pH 5.2) was performed. After 30 min at 4 °C, the medium was removed, and 86Rb+ release was measured. Values are given as mean ± S.D. (n = 3). C, cells were treated as described above. Additionally, toxin B (100 ng/ml) was bound to CHO cells for 2 h at 4 °C, anti-Tox B was added, and cells were incubated for 1 h. After pH shift (pH 7.5 and pH 5.2, respectively), the cells were incubated for 30 min at 4 °C, and the medium was removed and assayed for 86Rb+. Values are given as mean ± S.D. (n = 3).

Effect of Toxin B Fragment CDB 547-2366 on 86Rb+ Release from CHO Cells-- To determine the part of toxin B that mediates membrane permeabilization of cells after acidification, we cloned and expressed a toxin B fragment. CDB 547-2366 consists of residues 547-2366, i.e. of the putative receptor binding and membrane insertion domains, and lacks the catalytic domain (Fig. 6A). The proteins were allowed to bind to 86Rb+-preloaded CHO cells, and cells were subsequently exposed to pH 7.5 or pH 4.5, respectively. The release of 86Rb+ was determined. Fig. 6 shows that the release of 86Rb+ from preloaded CHO cells was increased when cells were treated with CDB 547-2366 and then exposed to acidic medium. It is noteworthy that the concentration of toxin B fragment CDB547-2366 (4 µg/ml) was higher than that of the full-length toxin B (200 ng/ml), which was applied in parallel.



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Fig. 6.   Effect of toxin B fragment CDB547-2366 on 86Rb+ release from CHO cells. A, SDS-polyacrylamide gel electrophoresis of recombinant CDB547-2366. CDB547-2366 was expressed in E. coli and purified as described. After cleavage with thrombin, 1 µg of protein was analyzed by 7% SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. B, release of 86Rb+ from CHO cells after treatment of cells with toxin B and CDB547-2366, respectively, and exposure to low pH medium. 86Rb+-preloaded (1 µCi/ml) cells were incubated for 2 h at 4 °C with toxin B (200 ng/ml) or with CDB547-2366 (4 µg/ml) in fresh medium without serum. For control, cells were incubated without toxin. Cells were washed, and prewarmed medium (37 °C; pH 7.5 or pH 4.5, respectively) was added. Cells were incubated at 37 °C for 5 min and then incubated at 4 °C for an additional 55 min. The medium (500 µl) was removed and assayed for 86Rb+. Values are given as mean ± S.D. (n = 3).

Effect of C. sordellii Lethal Toxin on 86Rb+ Release from Cells-- Lethal toxin of C. sordellii was tested for an influence on 86Rb+ release from cells after exposure to low pH medium. Therefore, the toxin was allowed to bind to preloaded CHO cells, and cells were subsequently shifted to pH 7.5 or pH 4.5, respectively, for 5 min at 37 °C and incubated for an additional 55 min at 4 °C. As shown in Fig. 7, treatment of cells with lethal toxin of C. sordellii and subsequent exposure to low pH medium increased the efflux of 86Rb+ from cells. Similar results were obtained when RBL cells were used.



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Fig. 7.   pH-induced release of 86Rb+ from CHO cells after treatment with lethal toxin (LT). 86Rb+-preloaded CHO cells were incubated for 2 h at 4 °C in serum-free medium with lethal toxin (400 ng/ml). Cells were washed to remove unbound toxin, and prewarmed medium (37 °C; pH 7.5 or pH 4.5) was added. Cells were incubated at 37 °C for 5 min and then incubated at 4 °C for an additional 55 min. The medium (500 µl) was removed to determine released 86Rb+. Values are given as mean ± S.D. (n = 3).

Toxin B Leads to Channel Formation in Artificial Lipid Bilayer Membranes under Conditions of Low pH-- The toxin B-mediated efflux of 86Rb+ from cells indicated that toxin B formed channels, which were at least permeable to ions. Therefore, we studied whether ion-permeable channels are also formed in lipid bilayer membranes. In a first set of experiments, we studied the effect of toxin B on membranes formed from different lipids such as phosphatidylcholine and asolectin at pH 6. Under these conditions, we observed only rare current fluctuations in single-channel recordings (see Fig. 8A). The fluctuations were very rapid as Fig. 8A demonstrates and had only a short lifetime on the order of seconds. Their single-channel conductance was ~0.2-1.5 nS in 1 M KCl. Enhanced single-channel activity was observed, however, when the pH was lowered to pH 5, and many more channels were recorded under the conditions of the lowered pH (see Fig. 8B). The current fluctuations were again very rapid, and it was difficult to provide a precise value for the single-channel conductance. Histograms of the fluctuations that could be resolved suggest that the pH did not influence the size of the channels as compared with pH 6. The low pH seemed to increase simply the channel-forming activity. Measurement with other salts such as LiCl suggested that a variety of ions were permeable through the toxin B channel, but it seemed to have generally a higher permeability for cations than for anions.



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Fig. 8.   Channel formation of toxin B in artificial lipid bilayer membranes. A, single-channel recording of an asolectin/n-decane membrane in the presence of toxin B from C. difficile. 10 min after the formation of the membrane, 80 ng/ml toxin B was added to the aqueous phase on one side of the membrane. The aqueous phase contained 1 M KCl and 10 mM MES, pH 6. B, an experiment similar to that described in A. The left side of the record demonstrates the small channel-forming activity of toxin B at pH 6. About 30 min after membrane formation, the pH was lowered to pH 5 (right side, arrow). Shortly after the decrease of the pH, the membrane conductance increased due to the formation of many channels, which had a small lifetime. The applied membrane potential was 50 mV; temperature = 20 °C.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial toxins that modify intracellular substrates must translocate across a lipid bilayer membrane to reach their targets (26). After endocytosis, two pathways are described that are used by bacterial toxins to reach their cytosolic substrates. One possible route for toxin uptake (e.g. cholera toxin (13)) is the retrograde transport from endosomal compartments via the Golgi apparatus to the endoplasmic reticulum, from which the toxins translocate into the cytoplasm. Other toxins (e.g. diphtheria toxin (16) or the binary anthrax toxin (17)) translocate directly from the acidic endosomal compartment into the cytosol. Inhibition of the cytotoxic action of toxin B by bafilomycin, as reported recently and shown in this report, indicates that toxin B also requires an acidic endosome for cellular uptake (19). In line with this notion is the finding that the lowering of the extracellular pH allows uptake of the toxin even in the presence of bafilomycin. Thus far, our knowledge of the events that cause translocation of toxin B is very limited. In the present study, we report that toxin B is capable of inducing ion-permeable channels, a process that might reflect molecular mechanisms closely related to translocation of the toxin into the cytosol. For detection of channel formation, the release of 86Rb+ from preloaded cells was measured, and conditions were chosen that mimic the endosomal compartment, e.g. cell-bound toxin B was exposed to an acidic buffer (pH < 5.6) for a short time (5 min) at 37 °C to induce channel formation by the toxin. Similarly, we observed a strong induction of channel formation in lipid bilayer membranes when the pH was lowered to pH 5 (see Fig. 8). At pH 6, only a very small channel-forming activity was observed. It is noteworthy that pH-dependent channel formation has been reported for several protein toxins that are taken up from endosomes (18, 21). It is believed that the low pH present in endosomal compartments causes conformational changes of the toxin, which are accompanied by surface exposition of the otherwise intramolecularly located hydrophobic region of the toxins. The hydrophobic regions are inserted into the bilayer membrane to form channels and/or to allow protein translocation. Recently, Qa'Dan et al. (19) analyzed the pH-induced conformational changes occurring in toxin B. Using various fluorescence methods, it was shown that the hydrophobicity of toxin B was increased at pH <=  5.0 but not at pH >=  5.5 and concluded that toxin B undergoes pH-induced structural changes that finally allow membrane insertion and translocation of the toxin from the acidic endosome into the cytosol.

Thus far, the structural basis of the pH-dependent channels is not clear. Our studies show that the N-terminal catalytic domain (toxin B fragment consisting of amino acids 1-546) is not necessary for channel formation. In contrast, a N-terminal truncated toxin B fragment that consisted of amino acid residues 547-2366 was capable of channel formation. However, because the C-terminal fragment was only active at concentrations higher than that of the holotoxin, it is suggested that the N terminus might also play a minor role in binding/insertion or stabilization of the holotoxin. Nevertheless, the results are in line with the view that the C terminus and the middle part of large clostridial toxins are involved in receptor binding and translocation. This hypothesis is supported by the findings that an antibody directed against the holotoxin, which recognizes mainly the receptor binding domain of toxin B (25), inhibited channel formation and intoxication, whereas an antibody against the N-terminal catalytic domain of toxin B was not able to prevent the increase in Rb efflux. In experiments with artificial black lipid bilayer membranes, membrane permeabilization by toxin B was detected at pH 6.0 and increased under more acidic conditions (pH 4.5). Here, the antibody against the holotoxin had no effect on toxin B-induced membrane permeabilization. This indicates that the antibody did not affect membrane insertion of toxin B but might inhibit the toxin binding to the cell surface. However, it remains to be clarified whether insertion into artificial membranes and insertion into cell membranes follow identical mechanisms.

Moreover, the functional dissociation of channel formation and cytopathic effects induced by the toxin is corroborated by the finding that despite increased 86Rb+ efflux, the cells still showed the control morphology when experiments were performed at 4 °C and only the acidic shift was done at 37 °C.

An increased 86Rb+ efflux was measured when CHO, Vero, or RBL cells were studied. These cell lines show high sensitivity toward toxin B and appear to have a sufficient amount of cell membrane receptor molecules. In contrast, we were not able to detect toxin B-induced channel formation in some other cell lines (HT-29 and Caco-2). In agreement with the possible destruction of the barrier function of the cytoplasmic membrane of CHO, Vero, or RBL cells, we were able to identify channel formation in artificial lipid bilayer membranes. Channels were formed with the two lipids tested. This result seems to represent a contradiction to the obvious receptor-mediated permeabilization of cells. However, it has to be kept in mind that other toxins, such as C2 toxin (20) or the repeat in toxin toxins (27-29), form channels in lipid bilayer membranes without the need for receptors, whereas they all need a receptor for biological activity. An important role of the toxin receptor in events finally resulting in channel formation is supported by the concentration effect studies at high concentration of toxin, which show that limitation of channel formation is most likely due to reaching the binding capacity of cells. In contrast to toxin B, toxin A from C. difficile showed no detectable channel-forming activity in the 86Rb+ release assay with CHO cells. This might be due to the relative insensitivity of CHO cells to toxin A and to their low content of toxin A receptor molecules. Toxin A, which is also termed enterotoxin, is known to exhibit much less cytotoxic effect on many cultured cells than toxin B. Similar to toxin B, C. sordellii lethal toxin caused channel formation in CHO and RBL cells, which are especially sensitive to the lethal toxin, suggesting that channel formation is a general activity of large clostridial cytotoxins.


    ACKNOWLEDGEMENTS

We thank Katrin Thoma and Otilia Wunderlich for expert technical assistance. We thank Dr. Ingo Just for the polyclonal anti-toxin B antibody and for discussion of the results.


    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 388 and 487) and by the Fonds of the Chemische 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.

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, January 4, 2001, DOI 10.1074/jbc.M009445200


    ABBREVIATIONS

The abbreviations used are: CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; RBL, rat basophilic leukemia.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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