From the Microbiology and Tumorbiology Center,
Karolinska Institutet, S-171 77 Stockholm, Sweden, the
§ Unidad de Microscopía Electrónica,
Universidad de Costa Rica, San José, Costa Rica, the
Nobel
Institute of Neurophysiology, Department of Neuroscience, Karolinska
Institutet, S-171 77 Stockholm, and the ** Institut für
Medizinische Mikrobiologie und Hygiene, Verfügungsgebaude
für Forschung und Entwicklung, Obere Zahlbacher Strasse 63, Johannes Gutenberg-Universität Mainz, 55101 Mainz, Germany
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ABSTRACT |
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The large clostridial cytotoxins (LCTs)
constitute a group of high molecular weight clostridial cytotoxins that
inactivate cellular small GTP-binding proteins. We demonstrate that a
novel LCT (TcdB-1470) from Clostridium difficile strain
1470 is a functional hybrid between "reference" TcdB-10463 and
Clostridium sordellii TcsL-1522. It bound to the same
specific receptor as TcdB-10463 but glucosylated the same GTP-binding
proteins as TcsL-1522. All three toxins had equal enzymatic potencies
but were equally cytotoxic only when microinjected. When applied
extracellularly TcdB-1470 and TcdB-10463 were considerably more potent
cytotoxins than TcsL-1522. The small GTP-binding protein R-Ras was
identified as a target for TcdB-1470 and also for TcsL-1522 but not for
TcdB-10463. R-Ras is known to control integrin-extracellular matrix
interactions from inside the cell. Its glucosylation may be a major
determinant for the cell rounding and detachment induced by the two
R-Ras-attacking toxins. In contrast, fibroblasts treated with
TcdB-10463 were arborized and remained attached, with phosphotyrosine
containing structures located at the cell-to-cell contacts and
Bacteria of the genus Clostridium produce a wide
variety of toxins displaying different enzymatic activities. Among
them, the large clostridial
cytotoxins
(LCTs)1 comprise the largest
bacterial protein toxins known, ranging in size from 250 to 308 kDa
(1). So far, five toxins belong to this group: the Clostridium
difficile toxins A and B (TcdA and TcdB), the Clostridium
sordellii hemorrhagic and lethal toxins (TcsH and TcsL) and
A new member of the LCT family was recently identified in a sero group
F strain of C. difficile and named TcdB-1470 (the number indicates the strain from which it was isolated) (8, 9). The amino acid
sequence of this toxin shows an overall identity of 93 and 75% with
TcdB-10463 ("reference" TcdB) and TcsL-1522, respectively. Despite
the high overall identity of TcdB-1470 with TcdB-10463, it is only 79%
in the first 560 amino acids comprising the catalytic domain (Fig.
1). On the other hand, the identity is
99% in the carboxyl-terminal domain where the receptor-binding region
is located (10). In contrast to this polarized identity of TcdB-1470
with TcdB-10463, the sequence comparison with TcsL reflects the overall
identity in both domains (Fig. 1). In this work we have characterized
TcdB-1470, in terms of its cell surface binding, cytotoxic and
enzymatic potencies, substrate pattern, and type of CPE. Comparing
these parameters with those of TcdB-10463 and TcsL-1522 established the
hybrid character of TcdB-1470. Importantly, R-Ras was identified as a
major substrate for TcdB-1470 and TcsL-1522. The significance of this
novel substrate for development of the TcsL-like CPE is discussed.
Materials--
TcdB-10463, TcsL-1522, and TcdB-1470 were
prepared as described (11). The toxin preparations showed >95% purity
as assessed by SDS-PAGE. UDP-[14C]Glc (specific activity,
318 mCi/mmol) was from NEN Life Science Products and
N-succinimidyl 3-(4-hydroxy,
5[125I]iodophenyl)propionate (specific
activity, 2000 Ci/mmol) from Amersham Pharmacia Biotech. Anti-vinculin
antibody was from Sigma, and anti-ERK1 antibody and
anti- Amino Acid Sequence Analysis--
Binary alignment was performed
by the SIM software (ExPASy Molecular Biology Server) (12) after
translation of DNA sequences with the following accession numbers:
TcdB-10463, X53138; TcsL-1522, X82638; and TcdB-1470, Z23277.
Cell Culture and Preparation of Lysates--
Chinese hamster
lung fibroblasts (Don cells, CCL-16; American Type Culture Collection,
Rockville, MD) were cultured in Eagle's minimum essential medium
supplemented with 10% fetal bovine serum, 5 mM
L-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml) (Life Technologies, Inc.). Mouse Swiss 3T3 fibroblasts were obtained from Dr. Crister Höög (Department of Cell and Molecular Biology, Karolinska Institutet, Sweden) and cultured in
Dulbecco's medium supplemented as above. Both cell lines were incubated at 37 °C in a humid atmosphere containing 5%
CO2. Cytotoxic activities were titrated on cells cultivated
to 90% confluence in 96-well plates. The initial toxin concentration
was 2 µg/ml, and subsequent 1:10 dilutions were made. Cytotoxicity
was scored microscopically, and the results were expressed as
percentages of affected cells. Cell lysates were prepared as described
previously (13). Briefly, confluent cells in 75-cm2 flasks
were rinsed, mechanically removed, and washed twice with ice-cold
Hanks' balanced salt solution. Cell pellets were resuspended in 200 µl of lysis buffer (50 mM triethanolamine, 150 mM KCl, 2 mM MgCl2, 0.5 mM GDP, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, pH 7.8) and
sonicated five times for 5 s. After centrifugation (14,000 g, 3 min), the supernatant was used as postnuclear cell lysate. The amount
of protein in lysates was determined by Bio-Rad protein assay with
bovine serum albumin as a standard.
Scanning Electron Microscopy--
Subconfluent Don and Swiss 3T3
cells were treated with TcdB-10463 (50 ng/ml for 1 h), TcdB-1470
(50 ng/ml for 1 h), and TcsL-1522 (600 ng/ml for 6 h).
Control cells were not treated. After treatment the cells were
chemically immobilized with 2% glutaraldehyde in 0.1 M
phosphate buffer, at 4 °C for 2 h and post-fixed in 1% osmium tetroxide (Agar Scientific Lta., Cambridge, UK) in phosphate buffer for
30 min. The fixed cells were dehydrated in increasing concentrations of
ethanol and frozen in tert-butyl alcohol (T. J. Baker,
Inc., Phillisburg, NJ). Cells were freeze-dried by sublimation and
coated with 20 nm of gold/palladium (10 mAmp, 2 min). Cells were
observed and photographed with a S-570 (Hitachi, Tokyo, Japan) Scanning Electron Microscope operating at 15 kV.
Microinjection Experiments--
Don cells were cultivated on
13-mm slides for 48 h. Toxin antiserum (1:100) was added to the
medium to neutralize any toxin molecules leaking from the injection
needle. Goat anti-TcdB-10463 was used for both TcdB-10463 and TcdB-1470
injections. Mouse anti-TcsL was used for TcsL-1522 injections.
Semiconfluent cells were microinjected (Automatic Eppendorf
Microinjector) with TcdB-10463, TcsL-1522, or TcdB-1470 diluted in 0.01 M phosphate-buffered saline (PBS) at the concentrations
defined in figure legends. The solution contained 2% fluorescein
dextran to localize microinjected cells. After microinjection, cells
were incubated 2 h at 37 °C, washed with PBS, and fixed with
3.7% paraformaldehyde (Fisher). The CPE of microinjected cells was
determined by phase contrast microscopy. Three different fields (400×)
were analyzed per experiment, and the activity of the toxins was
expressed as the percentage of affected cells/microinjected
(fluorescent) cells.
Glucosyltransferase Activity--
5 µl of
UDP-[14C]Glc dissolved in ethanol were vacuum dried, and
10 µl of cell lysate (5 mg protein/ml) or 10 µl of lysis buffer containing recombinant Rho, Rac, Cdc42, Rap1, Rap2, Ras, Ral, or R-Ras
(1-3 µg) were added. The final concentration of
UDP-[14C]Glc was 30 µM. The mixtures were
incubated 1 h at 37 °C with TcdB-10463, TcsL-1522, or TcdB-1470
at the concentrations defined in figure legends. The reaction was
stopped by heating at 95 °C in sample buffer. Proteins were
separated by 12.5% SDS-PAGE (14) or by two-dimensional gel
electrophoresis (15). Radiolabeled bands or spots were detected by
PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). The
intensity of the bands was calculated using the ImageQuant software
(Molecular Dynamics). The result was expressed as a percentage of the
labeling obtained with the highest toxin concentration (5 µg/ml). For
differential glucosylation experiments, cells were treated in
vivo with the indicated toxins until 100% CPE was achieved. Cell
lysates were then prepared and processed for glucosylation as described above.
Specific Binding and Competition with
125I-TcdB-10463--
TcdB-10463 was labeled with
125I as described previously (16). Total binding of
125I-TcdB-10463 was determined by the addition of ice-cold
125I-TcdB-10463 (2 µg/ml) to precooled (15 min at
4 °C) Don cells in 96-well plates. After 2 h of incubation at
4 °C, cells were washed three times with PBS and lysed with 50 µl
of 1% Nonidet P-40, and the radioactivity was measured in an automated
EGF-induced ERK Phosphorylation--
Experiments examining the
effects of LCTs on EGF-stimulated phosphorylation and activation of ERK
were performed as follows. Confluent Swiss 3T3 cells were serum-starved
overnight in medium with 0.1% fetal bovine serum. These cells were
treated with TcdB-10463 (50 ng/ml) or TcdB-1470 (50 ng/ml) for 30 min
or with TcsL-1522 (600 ng/ml) for 6 h. When 100% of the cells
showed a clear CPE, they were exposed to EGF (100 ng/ml) for 5 min.
This step was omitted in control cells. Then cells were lysed with
Laemmli sample buffer (14), and 20 µg of total protein/lane were
loaded. Proteins were resolved by SDS-PAGE and electrotransferred to
nitrocellulose membranes, which were then incubated with monoclonal
anti-ERK1 antibodies (1:250). Finally, blots were developed with a
Chemiluminiscence Western blotting kit (Boehringer Mannheim) using a
horseradish peroxidase-labeled secondary antibody. The cross-reaction
of the anti-ERK1 antibody with ERK2 (44 kDa) has been documented in rat cells (catalog of Transduction Laboratories) and was also observed in a
previous study (5) with 3T3 fibroblasts.
Measurement of Toxin-induced Cell Detachment--
Triplicate
cultures of Swiss 3T3 fibroblasts in 24-well plates were incubated with
TcdB-10463 or TcdB-1470 (50 ng/ml) 1 h at 37 °C. Control cells
were not treated. Then the cells were washed three times with PBS to
remove detached cells. Remaining cells were collected with 0.1%
trypsin (10 min at 37 °C), suspended in 200 µl of PBS, recovered
by centrifugation (3000 × g for 10 min), and lysed in
30 µl of 1% SDS. The protein concentration in lysates was taken as a
measure of the number of cells remaining attached after toxin treatment.
Immunofluorescence Experiments--
Swiss 3T3 cells on 13-mm
glass coverslips were left untreated (control cells) or treated with 50 ng/ml of TcdB-10463 or TcdB-1470 for 30 min at 37 °C. Cells were
fixed with 3.7% paraformaldehyde in PBS, permeabilized with 0.5%
Triton X-100 in PBS and incubated 1 h at room temperature with the
antibodies specified in figure legends. Then the cells were washed
three times in PBS/0.5% Triton X-100 and incubated for 30 min at
22 °C with rhodamine-conjugated antibody, followed by three
washes with PBS. The cells were analyzed at 600 × using a
confocal microscope (Molecular Dynamics).
Cytopathic Effect and Cell Detachment Induced by TcdB-1470--
As
viewed by scanning electron microscopy, TcdB-1470 induced 3T3 cell
rounding with a mitotic-like morphology, indistinguishable from that
induced by TcsL-1522 (Fig. 2). All
cell-to-cell contacts disappeared after treatment with these toxins. In
contrast, TcdB-10463 induced the characteristic arborizing effect (Fig.
2), and cell-to-cell contacts were retained. Similar CPEs were induced
in Don cells (data not shown). Light microscopy indicated that
fibroblasts treated with TcdB-1470 or TcsL-1522 detached easily from
the plastic substrate, whereas cells treated with TcdB-10463 remained
attached. Indeed, the protein concentration in lysates of
TcdB-10463-treated cells (799+-21 µg/ml) was equal to control cells
(765+-70 µg/ml), whereas it was greatly diminished in cultures
exposed to TcdB-1470 (158+-51 µg/ml), reflecting a massive detachment
of cells.
Cytotoxic and Enzymatic Potency of TcdB-1470--
The cytotoxic
potency of extracellularly applied TcdB-1470 was determined in Don
cells by titration and compared with the potencies of TcdB-10463 and
TcsL-1522. TcdB-1470 was almost as strongly cytotoxic as TcdB-10463,
whereas TcsL-1522 showed a weak potency (Fig.
3a), in agreement with
previous reports on other cell lines (17, 18). However, when the
cytotoxic potencies were determined after intracellular application by
microinjection, all three toxins showed similar activities (Fig.
3b). In the presence of UDP-[14C]Glc TcdB-1470
modified 20-30-kDa target proteins in cell lysates confirming its
glucosyltransferase character. The activities of the toxins were
compared in terms of the glucosyltransferase reaction. All three toxins
exhibited similar enzymatic potency (see Fig. 5a). This
result, together with the similar cytotoxic potencies observed after
microinjection (Fig. 3b), indicates that the low cytotoxicity of TcsL-1522 is not a matter of a low enzymatic activity but rather due to an inefficient binding and/or internalization into
the cell.
TcdB-10463 and TcdB-1470 Share a Specific Receptor on the Cell
Surface--
To test the possibility that TcdB-1470 uses the same
receptor as TcdB-10463, competition experiments were performed.
Pretreatment with TcdB-1470 reduced the binding of
125I-TcdB-10463 to Don cell surfaces at 4 °C, almost to
the same extent as cold TcdB-10463 did (Fig.
4), suggesting that TcdB-10463 and
TcdB-1470 share the same receptor on these cells. In contrast, TcsL-1522 did not compete with 125I-TcdB-10463 (Fig.
4) and thus binds to a different receptor on Don cell
surfaces.
TcdB-1470 and TcsL-1522 Glucosylate Similar Cellular
Substrates--
The substrate pattern of TcdB-1470, as analyzed by
SDS-PAGE, was very similar to that of TcsL-1522, particularly with
respect to a 30-kDa band typically seen after TcsL-1522 modifications (Fig. 5). Accordingly, the patterns
obtained by two-dimensional electrophoresis showed great
similarity between TcsL-1522 and TcdB-1470, whereas a different pattern
of labeled GTPases was obtained with TcdB-10463 (Fig. 5a).
The only observable major difference between TcdB-1470 and TcsL-1522
was an acidic spot of approximately 20 kDa labeled by the latter. To
determine whether TcdB-1470 and TcsL-1522 modify the same substrates
also in intact cells, a differential glucosylation experiment was
performed. TcsL-1522 labeled its substrates in lysates from
TcdB-10463-treated cells almost to the same extent as in lysates from
nontreated cells (Fig. 5b). The GTPase, which is obvious in
lane 4 but not labeled in lane 5, probably
represents Rac, the only known common substrate of TcdB-10463 and
TcsL-1522. In lysates from TcdB-1470-treated cells, however, TcsL-1522
was no longer able to glucosylate its substrates (Fig. 5b).
On the other hand TcdB-10463 labeled its substrates in lysates from
TcdB-1470-treated cells (Fig. 5b), demonstrating that
TcdB-1470 in intact cells does not modify the substrates recognized by
TcdB-10463. The actual substrates of TcdB-1470 were determined with a
panel of recombinant small GTPases. Rac, Rap1, Rap2, and RalA were
labeled by TcdB-1470, whereas Cdc42 was modified to a lower extent
(Fig. 6a). Interestingly,
R-Ras, a small GTP-binding protein important in integrin activation
(19), was glucosylated both by TcdB-1470 and TcsL-1522 but not by
TcdB-10463 (Fig. 6b). Thus, inactivation of R-Ras might play
a role in the cell rounding (Fig. 2) and detachment induced by
TcdB-1470 and TcsL-1522.
TcdB-1470 Does Not Block EGF-induced Phosphorylation of
ERKs--
To determine the cellular effect of TcdB-1470 on activation
of ERKs, serum-starved 3T3 cells were treated with toxin until 100%
CPE was observed. After stimulation with EGF for 5 min, phosphorylation of ERKs was assessed as a shift in their molecular weights. This shift
occurred in TcdB-1470- and TcdB-10463-treated cells to the same extent
as in nontreated control cells (Fig. 7).
As an additional control we included TcsL-1522, which is known to
inhibit the EGF stimulation of ERK (5). We conclude that inactivation
of R-Ras did not affect the Ras-dependent mitogen-activated
protein kinase pathway.
Effect of TcdB-10463 and TcdB-1470 on Focal Adhesion
Components--
The different CPEs induced by TcdB-10463 and TcdB-1470
might reflect a differential effect of these toxins on focal adhesion complexes. Thus, toxin-treated 3T3 cells were probed with
anti-vinculin, anti- Some C. difficile sero group F strains do not elaborate
TcdA (9), although they produce the cytotoxin TcdB-1470 (8), which
differs somewhat from the classic cytotoxin TcdB-10463. Here we
characterize the enzymatic properties of this novel toxin and compare
its cytotoxic effects with those of the most closely related LCTs,
i.e. TcdB-10463 and TcsL-1522. As discussed below TcdB-1470
turns out to be an interesting addition to the family of LCTs, behaving
functionally as a TcsL-1522/TcdB-10463 hybrid, with the same cytotoxic
potency as TcdB-10463 but inducing a TcsL-like CPE.
All three toxins had equal cytotoxic potencies upon microinjection,
i.e. when the normal receptor binding and internalization routes were bypassed. In contrast, extracellularly applied TcsL-1522 was remarkably less cytotoxic than TcdB-10463 or TcdB-1470. In view of
the equal enzymatic potencies when tested in vitro, we propose that this difference is mainly determined by the receptor binding event of each toxin. The high cytotoxic potency of
extracellularly applied TcdB-1470 probably depends on its specific
binding to the same receptor as used by TcdB-10463. This was predicted
from the high identity (99%) between the receptor-binding domains of TcdB-1470 and TcdB-10463 (8) and here substantiated by cell surface
binding experiments, demonstrating a competition between TcdB-1470 and
125I-TcdB-10463. That this competition was somewhat lower
than with TcdB-10463 could be due to a slightly lower affinity of
TcdB-1470 for the receptor. In contrast, TcsL-1522 could not compete
out 125I-TcdB-10463 at all, suggesting that it binds to a
different receptor. The nature of the receptors is not known, but the
existence of a specific receptor for TcdB-10463 on the fibroblast
surface was recently demonstrated (16). In Don cells this
TcdB-1470/TcdB-10463 receptor should be present in considerably higher
numbers than the putative receptor for TcsL-1522. Alternatively
TcsL-1522 might have a very weak affinity for its own receptor.
Despite the similar cytotoxic potencies of TcdB-10463 and TcdB-1470,
they induce different types of CPE. TcdB-10463 induces a collapse of
the actin cytoskeleton with retraction of the cell body. Because
protrusions remain attached to the extracellular matrix, this
morphology is designated arborizing, actinomorphic, or neurite-like
(2). TcdB-1470, in contrast, induced a complete rounding of the
fibroblast body, conferring a mitotic-like appearance indistinguishable
from that induced by TcsL-1522. The respective CPEs should somehow
correlate with the substrates modified. Indeed, Rho was previously
shown to be the crucial substrate for the arborizing CPE, because
transient transfection with Rho could protect cells against TcdB-10463
but not TcsL-82 (18). The crucial substrate(s) for the rounding CPE by
TcsL-1522 and TcdB-1470 is not known, but we hypothesized that these
two toxins modify the same or similar substrates. This concept was
substantiated by two-dimensional electrophoresis showing similar
in vitro substrate patterns and by the differential
glucosylation experiments (Fig. 5), which confirmed that the same
substrates are also modified in vivo. Thus, the substrate
pattern of TcdB-1470 is similar to that of TcsL-1522 with the only
difference that TcsL-1522 is also able to modify Ras (5, 6).
The possibility of an additional substrate for TcdB-1470 and TcsL-1522
was considered because none of the known substrates could adequately
explain the TcsL-like rounding CPE: (i) Rac is a common substrate for
all LCTs, including those that induce the arborizing effect, (ii) Rap
has been shown to be also modified by TcdA (16), which induces the
arborizing effect, (iii) Ras is modified only by TcsL-1522 and not by
TcdB-1470, and (iv) Ral is mainly present in neuronal tissue and was
almost undetectable with specific polyclonal antibodies in the
fibroblasts used here (data not shown). R-Ras, being a member of the
Ras subfamily of small GTPases, shows a high sequence identity with Ras
(20) but has partially differing downstream targets (21).
Interestingly, R-Ras was found recently to control the activation of
integrins from inside the cell (19). GTP-loaded R-Ras activates
integrins, whereas GDP-loaded R-Ras inactivates them, thereby inducing
detachment from the extracellular matrix (19). This new function of
R-Ras suggested it could be involved in the rounding CPE induced by TcdB-1470 and TcsL-1522. Indeed, R-Ras turned out to be a major substrate for both toxins, whereas TcdB-10463 did not modify this GTPase (Fig. 6b). The molecular weight of the uppermost band
labeled by TcdB-1470 and TcsL-1522 in Fig. 5 agrees with the reported molecular weight of R-Ras (20).
We propose that modification of R-Ras in fibroblasts treated with
either TcdB-1470 or TcsL-1522 inactivates this small GTP-binding protein and that this in turn leads to inactivation of integrins from
inside the cell. This would induce the loss of integrin adhesiveness to
extracellular matrix proteins, resulting in the observed rounding and
detachment of cells. This hypothesis is supported by the
TcdB-1470-induced efficient fibroblast detachment, whereas
TcdB-10463-treated cells remained attached. Inactivation of integrins
is also known to induce a disassembly of focal adhesion complexes.
Indeed, cells treated with either toxin showed a disappearance of
vinculin from focal adhesion complexes. However, TcdB-10463-treated
cells showed two peculiarities that did not occur in TcdB-1470-treated
cells: (i) Based on these findings we speculate that inactivation of R-Ras in
cells treated with TcdB-1470 or TcsL-1522 causes a collapse of the
entire focal adhesion complexes. In contrast, the inactivation of Rho
by TcdB-10463 probably induces only a partial disassembly of the focal
adhesion complexes. The disappearance of vinculin may be initiated at a
point "downstream" the cytoplasmic tail of integrins, closer to the
actin filaments. Therefore, integrins remain in place and active,
explaining the persisting attachment of TcdB-10463-treated cells to the
substrate, and the remaining protrusions that confer the arborizing or
neurite-like appearance. Furthermore, glucosylation of Rho apparently
does not destroy the cell-to-cell contacts because TcdB-10463-treated
cells retained phosphotyrosine-containing structures at these sites
(Fig. 8).
Thanks to their highly specific mechanisms of action, bacterial toxins
can be used as exquisite tools in cell biology. The addition of
TcdB-1470 to the panel of already known LCTs broadens the possibilities
to apply this family of toxins for elucidating the functions of several
small GTPases, now including also R-Ras for two of the LCTs. TcdB-10463
and TcdB-1470 together form a particularly useful pair because they
have the same enzymatic potency and bind the same (or a similar)
receptor. Thus they enter cells by the same internalization pathway,
implying that the only difference in cells treated with these toxins is
the GTPase substrate attacked. These features allow accurate
comparisons of the cellular consequences at various levels of
inactivation of the different GTPases. The fact that these two toxins
act rapidly is another advantage for their use as cell biology tools,
because secondary pleiotropic effects due to long incubation times are
avoided. Finally, we also want to emphasize the potential utility of
TcdB-1470 for understanding the pathogenic significance of the various
toxin domains. The use of this natural hybrid toxin might enable
clarification of the respective roles of the binding and catalytic
regions in the action of TcdB on host tissues, whether in the induction
of colitis or in its lethal effect.
3-integrin remaining at the tips of cellular
protrusions. These components were absent from cells treated with the
R-Ras-inactivating toxins. The novel hybrid toxin will broaden the
utility of the LCTs for clarifying the functions of several small
GTPases, now including also R-Ras.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-toxin (Tcn
) from Clostridium novyi. TcdA and TcdB are
responsible for the symptoms in C. difficile-induced antibiotic-associated diarrhea and pseudomembranous colitis, whereas TcsH, TcsL, and Tcn
are virulence factors in gas gangrene (1). In
cultured cells the LCTs elicit cytopathic effects (CPEs) characterized by collapse of the actin cytoskeleton followed by arborization and/or
rounding up of the cells. This effect is due to a glycosylation of
different small GTP-binding proteins (2). The sugar moiety from either
UDP-glucose or UDP-N-acetylglucosamine is transferred to a
conserved threonine in the effector region of the target protein. The
GTP-binding protein is inactivated, its proper interaction with
immediate downstream effectors is impaired, and normal cell signaling
is interrupted (3). The CPEs induced by the different LCTs depend on
the targets attacked. In fibroblasts TcdA, TcdB, and Tcn
induce an
arborizing effect (1) reported to arise upon modification of Rho alone,
although these toxins also affect Rac and Cdc42 (3, 4). TcsL modifies
Ras, Rap, Ral, and Rac but not Rho (5-7) and induces a complete
rounding of the cell body without arborization (1). It is not known
which GTP-binding protein target(s) is crucial for the TcsL-induced morphology.
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Fig. 1.
Schematic representation of TcdB-10463,
TcdB-1470, and TcsL-1522. The numbers indicate the
percentage of identity in different domains comparing TcdB-1470 with
both TcdB-10463 and TcsL-1522. The upper scale indicates the
amino acid numbers.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-integrin antibody were from Transduction
Laboratories (Lexington, KY). Goat antiserum against TcdB was obtained
from Dr. D. M. Lyerly (Virginia Polytechnic Institute, Blacksburg,
VA). Mouse antiserum against TcsL was provided by Dr. M. R. Popoff
(Institut Pasteur, Unité de Toxines Microbiennes, Paris, France).
Recombinant proteins Rho, Rac, Cdc42, Ras, and Rap were provided by Dr.
P. Boquet (INSERM, Nice, France). A GST-Ral fusion protein was made as
follows: The rat RalA clone (accession number L19698, a gift from Gary
M. Wildey, Cleveland Clinic Foundation) and the primers:
5'-CGGGATCCATGGCTGCAAACAA-3' and 5'-CGGGATCCCGTTATAAAATGCAGCA-3' were
used for polymerase chain reaction. The resulting polymerase chain
reaction fragment was cloned into the BamHI site of pGEX-KT
and verified by sequencing. The plasmid was transformed into
Escherichia coli BL-21 strain, and expression of the
RalA-GST fusion protein was induced with isopropyl-1-thio-
-D-galactopyranoside. The GST-R-Ras
fusion protein construct was a generous gift from Adrienne Cox
(University of North Carolina). All recombinant proteins were purified
with glutathione-Sepharose 4B (Amersham Pharmacia Biotech, Uppsala,
Sweden) and cleaved with the appropriate protease. Proteases were
removed with benzamidine Sepharose 6B. All other reagents were of
analytical grade and obtained from local commercial sources.
counter. Competition experiments were performed following the same
protocol but using a 50-fold excess of the indicated cold competitor.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Morphological effects induced by TcdB-1470,
TcdB-10463, and TcsL-1522. Cytopathic effect induced by TcdB-1470
(b), TcdB-10463 (c), and TcsL-1522 (d)
on Swiss 3T3 fibroblasts as analyzed by scanning electron microscopy.
Nontreated control cells are shown in a. Bar, 15 µm.
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Fig. 3.
Cytotoxic potencies of TcdB-10463, TcdB1470,
and TcsL-1522 applied extracellularly and intracellularly.
a, Don cells in 96-well plates were exposed to TcdB-10463
(filled circles), TcdB-1470 (filled squares), and
TcsL-1522 (empty circles) at the indicated concentrations.
After 24 h incubation the percentage of rounded cells was
calculated. b, TcdB-10463 (filled circles),
TcdB-1470 (filled squares), and TcsL-1522 (empty
circles) were microinjected at the indicated concentrations in Don
cells together with fluorescent dextran as a marker for injected cells.
After 2 h of incubation the cells were fixed, and the percentage
of affected cells was calculated.
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Fig. 4.
Competition of 125I-TcdB-10463
binding by TcdB-10463, TcsL-1522, and TcdB-1470. Don cells in
96-well plates were incubated with 125I-TcdB-10463 (2 µg/ml) alone or in the presence of a 50-fold excess of the indicated
unlabeled competitor. After 2 h of incubation at 4 °C, nonbound
125I-TcdB was removed by three washes with ice-cold PBS.
Bound toxin was released by lysis of the cells with 1% Nonidet, and
radioactivity was determined by counting. The means and standard
deviations of triplicate samples expressed as total counts are
shown.
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Fig. 5.
In vitro and in vivo
glucosylation of cellular substrates by TcdB-10463, TcsL-1522,
and TcdB-1470. a, Don cell lysates were incubated with
the indicated concentrations of TcdB-10463, TcsL-1522, and
TcdB-1470 in the presence of 30 µM
UDP-[14C]Glc. After 1 h of incubation at
37 °C, the reaction was stopped by the addition of Laemmli
sample buffer. Cellular proteins were resolved by 12.5% SDS-PAGE
(left panel) or two-dimensional electrophoresis (right
panel). Labeled proteins were detected by PhosphorImager analysis.
b, confluent Don cells in 75-cm2 flasks were
left untreated (control) or treated with either TcdB-10463 or TcdB-1470
until 100% of the cells showed a visible CPE. The cells were lysed,
and the lysate proteins were glucosylated in vitro with
either TcdB-10463 or TcsL-1522 (5 µg/ml) in the presence of
UDP-[14C]Glc (30 µM). Proteins were
resolved by 12.5% SDS-PAGE and detected by PhosphorImager
analysis.
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Fig. 6.
Substrate specificity of TcdB-1470.
a, purified recombinant small GTPases (1-3 µg) were
incubated with TcdB-1470 (5 µg/ml) in the presence of
UDP-[14C]Glc (30 µM). After 1 h of
incubation at 37 °C, the reaction was stopped by the addition of
Laemmli sample buffer. Proteins were resolved by 12.5% SDS-PAGE, and
labeled bands were detected by PhosphorImager analysis. b,
purified recombinant R-Ras in GST fusion form was incubated with
TcdB-10463, TcsL-1522, or TcdB-1470 (5 µg/ml) in the presence of
UDP-[14C]Glc (30 µM) and processed
following the same procedure as in A. Coomassie-stained gel
and PhosphorImager analysis of the same gel are shown.
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Fig. 7.
Effects of TcdB-10463, TcsL-1522, and
TcdB-1470 on EGF-mediated ERK activation. Confluent 3T3 cells,
serum starved for 16 h, were left untreated (lanes 1,
3, 4, and 6) or treated with
TcdB-10463 (lane 2), TcdB-1470 (lane 5), or
TcsL-1522 (lane 7) until 100% of the cells were affected.
Cells were then stimulated with EGF (100 ng/ml) 5 min (lanes
2, 3, 5, 6, and 7) or
left unstimulated (lanes 1 and 4). Cells were
washed with ice cold PBS and lysed with Laemmli sample buffer. Equal
amounts of proteins were loaded per lane, resolved on 7.5% SDS-PAGE,
and transferred to nitrocellulose membranes. Membranes were probed with
anti-ERK antibodies, and recognized bands were detected by
chemiluminescence.
3-integrin, and anti-phosphotyrosine
antibodies. Both TcdB-10463 and TcdB-1470 caused vinculin to disappear
from focal adhesions after only 20 min (Fig.
8, a-c). However,
3-integrin-containing protrusions remained at the
ruffling edges of the fibroblasts after treatment with TcdB-10463 (Fig.
8e), whereas TcdB-1470 completely disrupted these structures
(Fig. 8f). Tyrosine-phosphorylated proteins remained at most
cell-to-cell contacts (Fig. 8h) in TcdB-10463-treated cells,
whereas TcdB-1470 completely disrupted also these structures. (Fig.
8i). Thus, TcdB-10463 and TcdB-1470 affect focal adhesion components differentially.
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Fig. 8.
Effects of TcdB-10463 and TcdB-1470 on the
distribution of vinculin,
3-integrin, and tyrosine phosphorylated
proteins. 3T3 cells growing on 13-mm round glass coverslips were
treated 20 min with TcdB-10463 (b, e, and
h) or TcdB-1470 (c, f, and
i) (50 ng/ml for both toxins) or left untreated
(a, d, and g). Cells were fixed with
3.5% paraformaldehyde and stained with anti-vinculin
(a-c), anti-
3-integrin (d-f), or
anti-phosphotyrosine (g-i) antibodies.
3-Integrin and phosphotyrosine accumulations are
indicated with large (control cells) or small
(TcdB-10463-treated cells) arrowheads.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-integrin remained at the tip of the
protrusions surrounding the cells and (ii) phosphotyrosine containing
structures were detected at the cell-to-cell contacts.
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ACKNOWLEDGEMENTS |
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We are grateful to Caterina Guzman-Verri and Alberto Alape for critical reading of the manuscript and Edgardo Moreno for stimulating discussions and laboratory facilities.
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FOOTNOTES |
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* This work was supported by Swedish Medical Research Council Grant 05969 and grants from Magnus Bergvalls Stiftelse and Karolinska Institutet.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.
¶ Recipient of a grant from the Swedish International Development Agency (Sida-SAREC), as a part of the Karolinska International Research Training program.
Supported by Grants Ei 206/3-2 and Ei 206/9-1 from the Deutsche Forschungsgemeinschaft.
§§ To whom correspondence should be addressed: Microbiology and Tumorbiology Center (MTC), Box 280, Karolinska Institutet, S-171 77 Stockholm, Sweden. Tel.: 46-8-728-71-62; Fax: 46-8-33-15-47; E-mail: monica.thelestam{at}mtc.ki.se.
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ABBREVIATIONS |
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The abbreviations used are:
LCTs, large
clostridial cytotoxins;
TcdA, C. difficile toxin A;
TcdB, C. difficile toxin B;
TcsH, C. sordellii
hemorrhagic toxin;
TcsL, C. sordellii lethal toxin;
Tcn, C. novyi
-toxin;
CPE, cytopathic effect;
GST, glutathione
S-transferase;
EGF, epidermal growth factor;
ERK, extracellularly regulated kinase;
PAGE, polyacrylamide gel
electrophoresis;
PBS, phosphate-buffered saline.
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REFERENCES |
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