From the
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
[
The enterotoxin (ToxA)
C. difficile ToxA
(15) and C. botulinum C3 exoenzyme
(16) were purified as described.
As Thr
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
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.
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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.