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J. Biol. Chem., Vol. 278, Issue 45, 44535-44541, November 7, 2003
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From the Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Otto-Krayer-Haus, Albertstrasse 25, D-79104 Freiburg, Germany
Received for publication, July 14, 2003 , and in revised form, August 22, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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65 kDa) was recognized by an antibody against the N terminus of toxin B and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis as the catalytic domain of toxin B. The toxin fragment located in the cytosol possessed glucosyltransferase activity that could modify RhoA in vitro, but it was not able to intoxicate intact cells. After treatment of Vero cells with a radiolabeled fragment of toxin B (amino acid residues 547–2366), radioactivity was identified in the membrane fraction of Vero cells but not in the cytosolic fraction of Vero cells. Furthermore, analysis of cells by fluorescence microscopy revealed that the C terminus of toxin B was located in endosomes, whereas the N terminus was detected in the cytosol. Protease inhibitors, which were added to the cell medium, delayed intoxication of cells by toxin B and pH-dependent translocation of the toxin from the cell surface across the cell membrane. The data indicate that toxin B is proteolytically processed during its cellular uptake process. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To reach their substrates in the cytosol, the catalytically active domain of the toxin must cross the cellular membrane. It has been proposed that toxins A and B have a tripartite functional organization (see Fig. 1). The catalytic domain is located at the N terminus of the toxin (9), and the C terminus is believed to bind to the cellular receptor (10–12). The middle part of the protein harbors a hydrophobic region that is proposed to be involved in the translocation of the toxin across cellular membranes.
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Over the past few years, some progress has been made in understanding the cellular uptake mechanism of the large clostridial cytotoxins. The toxins, or at least their enzymatic domains, translocate from early endosomal compartments into the cytosol (13, 14). This step requires acidification of the endosomal lumen and can be blocked by the vesicular H+-ATPase inhibitor bafilomycin A1, which prevents acidification of endosomes (13). This translocation of the toxin across endosomal membranes can be mimicked at the cell surface by a decrease in the pH of the culture medium. Extracellular acidification (pH 5.2) induces uptake of toxin B into the cytosol, even in the presence of bafilomycin A1 (13, 14). However, the mechanism of toxin translocation across membranes is still unclear. Moreover, it is not known whether the complete holotoxin or only its catalytic domain is transported into the cytosol. Recently, we reported that both full-length toxin B and its putative receptor binding/translocation domain (amino acid residues 547–2366, see Fig. 1) form pores in membranes of intact Chinese hamster ovary cells (13). This pore formation depends on an acidic pulse (5 min at pH 5.2). In addition, toxin B induces pH-dependent channels in artificial black lipid bilayers. Most probably, the low pH of endosomes leads to a conformational change of toxin B, which is accompanied by membrane insertion and pore formation (13). However, the role of the pores in translocation of the toxin or its catalytic domain is not clear.
Here we cloned and expressed fragments of toxin B and used radiolabeling to study the translocation of the toxin into the cytosol of target cells. We found that during cellular uptake of toxin B, the binding/translocation domain (amino acid residues 547–2366) remained in endosomes of intoxicated cells and was not delivered into the cytosol. When cells were treated with the holotoxin and subsequently cytosolic and membrane fractions were separated, only an N-terminal catalytically active fragment of toxin B, which showed in vitro glucosyltransferase activity, was detected in the cytosol. This polypeptide was identified as the catalytic domain of toxin B by immunoblot analysis, fluorescence microscopy, and mass spectrometry.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cloning and Expression of Toxin B—The toxin B gene was amplified by using polymerase chain reaction from genomic DNA from C. difficile VPI 10463 with the following primers: CDB1C (5'-AGATCTATGAGTTTAGTTAATAGAAAAC-3') and CDB3sal1-anti (5'-GTCGACTTATTCACTAATCACTAATTGAGC-3'). The resulting PCR product was cloned into the pBAD-TOPO vector (Invitrogen), and subsequently, the toxin B gene was excised with BglII/SalI (New England Biolabs) and cloned in the BamHI/SalI digested pGEX4T-1 vector (Amersham Biosciences). Proteins were expressed in E. coli-BL21 as GST fusion proteins and purified by affinity chromatography with glutathione-Sepharose 4B according to the manufacturers' instructions. GST-toxin B was released from the beads with glutathione, and proteins were analyzed by SDS-PAGE.
Cell Culture—Vero cells (kidney cells from African green monkeys, ATCC CCL 81) were cultivated in a 1:1 ratio of Ham's F-12 and Dulbecco's modified Eagle's medium (Biochrom, Berlin, Germany) supplemented with 5% fetal calf serum (PAN Systems, Aidenbach, Germany), 2 mM L-glutamate, 100 units/ml penicillin, and 100 µg/ml streptomycin in 12-well plates at 37 °C and 5% CO2. Cells were routinely trypsinized and reseeded twice a week. Astrocyte cultures were obtained from newborn Wistar rats (15). After isolation of the cortices of rat brains, the surrounding meninges were removed prior to mincing. The tissue pieces were incubated with 0.25% trypsin in phosphate-buffered saline (PBS) (8.1 mM Na2HPO4, 1.5 mM KH2PO4, 0.137 mM NaCl, and 2.7 mM KCl, pH 7.5) at 37 °C for 10 min followed by dissociation into single cells by pipetting. The cells were then suspended in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 5 mM HEPES buffer, and 0.2 mg/liter lipopolysaccharide from Salmonella typhimurium from Sigma. The cells were maintained at 37 °C in 8.6% CO2 for 10 days.
Immunocytochemistry—Cells were fixed with methanol (–20 °C) for 10 min, washed with PBS, and permeabilized with 0.1% (v/v) Triton X-100 for 30 min. Normal goat serum was used to block nonspecific binding. Thereafter, cells were incubated with the primary monoclonal mouse anti-CDB1–546 and the primary polyclonal rabbit anti-CDB547–2366 antibody or together with the monoclonal mouse Rab5 antibody (BD Biosciences). The anti-CDB547–2366 immune complexes were visualized by using a CyTm 3-conjugated F(ab')2 fragment of goat anti-rabbit IgG (Dianova, Hamburg, Germany). The anti-CDB1–546 or the Rab5 immune complexes were visualized by using an Alexa-488-conjugated F(ab')2 fragment of goat anti mouse IgG (Molecular Probes). For actin staining, cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and permeabilized with 0.1% (v/v) Triton X-100 for 30 min. Afterward, the cells were incubated with TRITC-conjugated phalloidin (Biozol, München, Germany) and washed again with PBS. Cells were visualized by using a Bio-Rad (Hercules, CA) MRC 1024, version 3.2, confocal system with a krypton-argon laser and a Zeiss (Oberkochen, Germany) Axiovert 135TV microscope. Images were obtained by using laser sharp 2.1T software and processed by using the Metamorph 6.1 software (Visitron, Munich, Germany). The intensity of CDB547–2366 in the endosomes and the intensity of CDB1–546 staining in the endosomes and cytosol were compared by using the Metamorph 6.1 software. Single z-stock micrographs were obtained with identical settings from the confocal system. The intensity of staining of five endosomes per cell was measured and compared with the intensity of five randomly chosen areas per cell in the cytosol. 10 cells for each treatment group were analyzed in this way. The average of the intensity of the endosome staining was set as 100% for each group and was compared with the cytosol group.
Cytosol/Membrane Fractionation—For separation of cytosol and membranes, cells were washed with PBS (containing Complete protease inhibitor mixture), scraped off the cell culture dish, lysed with a syringe (0.45 x 12 mm, 10 passes through the needle), and ultracentrifugated for 1 h at 165.000 x g.
Radioactive Labeling of CDB547–2366—100 µg of CDB547–2366 was labeled with 100 µCi of radioactive Na-125I by using IODO-BEADS iodination reagent according to the manufacturers' instructions.
Binding of [125I]CDB547–2366 to Vero Cells—Vero cells were incubated with 5 µg/ml [125I]CDB547–2366 either on ice or at 37 °C for various time points followed by rinsing three times with cold PBS. Cytosol and membranes were separated by ultracentrifugation as described above and subjected to SDS-PAGE. [125I]CDB547–2366 was detected by autoradiography. For competition assays, Vero cells were preincubated with 50 µg/ml unlabeled CDB547–2366 for 30 min on ice and washed with cold PBS. Cells then were incubated with 5 µg/ml radioactively labeled [125I]CDB547–2366 for 3 h on ice, washed three times with PBS, and lysed. Proteins were separated by SDS-PAGE.
Intoxication of Vero Cells with Toxin B and Glucosylation Assay with Cytosol/Membrane Fractions—Vero cells were incubated with 1 µg/ml native toxin B for 90 min and rinsed with PBS, and cytosols and membranes were separated (total volume of each fraction = 50 µl). Glucosylation assays were performed in a total volume of 30 µl with 25 µl of cytosolic or membrane fractions, 1 µg of GST-RhoA, and 10 µM [14C]UDP-glucose in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.1 mg/ml bovine serum albumin, 2 mM MgCl2, and 2 mM MnCl2) for 30 min at 37 °C. Samples were subjected to SDS-PAGE, and labeled proteins were detected with PhosphorImager. Also, fresh Vero cells were incubated with 25 µl of the cytosolic or membrane fraction of the cells intoxicated before.
Precipitation of the N-terminal Fragment of Toxin B from Intoxicated Vero Cells—Cells were incubated with recombinant GST-toxin B (10 µg/ml for detection by Western blot, 16 µg/ml for MALDI-TOF analysis) for 90 min. Cytosol and membrane fractions were prepared and incubated separately with glutathione-Sepharose 4B beads at 4 °C overnight. The precipitated proteins were eluted with glutathione-containing buffer and analyzed by SDS-PAGE and subsequent immunoblotting with a monoclonal antibody directed against CDB1–546.
Mass Spectrometry—The protein of interest was excised from the gel and destained for 1 h at 50 °C in 40% acetonitrile and 60% ammonium hydrogen carbonate (50 mM, pH 7.8) to remove stain, gel buffer, SDS, and salts. The gel plug was subsequently dried by vacuum centrifugation. Thereafter, 0.2 µg of trypsin in 20 µl of ammonium hydrogen carbonate solution (50 mM, pH 7.8) was added and digestion was carried out for 12 h at 37 °C. A saturated matrix solution of 4-hydroxy-
-cyanocinnamic acid in a 1:1 solution of acetonitrile/aqueous 0.1% trifluoroacetic acid was prepared and mixed with 2 µl of the proteolytic peptide mixture in equal parts. For internal calibration, 5 pmol of human ACTH-(18–39) clip (molecular mass = 2465.20 Da, Sigma) and 5 pmol of human angiotensin II (molecular mass = 1046.54 Da, Sigma) were added to the matrix. Using the dried-droplet method of matrix crystallization, 1 µl of the sample matrix solution was placed on the mass spectrometer target and dried at room temperature, resulting in a fine granular matrix layer. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF mass spectrometry) was performed on a Bruker Biflex mass spectrometer equipped with a nitrogen laser (
= 337 nm) to desorb and ionize the samples. Mass spectra were recorded in the reflector-positive mode in combination with delayed extraction. Measured peptide masses were assigned to peptide masses of the predicted trypsin-digested protein CDB1–546 ("peptide mapping").
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Taken together, only the enzymatic domain was translocated into the cytosol, whereas the translocation/binding domain as well as part of the holotoxin apparently remained associated with membranes, presumably in endosomes. To further test this hypothesis, we next determined whether a protein of the size of the putative enzyme domain could be detected in the cytosol of intoxicated Vero cells. For this reason, the cytosol and membranes of Vero cells, which have been intoxicated by recombinant GST-toxin B, were incubated with glutathione-Sepharose beads to capture the GST-tagged proteins. After incubation, the beads were washed and subsequently the bound proteins were eluted with glutathione and subjected to SDS-PAGE and Western blot analysis with a monoclonal antibody directed against the putative toxin B catalytic domain (amino acids 1–546). As shown in Fig. 3B, a protein band with the size of 65 kDa was detected only in the cytosolic fraction. Interestingly, this mass corresponds to the mass of the predicted enzyme component (residues 1–546, 63.5 kDa). Additionally, the precipitated protein was identified by MALDI-TOF analysis combined with peptide mapping as an N-terminal fragment of toxin B. The masses of six measured tryptic peptides could be assigned to the N-terminal region of toxin B (Table I). Replication of the MALDI-TOF experiment yielded similar results.
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Localization of CDB547–2366 in Vero Cells—Because we detected the N-terminal catalytic domain but not the middle or C-terminal part of toxin B in the cytosol, we analyzed the cellular distribution of the putative translocation/binding domain of toxin B using a recombinant fragment (residues 547–2366, CDB547–2366) expressed in Escherichia coli and subsequently radiolabeled by iodination. It is noteworthy that we used this recombinant fragment for the experiments because radiolabeling of the holotoxin by iodination resulted in an oligomeric and inactive toxin. Fig. 5A shows the binding of [125I]CDB547–2366 to Vero cells over time. Maximal binding was observed after 90 min (Fig. 5A). Preincubation of Vero cells with unlabeled CDB547–2366 for 30 min on ice markedly decreased the amount of bound [125I]CDB547–2366 (data not shown), indicating specific and saturable binding of the fragment. These results are in agreement with our recent findings that CDB547–2366 binds specifically to the surface of eukaryotic cells and forms pores under acidic conditions (13). Vero cells were then incubated with 125I-labeled CDB547–2366 either at 4 °C to prevent endocytosis of proteins or at 37 °C to allow endocytosis of CDB547–2366. This was followed by the washing of the cells to remove unbound protein. Complete cell lysates as well as cytosolic and membrane fractions were analyzed for 125I-labeled CDB547–2366. [125I]CDB547–2366 was detected only in the membrane fraction of the cells, indicating that the putative translocation/binding domain was not translocated into the cytosol and most probably remained in the endosomes (Fig. 5B).
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Intracellular Localization of the Catalytic Domain and the Binding/Translocation Domain of Toxin B—To strengthen our biochemical findings on the subcellular localization of fragments of toxin B, we investigated the distribution of the toxin B fragments in intact cells by immunocytochemistry. Astrocytes were used as an established primary cell culture, enabling easy acquisition of immunocytochemical data. Astrocytes were treated with toxin B (100 ng/ml) for 2 h and subsequently stained with antibodies raised against CDB1–546 or CDB547–2366. After 2 h, the C-terminal domain of toxin B was detected by the antibody directed against CDB547–2366 only in endosomal-like compartments. However, the N-terminal domain of toxin B was detected in the cytosol as well as in the endosomal-like compartments by the monoclonal antibody directed against CDB1–546. The results shown in Fig. 4B indicate a diffuse distribution of the toxin B catalytic domain in the cytosol. We next used the Metamorph software to measure the intensity of CDB1–546 or CDB547–2366 staining in the endosomal compartments and compared it to the respective intensity in the cytosol. The intensity in the endosomes was set as 100%. Compared with the endosomes, the intensity of CDB1–546 in the cytosol of treated cells was 21.7 ± 5.1% after 2 h and the intensity of CDB547–2366 in the cytosol was 0.6 ± 0.4% after 2 h. These findings confirm our biochemical data in which only the enzymatic domain of toxin B is translocated to the cytosol. Note that because of the destruction of the actin cytoskeleton by toxin B, the endosomes fused more easily (Fig. 6, A and B, a single astrocyte is shown in each picture). No cross-reactivity of the CDB1–546 and CDB547–2366 antibodies was detectable (data not shown). Additionally, colocalization studies of CDB547–2366 with the early endosomal marker Rab5 indicated that CDB547–2366 was located in endosomes (Fig. 6C).
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Protease Inhibitor Mixture Complete Blocks Cytotoxic Effects of Toxin B and Its Translocation across Cell Membranes—We studied processing of the toxin during the intoxication process by adding the protease inhibitor mixture Complete to the cell medium. In the presence of Complete, intoxication of Vero cells by toxin B was dramatically delayed (Fig. 7A). Complete did not decrease binding of toxin B. This was demonstrated with cells, which were incubated at 4 °C with toxin B in the presence of Complete. Subsequently, cells were washed to remove Complete. When these cells were incubated in fresh medium without Complete, the cytotoxic effects of toxin B (detected as cell rounding) were comparable to cells, which have been treated with toxin B without Complete (Fig. 7A). In contrast, Complete inhibited intoxication of Vero cells by toxin B when it was added after binding of the toxin to the cells (Fig. 7A). This finding suggests that the cleavage of toxin B occurs after its binding to the cell receptors.
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We next tested whether the proteolytic cleavage of toxin B occurs at the cell surface or in acidified endosomes. Therefore, we used a well established experimental approach, which mimics the conditions of acidic endosomes. An acidic pulse induces the direct translocation of the toxin across cell membranes into the cytosol (13). In these experiments, the inhibitor bafilomycin A1 blocks the v-type H+-ATPase of endosomes and thereby prevents acidification of endosomes and the physiological uptake pathway of toxin B. When cells were shifted for 5 min to acidic medium (pH 5.0) after binding of toxin B to the cell surface, cells rounded up within an additional hour of incubation in neutral medium at 37 °C (Fig. 7B). In contrast, no cell rounding was observed when cells were exposed for 5 min to neutral medium. We tested the effect of Complete on the direct translocation of toxin B and found that it blocked intoxication of cells after exposure of the cells to acidic medium (Fig. 7B). This finding suggests that proteolytic cleavage of toxin B is essential for the delivery of active toxin into the cytosol.
Finally, it was tested whether the separated binding/translocation domain of toxin B was able to deliver the catalytically active domain into the cytosol of target cells. We incubated Vero cells with recombinantly expressed fragment CDB547–2366 (10 µg/ml medium) together with CDB1–546 (1 µg/ml). Neither the addition of the single proteins nor the combination of both fragments in the cell medium induced any alterations in cell morphology (data not shown). This observation indicates that both toxin domains must be linked during binding to cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In this report, we demonstrate for the first time that the C. difficile toxin B is processed during cellular uptake and that the binding/translocation domain remains in early acidic endosomes, whereas the catalytically active N-terminal domain translocates from acidic endosomes into the cytosol of target cells.
Previous studies showed that the N terminus of toxin B harbors the catalytically active domain of toxin B. This recombinant toxin fragment (CDB1–546) showed the same substrate (Rho, Rac, and Cdc42) and co-substrate specificity as the holotoxin (9) and did not glucosylate additional proteins from cell lysates. Moreover, the N-terminal domain of toxin B was sufficient to induce cytotoxic effects when delivered into the cytosol of cells by microinjection. This finding was confirmed using a chimeric toxin B-anthrax lethal toxin fusion protein (16, 17). These data indicate that for cytotoxicity of toxin B, delivery of the N-terminal domain into the cytosol is sufficient and translocation of the holotoxin is not required.
Here we present evidence that the N terminus of toxin B, but not the middle or C terminus, is translocated into the cytosol during the intoxication of cells. First, we showed that the cytosolic fraction obtained from intoxicated Vero cells contained the RhoA-glucosylation activity. Second, we were able to intoxicate naive cells with the membrane fraction of intoxicated cells, but not with the cytosolic fraction that contained the Rho-modifying activity. Third, we were able to isolate a peptide from the cytosolic fraction of toxin-treated cells by GST-affinity precipitation, which cross-reacted with an antibody directed against the N terminus of toxin B. SDS-PAGE analysis revealed that this peptide migrated with an apparent molecular mass of 65 kDa. Moreover, MALDI-TOF mass spectrometry analysis of the isolated 65-kDa peptide showed that it contained exclusively amino acid sequences corresponding to the N terminus of toxin B. It is important to note that the minimal N-terminal fragment of toxin B (amino acid residues 1–546), which could be expressed as an enzymatically active protein in E. coli (9), has a molecular mass of 63.5 kDa. Therefore, we believe that the cleavage product of toxin B, which we detected in the cytosol of toxin-treated cells, corresponds very closely in size to the minimal active domain of toxin B reported recently (9). Fourth, immunostaining of toxin-treated cells with antibodies directed against the N terminus or against the middle and C terminus of toxin B revealed a diffuse distribution of the N terminus of toxin B probably in the cytosol, whereas the middle and C terminus was exclusively detected in endosome-like structures that colocalized with the early endosome marker Rab5. Finally, using protease inhibitors, we show that cleavage of toxin B is involved in the intoxication process.
Because we were not able to detect the middle or C terminus of toxin B in the cytosol of intoxicated cells, we decided to analyze the cellular distribution of these regions of the toxin by using 125I-radiolabeled versions of the recombinant proteins expressed in E. coli. The toxin fragment CDB547–2366 bound to cell membranes in an apparently specific and saturable manner. In line with our finding with full-length toxin B, the radiolabeled binding/translocation domain CDB547–2366 was exclusively detected in the membrane fraction (most probably in early endosomal vesicles) and not in the cytosol. Therefore, we suggest that this part of the toxin participates in the translocation of the catalytic domain from acidic endosomes into the cytosol; however, it does not freely enter the cytosol.
Taken together, our findings indicate that toxin B is processed during cellular uptake. The N-terminal catalytically active domain is cleaved off and is delivered into the cytosol without the binding/translocation domain. This may explain earlier reports that toxin B needs processing in endosomal vesicles. When the holotoxin was delivered into the cytosol of cells via microinjection, cytotoxic effects were observed but much higher amounts of toxin B were necessary to achieve these effects (18). From these findings, the conclusion can be drawn that toxin B is also processed in the cytosol but to a lesser extend than during "normal uptake" via acidic endosomes. So far, it is not entirely clear where the processing of toxin B occurs. Our studies show that the delivery across the cell membrane by mimicking the low pH of endosomes is blocked by protease inhibitors. Because we were not able to detect any autocatalytically cleavage of toxin B when it was treated for an extended time (1 h at 37 °C) at pH 7.5, 5.6, and 4.2 (data not shown), we suggest that a host cell protease present in the endosome and/or at the cell membrane but not an autocatalytically processing is involved in toxin B cleavage.
The binding/translocation domain of toxin B includes the C terminus of the protein, which contains units of repetitive oligopeptides (10). The middle part of toxin B contains hydrophobic domains, which are thought to be essential for membrane insertion of the protein and for the translocation across the endosomal membranes. When this translocation domain is deleted, cytotoxicity is decreased by four orders of magnitude. It was shown by Qa'Dan et al. (14) that toxin B undergoes pH-induced conformational changes and translocates across cell membranes under acidic conditions. This is in line with our recent findings that acidic pH is absolutely essential for membrane insertion and pore formation of toxin B (13). When cell-bound toxin B was exposed to an acidic impulse (5 min at pH 5.2), ion-permeable pores were formed in membranes of intact cells. Pore formation was dependent on binding of the toxin to its cellular receptor prior to acidification of the extracellular medium. Moreover, the fragment CDB547–2366 behaved similar to the holotoxin with respect to pore formation, suggesting that the binding/translocation domains are sufficient for this process (13). Although it is still an open question whether the catalytically active domain translocates directly through the pore formed by the binding/translocation domain, the present results show for the first time that only a part of toxin B is translocated into the cytosol of target cells. It will be of great interest to analyze whether the other members of the family of large clostridial cytotoxins, including C. difficile toxin A and Clostridium sordelli lethal toxin, share the same cleavage process as toxin B during their cellular uptake.
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FOOTNOTES |
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Both authors contributed equally to this work.
To whom correspondence may be addressed: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Otto-Krayer-Haus, Albertstr. 25, D-79104 Freiburg, Germany. Tel.: 49-761-2035301 (K. A.) or 49-761-2035308 (H. B.); Fax: 49-761-2035311; E-mail: klaus.aktories{at}pharmakol.uni-freiburg.de (K. A.) or Holger.Barth{at}pharmakol.uni-freiburg.de (H. B.).
1 The abbreviations used are: CDB1–546, catalytic domain of C. difficile toxin B (amino acid residues 1–546); PBS, phosphate-buffered saline; GST, glutathione S-transferase; CDB547–2366, enzymatic inactive fragment of C. difficile toxin B (amino acid residues 547–2366); MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight; TRITC, tetramethylrhodamine isothiocyanate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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