©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Enterotoxin from Clostridium difficile (ToxA) Monoglucosylates the Rho Proteins(*)

Ingo Just (§) , Matthias Wilm (1), Jörg Selzer , Gundula Rex , Christoph von Eichel-Streiber (2), Matthias Mann (1), Klaus Aktories(§)

From the (1) Institut für Pharmakologie und Toxikologie der Universität des Saarlandes, D-66421 Homburg/Saar, and Institut für Pharmakologie und Toxikologie, D-79104 Freiburg, European Molecular Biology Laboratories, D-69012 Heidelberg, and (2) Institut für Medizinische Mikrobiologie der Johannes-Gutenberg-Universität, D-55101 Mainz, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The enterotoxin from Clostridium difficile (ToxA) is one of the causative agents of the antibiotic-associated pseudomembranous colitis. In cultured monolayer cells ToxA exhibits cytotoxic activity to induce disassembly of the actin cytoskeleton, which is accompanied by morphological changes. ToxA-induced depolymerization of actin filaments is correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins (Just, I., Selzer, J., von Eichel-Streiber, C., and Aktories, K.(1995) J. Clin. Invest. 95, 1026-1031). Here we report on the identification of the ToxA-induced modification of Rho. Applying electrospray mass spectrometry, the mass of the modification was determined as 162 Da, which is consistent with the incorporation of a hexose into Rho. From several hexoses tested UDP-glucose selectively served as cosubstrate for ToxA-catalyzed modification. The acceptor amino acid of glucosylation was identified from a Lys-C-generated peptide by tandem mass spectrometry as Thr-37. Mutation of Thr-37 to Ala completely abolished glucosylation. The members of the Rho family (RhoA, Rac1, and Cdc42Hs) were substrates for ToxA, whereas H-Ras, Rab5, and Arf1 were not glucosylated. ToxA-catalyzed glucosylation of lysates from ToxA-pretreated rat basophilic leukemia (RBL) cells resulted in a decreased incorporation of [C]glucose, indicating previous glucosylation in the intact cell. Glucosylation of the Rho subtype proteins appears to be the molecular mechanism by which C. difficile ToxA mediates its cytotoxic effects on cells.


INTRODUCTION

The enterotoxin (ToxA)() and the cytotoxin (ToxB) secreted by pathogenic Clostridium difficile strains are the virulence factors causing antibiotic-associated diarrhea and the potentially fatal form, pseudomembranous colitis (1, 2, 3) . Genetic analysis demonstrated an overall 63% homology of the amino acids of both toxins (4) . Nevertheless the activities of both toxins differ. In the hamster model ToxA induces the typical clinical picture resembling pseudomembranous colitis, whereas ToxB does not cause any kind of disease (5) . However, in cultured cell lines both toxins exhibit potent cytotoxic activities, which are characterized by a disaggregation of the actin cytoskeleton followed by rounding of the cells (1, 6) . Recently, we showed that ToxA (7) as well as ToxB (8, 9) act on the low molecular mass GTPase Rho to inhibit the C3-catalyzed ADP-ribosylation. Rho proteins, which are involved in the formation of focal adhesions and stress fibers (10) , are the substrates of Clostridium botulinum C3 ADP-ribosyltransferase (11, 12) . C3-catalyzed ADP-ribosylation of Rho at Asn-41 renders the small GTPase inactive, resulting in depolymerization of the actin filaments (13, 14) . Because C. difficile toxins induced a decrease in Rho-ADP-ribosylation, which correlated with the toxin-caused depolymerization of actin filaments, it was suggested that ToxA and ToxB act on the regulatory Rho proteins. Several findings indicate a covalent modification of Rho (7, 8). Toxin-induced modification of recombinant Rho depends on the presence of a consumable cytosolic factor, which is heat stable, nonproteinacious, and exhibits a molecular mass between 500 and 3000 Da (7) . Here we report on the identification of the ToxA-induced modification by applying electrospray mass spectrometry.


EXPERIMENTAL PROCEDURES

C. difficile ToxA (15) and C. botulinum C3 exoenzyme (16) were purified as described. C-Labeled UDP-galactose, UDP-N-acetylglucosamine, UDP-glucose, and [P]NAD were purchased from DuPont NEN, Dreieich, Germany.

Recombinant Proteins

Recombinant proteins (RhoA, Rac1, Cdc42Hs, and the mutant RhoA) were purified from Escherichia coli transformed with a plasmid expressing GST-RhoA (RhoA, Rac1, and Cdc42Hs) fusion protein. RhoA, RhoA, and Rac1 plasmids were kindly donated by Dr. A. Hall (MRC Laboratory for Molecular Cell Biology, London) and Cdc42Hs by Dr. M. Aepfelbacher (Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Munich, Germany). The glutathione fusion proteins were isolated by affinity purification with glutathione-Sepharose beads (Pharmacia, Germany) followed by proteolytic cleavage with thrombin (100 µg/ml for 30 min at 22 °C). Thrombin was removed by precipitation with benzamidine-Sepharose beads (Pharmacia, Germany). Rab5 was kindly donated by Dr. M. Zerial (EMBL, Heidelberg, Germany) and Arf1 by Dr. M. Schmidt (Institut für Pharmakologie, Essen, Germany).

Cell Culture

Rat basophilic leukemia (RBL) cells were cultured as described (7) . For intoxication the cells were incubated with ToxA (500 ng/ml) for 4 h followed by lysis as described below.

Preparation of the Cytosolic Fractions

RBL cells were rinsed with ice-cold phosphate-buffered saline and were then mechanically removed from the dishes in the presence of lysis buffer (2 mM MgCl, 1 mM EGTA, 1 mM dithiothreitol, 0.3 mM phenylmethylsulfonyl fluoride, 30 µg/ml leupeptin, 50 mM triethanolamine HCl, pH 7.5), sonicated 5 times on ice, and centrifuged (10 min at 2,000 g). The supernatant was centrifuged for 60 min at 100,000 g to give the cytosolic fraction. For preparation of the cytosolic subfraction, the cytosol was first incubated for 15 min at 95 min and then centrifuged (15 min at 30,000 g). Thereafter, the supernatant was passed through a filter membrane (ultrafiltration membranes YM3, 3,000-Da cutoff, Amicon Corp.).

ADP-ribosylation Reaction

Recombinant RhoA (50 µg/ml) was incubated with ToxA (10 µg/ml) in the presence of either cytosol from RBL cells, a cytosolic fraction, or various UDP-hexoses (as indicated) (50 µM) for 45 min at 37 °C. Thereafter, the samples were incubated for 15 min at 37 °C in a buffer (3 mM MgCl, 1 mM dithiothreitol, 50 mM triethanolamine HCl, pH 7.5) containing 10 µM [P]NAD (0.5 µCi) and C3 exoenzyme (0.5 µg/ml) in a total volume of 50 µl. The reaction was terminated by addition of Laemmli sample buffer.

Glucosylation Reaction

The glucosylation reaction was performed in a medium containing 30 µM UDP-[C]glucose (50 nCi), 3 mM MgCl, 0.3 mM GDP, 150 mM KCl, 50 mM triethanolamine HCl, pH 7.5, 10 µg/ml ToxA, and the indicated recombinant GTPases (50 µg/ml) or cell lysate (2 mg/ml protein) in a total volume of 25 µl. The incubation was performed at 37 °C for 45 min or as indicated. For the differential glucosylation 30 µM UDP-[C]glucose (500 nCi) was used. After the incubation, Laemmli sample buffer was added and the proteins were separated on 12.5% SDS-PAGE.

Gel Electrophoresis

Proteins were separated by 12.5% SDS gel electrophoresis according to Ref. 17. The gels were analyzed by the PhosphorImager SF from Molecular Dynamics.

Proteolytic Peptide Map

RhoA (100 µg) was treated without or with ToxA (10 µg/ml) in the presence of the cytosolic subfraction (<3,000 Da) for 90 min at 37 °C and was then separated by HPLC chromatography on a Sephasil RP-C18 column (Pharmacia). The proteins were eluted with a linear gradient from 0.1% trifluoroacetic acid in water to 70% acetonitrile and 0.1% trifluoroacetic acid. After freeze drying, RhoA was dissolved in 200 µl of a medium containing 1 M urea, 1 mM EDTA, and 25 mM triethanolamine HCl, pH 8.5 and incubated with Lys-C (10 µg/ml, Boehringer Mannheim, Germany) for 16 h at 30 °C. The digestion was stopped by addition of 0.1% trifluoroacetic acid, and the peptides were separated on the Sephasil RP-C18 column with the same gradient as described above.

Mass Spectrometry

For mass spectrometric analyses RhoA in its unmodified and modified form was purified with HPLC as described above. The masses of control and modified RhoA protein were measured on a electrospray triple quadrupole instrument (API III from Sciex-Perkin Elmer, Ontario, Canada) equipped with a modified electrospray source (18). One peptide from Lys-C digestion showed an altered retention time for the modified RhoA in HPLC separation. For identification it was subjected to tandem mass spectrometry in its modified and unmodified form. To localize the acceptor amino acid, the peptide was subdigested with pepsin. The resulting petide mixture was analyzed with mass spectrometry. A modified peptide in the digest of the original modified RhoA petide was selected for fragmentation. Its smallest fragment, which still contained the modification, identified the acceptor amino acid.

Glycohydrolase Activity

ToxA (250 µg/ml) was incubated in buffer (10 µl) containing 50 mM triethanolamine HCl, pH 7.0, 150 mM KCl, and 25 µM UDP-[C]glucose (80 nCi) for the indicated times at 37 °C. Samples were spotted on polyethyleneimine cellulose and run with 0.25 M LiCl. Evaluation of the TLC was performed with the PhosphorImager SF (Molecular Dynamics). For testing other hexoses, C-labeled UDP-galactose and UDP-N-acetylglucosamine were used.


RESULTS AND DISCUSSION

Identification of the Modification

Recombinant RhoA, which had been treated with ToxA in the presence of the cytosolic subfraction (<3,000 Da), was separated by HPLC and, subsequently, subjected to electrospray mass spectrometry. The mass of the complete protein was 162 ± 0.8 Da higher than the unmodified protein (not shown). These data are consistent with a monoglycosylation of RhoA (180-Da hexose minus 18 Da of released HO). To identify the type of sugar incorporated into Rho several hexoses were tested. Because the molecular weight of the cosubstrate is higher than 500 (7) , simple monohexoses (M 180) were ruled out and activated forms of hexoses (UDP- and GDP-hexoses, M 566) were tested (Fig. 1). Only in the presence of UDP-glucose, ToxA was able to inhibit the C3-catalyzed ADP-ribosylation of RhoA. Applying UDP-[C]glucose, ToxA catalyzed the incorporation of [C]glucose into RhoA (Fig. 2A). Hydrolysis of UDP-glucose to glucose 1-phosphate by treatment with phosphodiesterase or oxidation to UDP-glucuronic acid by dehydrogenase completely abolished incorporation of glucose, indicating that UDP-glucose is the actual cosubstrate (Fig. 2A). Monoglucosylation of RhoA depended on the native structure of both the substrate RhoA and the transferase ToxA because denaturation of either completely inhibited incorporation of glucose (not shown). Glucosylation of Rho is significantly enhanced in the presence of 150 mM KCl whereas 150 mM NaCl did not increase incorporation (not shown). The time course of glucosylation showed that incorporation of glucose did not exceed incorporation of 1 mol of glucose/mol of Rho (Fig. 2C), consistent with the findings of the mass spectrometry.


Figure 1: Influence of various hexoses on the C. difficile ToxA-induced decrease in C3-catalyzed ADP-ribosylation of Rho. Recombinant RhoA (50 µg/ml) was incubated with ToxA (10 µg/ml) in the presence of UDP-hexoses (50 µM) for 45 min at 37 °C. Thereafter, the samples were subjected to ADP-ribosylation with C3 in the presence of [P]NAD (10 µM, 0.5 µCi). PhosphorImager data from SDS-PAGE are shown. UDPGlc, UDP-glucose; UDPGal, UDP-galactose; UDPMan, UDP-mannose; UDPGlcA, UDP-glucuronic acid; UDPGlcNAc, UDP-N-acetylglucosamine; GDPMan, GDP-mannose; ADPGlc, ADP-glucose; Glc, glucose.




Figure 2: ToxA-catalyzed incorporation of glucose into Rho proteins. A, prior to the glucosylation reaction, 30 µM UDP-[C]glucose (50 nCi) was treated with either 50 µg/ml phosphodiesterase from Crotalus durissus (lane2) or with 500 µg/ml UDP-glucose dehydrogenase and 100 µM NAD (lane3) for 30 min at 30 °C followed by incubation for 15 min at 95 °C to inactivate the UDP-glucose-degrading enzymes. Lane1, control. Thereafter, the glucosylation reaction (25 µl) was started by the addition of RhoA and ToxA. B, substrate specificity of ToxA. The indicated recombinant low molecular mass GTPases (50 µg/ml, dissolved in 3 mM MgCl, 0.3 mM GDP, 150 mM KCl, 50 mM triethanolamine HCl, pH 7.5) or lysate from RBL cells were incubated with 10 µg/ml ToxA and 30 µM UDP-[C]glucose (50 nCi) in a total volume of 25 µl for 45 min at 37 °C. C, time course of ToxA-catalyzed incorporation of glucose in the Rho subtype proteins. The glucosylation reaction of RhoA (), Rac1 (), and Cdc42Hs () (each 50 µg/ml) was performed as described in B. After the indicated times, samples were separated by SDS-PAGE (12.5%). From the PhosphorImager data incorporation of glucose was calculated (mol of glucose/mol of G-protein). D, glucosylation of RhoA. RhoA (50 µg/ml, dissolved in 3 mM MgCl, 0.3 mM GDP, 150 mM KCl, 50 mM triethanolamine HCl, pH 7.5) was either incubated with C3 (0.5 µg/ml) and [P]NAD (10 µM) (lane1) or with ToxA (10 µg/ml) and UDP-[C]glucose (30 µM) (lane2) for 45 min at 37 °C. PhosphorImager data from SDS-PAGE are shown.



In cell-free systems, ToxA exhibits its enzymatic effects on Rho only at 50-100 times higher concentration than usually applied to intact cells. This finding indicates that ToxA is most likely activated intracellularly but not in the cell-free assay. Because ToxA is taken up via receptor-mediated endocytosis (1, 6, 19) , it is most likely that ToxA is activated in the postendosomes comparable with the reported mechanisms of diphtheria, pertussis, and cholera toxins (20) to elicit its transferase activity in the cytosol.

Glycohydrolase Activity

Bacterial ADP-ribosyltransferases catalyze incorporation of ADP-ribose into eukaryotic target proteins by glycosidic bonds, but in the absence of target proteins, they exhibit NAD glycohydrolase activity and cleave NAD to ADP-ribose and nicotinamide. We tested whether ToxA exhibits a comparable activity to cleave UDP-glucose into glucose and UDP. As shown in Fig. 3, ToxA cleaved UDP-glucose in a time-dependent manner. Other UDP-hexoses (UDP-galactose, UDP-N-acetylglucosamine) were not hydrolyzed. These data indicate that ToxA exhibits both transferase and glycohydrolase activity.


Figure 3: UDP-glucose hydrolase activity of ToxA. 250 µg/ml ToxA was incubated with 25 µM UDP-[C]glucose (80 nCi) in a buffer (10 µl) containing 150 mM KCl and triethanolamine HCl, pH 7.0, for the indicated times. Samples were analyzed by TLC as described under ``Experimental Procedures.'' The amount of UDP-glucose hydrolyzed (mol/mol of ToxA) was evaluated from TLC by the PhosphorImager.



Acceptor Amino Acid

To identify the acceptor amino acid of glucosylation, modified and unmodified RhoA was digested with endopeptidase Lys-C. The peptides of both digests were separated by HPLC on a Sephasil RP-C18 column. In the separation of the peptides from modified RhoA one additional peak appeared. Mass spectrometry of this HPLC fraction showed a mass of 2935.5 Da, which was 162 Da higher than that of a peptide fragment corresponding to D28-K51 (2774.0 Da). The assignment to peptide D28-K51 was confirmed by sequencing of the unmodified (Fig. 4A) and the modified peptide (Fig. 4B) with electrospray tandem mass spectrometry (ES MS/MS) using a NanoES source (18) . Both MS/MS spectra revealed the same peptide sequence of the residues Asp to Lys of the RhoA protein. The modification was localized within the N-terminal 16 residues (Fig. 4B). For final determination of the site of modification the modified peptide (Asp-Lys) was subdigested with pepsin, and the digest was analyzed directly by ES MS/MS (Fig. 4C). The peptide Asp-Glu in its modified form was identified in the digest by its mass (1730.9 Da) and by its sequence. ES MS/MS shows that the Thr residue carries the modification (Fig. 4C).


Figure 4: Localization of the modification by ES MS/MS. Sequencing was performed on a Sciex API III triple quadrupole mass spectrometer. A, measurement of the unmodified peptide. The measured mass (2773.6 Da, average mass) is consistent with peptide D28-K51 (expected monoisotopic molecular mass, 2777.0 Da). Sequencing by ES MS/MS confirmed the sequence by a series of B and Y" ions (fragments with charge retention on the N- and C-terminal part of the molecule, respectively) (25). In the figure, the Y" ion series covering C-terminal amino acids of the peptide sequence are marked. B, measurement of the modified peptide. The measured mass (2935.5Da) is consistent with a modification of 162 Da. The same sequence-specific fragment ions as in panelA were found. This observation shows that the bond between the conjugate and the peptide backbone is less stable than the backbone bond. Nevertheless, comparison of the fragment spectra of the modified and the unmodified peptide shows an additional fragment in the spectrum of the modified peptide 162 Da larger in mass than the Y" ion. This localizes the modification to the C-terminal 16 amino acids in the peptide sequence. C, MS/MS analysis of a peptide from a pepsin subdigest of the modified peptide Asp-Lys. A peptide of mass 1730.9 Da was sequenced and identified as peptide Asp-Glu in the modified form. A series of fragment ions with accompanying +162 Da peaks was observed. B is the lowest fragment ion for which the modification is observed localizing the modification to Thr.



As can be deduced from the crystal structure of H-Ras, Thr (Thr in Rho) is located in the GTP-binding and hydrolyzing domain (G-2) and participates in the coordination of Mg, which is the ligand for - and -phosphates of GTP (21) . In the GDP-bound state, loop L2 moves and Thr (Thr in Rho) is now accessible (22) . Consistent with this model is our finding that GTP-bound Rho is a poor substrate compared with the GDP-bound form (data not shown). To assess the specificity of the acceptor amino acid Thr, we tested whether the exchange of Thr in position 37 to Ala abolishes glucosylation (Fig. 2D). RhoA was still ADP-ribosylated by C3 exoenzyme indicating no gross alteration of the protein structure in this mutant form. However, glucosylation of RhoA by ToxA was completely blocked. There are no alternative acceptor amino acid residues that are modified when the acceptor Thr is eliminated by mutation. These findings prove that ToxA selectively catalyzes attachment of glucose to Thr in Rho.

As Thr is located in the putative effector domain of Rho (23), the hydrophilic glucose group in this domain might interfere with the interaction of activated Rho protein with the effector protein. This notion is in agreement with the observed disassembly of actin filaments, resulting from a blockade of the signal transduction due to Rho modification.

Substrate Specificity

Incubation of RBL cell lysates with ToxA in the presence of UDP-[C]glucose resulted in labeling of cellular proteins with a molecular mass in the range of 20-24 kDa. No additional proteins were labeled. These findings were observed with several cell lines (NIH3T3 fibroblasts and PtK2 kidney epithelial cells). To identify the target proteins of glucosylation, several recombinant low molecular mass GTP-binding proteins were tested. As shown in Fig. 2B, members of the Rho subfamily (RhoA, Rac1, and Cdc42Hs) were glucosylated, whereas the other Ras-related GTPases (H-Ras, Rab5, and Arf1) were not substrates for ToxA. Because all the members of the Ras superfamily share a threonine in domain G-2 at the same position (24) , it seems that structural requirements only present in Rho subtype proteins are essential for modification by ToxA. As shown for RhoA, incorporation of glucose did not exceed 1 mol/mol of Rac1 and Cdc42Hs, respectively, consistent with monoglucosylation of these proteins (Fig. 2C). The Rho subtype proteins seem to be the exclusive target proteins of ToxA.

In Vivo Glucosylation

To test whether Rho proteins were glucosylated in intact cells during the intoxication process, the method of differential glucosylation was applied. Assuming that in intact cells ToxA catalyzes glucosylation of Rho subtype proteins, subsequent C glucosylation of the lysates should result in a decreased incorporation of [C]glucose into the GTPases. As shown in Fig. 5pretreatment of cells with ToxA decreased glucosylation of two substrate proteins (doubleband). The upperband of the doublet was identified as RhoA and the lower one as Cdc42Hs by immunoblot of two-dimensional separated cell lysates C-glucosylated by ToxA (data not shown). As expected, C3-catalyzed [P]ADP-ribosylation of Rho was also inhibited (Fig. 5). These findings indicate that ToxA most likely catalyzes glucosylation of the Rho subtype proteins not only in cell lysates but also in intact cells.


Figure 5: In vivo glucosylation. RBL cells were treated with ToxA (500 ng/ml) until all cells were rounded. Lysates from control and ToxA-treated cells were either glucosylated or ADP-ribosylated. The lysates were incubated with 10 µg/ml ToxA and 30 µM UDP-[C]glucose (500 nCi) in 50 mM triethanolamine HCl (pH 7.5) for 45 min at 37 °C (25 µl, total volume). ADP-ribosylation of the lysates was performed with 0.5 µg/ml C3 and 10 µM [P]NAD (0.5 µCi) in triethanolamine HCl (50 mM, pH 7.5) for 45 min at 37 °C (25 µl, total volume). After incubation the proteins were separated by 12.5% SDS-PAGE. PhosphorImager data from SDS-PAGE are shown.



In summary, we demonstrate that C. difficile ToxA catalyzes the O-glucosylation of Rho subtype proteins in Thr. Substrate specificity of ToxA is restricted to the Rho family (Rho, Rac, and Cdc42Hs) whereas other subtypes of the Ras superfamily are not substrates. Because ToxA-catalyzed glucosylation of Rho proteins occurs in intact cells we suggest that monoglucosylation is the molecular mechanism by which ToxA mediates its cytotoxicity and probably its in vivo enterotoxicity. The identified modification of GTP-binding proteins is a novel mode of action by which bacterial toxins directly attack intracellular regulatory proteins of eukaryotic cells.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft (Project Ju231/3-1, SFB246 (B10), and Project Ei206/3-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-6841-166416; Fax: 49-6841-166402.

The abbreviations used are: ToxA, toxin A; C3, C. botulinum ADP-ribosyltransferase C3; RBL, rat basophilic leukemia; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; ES MS/MS, electrospray tandem mass spectrometry.


ACKNOWLEDGEMENTS

We gratefully acknowledge the excellent technical assistance of Monika Lerner and Gabriele Kiefer. We would like to thank Dr. M. Zerial (EMBL, Heidelberg, Germany) for the kind gift of Rab5, Dr. M. Aepfelbacher (Munich, Germany) for Cdc42Hs, Dr. M. Schmidt (Essen, Germany) for Arf1, and Dr. A. Hall (London, UK) for plasmids.


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