From the Microbiology and Tumorbiology Center, Box
280, Karolinska Institutet, S-17177 Stockholm, Sweden,
§ Centro de Investigación en Enfermedades Tropicales,
Facultad de Microbiologia, Universidad de Costa Rica, 1000 San
José, Costa Rica, ¶ Programa de Investigación en
Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad
Nacional, Aptdo 304-3000 Heredia, Costa Rica, and ** Centro
de Investigación en Estructuras Microscópicas, Universidad
de Costa Rica, 1000 San José, Costa Rica
Received for publication, September 9, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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Clostridium difficile induces
antibiotic-associated diarrhea through the production of toxin A and
toxin B; the former toxin has been assumed to be responsible for the
symptoms of the disease. Several toxin A-negative strains from C. difficile have recently been isolated from clinical cases and
have been reported to produce toxin B variants eliciting an atypical
cytopathic effect. Ultrastructural analysis indicated these toxins
induce a rounding cytopathic effect and filopodia-like structures.
Toxin B variants glucosylated R-Ras, and transfection with a
constitutively active mutant of this GTPase protected cells against
their cytopathic effect. Treatment of cells with toxin B variants
induced detachment from the extracellular matrix and blockade of the
epidermal growth factor-mediated phosphorylation of
extracellular-regulated protein kinases, demonstrating a deleterious effect on the R-Ras-controlled avidity of integrins. Treatment with
toxin B variants also induced a transient activation of RhoA probably
because of inactivation of Rac1. Altogether, these data indicate that
the cytopathic effect induced by toxin B variants is because of cell
rounding and detachment mediated by R-Ras glucosylation, and the
induction of filopodia-like structures is mediated by RhoA activation.
Implications for the pathophysiology of C. difficile-induced diarrhea are discussed.
Clostridium difficile induces clinical entities like
antibiotic-associated diarrhea and pseudomembranous colitis via the
production of two toxins, toxin A
(TcdA)1 and toxin B (TcdB).
TcdA is known as the enterotoxin, because when inoculated in animal
models in purified state, it is able to reproduce the clinical
manifestations induced by C. difficile (1). TcdB, on the
other hand, is known as the cytotoxin because of its potent cytotoxic
activity on cultivated cells. Although TcdA is also cytotoxic it is
1000 times less efficient than TcdB (2, 3). TcdB is not able to induce
clinical symptoms in animal models unless inoculated together with
sublethal amounts of TcdA (1). These two toxins belong to the large
clostridial cytotoxin (LCT) family (~300 kDa) and are
glucosyltransferases that modify GTPases of the RhoA subfamily by
transferring the glucose moiety from UDP-glucose to threonine 35 (37 in RhoA) (4, 5). This amino acid is located in the so-called
effector region, which transmits signals to downstream effector
molecules. Thus, it is predictable that a modification in this domain
will impair the signal transduction cascade controlled by the GTPase
(6). Members of the RhoA subfamily are well known key controllers of the actin cytoskeleton (7), thus one of the main characteristics of
cellular intoxication with LCTs is a collapse in this network structure.
In addition to TcdB and TcdA from the prototype strain VPI-10463, toxin
variants of C. difficile toxin B, TcdB-1470 and TcdB-8864, have been isolated from TcdA-negative strains (8, 9). These variants
have been reported to produce a somehow different cytopathic effect
(CPE) than the classical neurite-like morphology induced in fibroblasts
by TcdB-10463 (9, 10). Their role in pathogenic processes is uncertain
although the appearance of TcdA-negative C. difficile
strains producing gastrointestinal pathology (11, 12) has been
reported. More recently, the prevalence of toxin A-negative strains
producing C. difficile-associated diarrhea in 334 patients
in France was found to be 2.7% (13). In all these cases toxin B was
reported to produce an atypical CPE. Thus, cytotoxins from these
strains might be relevant virulence factors that take over functions
generally accepted to depend on TcdA. Of those variant cytotoxins,
TcdB-1470 was shown to be a functional hybrid possessing the
receptor-binding and internalization domain of TcdB-10463 and the
glucosyltransferase domain of Clostridium sordellii TcsL
(10). Because of these characteristics, TcdB-1470 and TcdB-10463 are
the prototypes of the two different groups of LCTs divided according to
substrate specificities as follows: (i) TcdA-10463 and TcdB-10463
modify RhoA, Rac1, and Cdc42 as common substrates; and (ii) TcdB-1470
(from C. difficile strain 1470) and TcsL (from C. sordellii) modify Rac1, Ral, Rap, and R-Ras (5, 10, 14, 15). R-Ras
is a particularly interesting substrate, because it has a unique
30-amino acid sequence in its N terminus differentiating it from H-,
K-, or N-ras oncogenes (16). Furthermore, whereas activated
H-Ras has been shown to suppress the activation of certain integrins
(17) R-Ras has the opposite activity and promotes
integrin-dependent adhesion (18, 19). Thus, its
inactivation could have dramatic consequences for cell morphology and adhesion.
The modification of RhoA has been shown to be the event responsible for
the collapse of the actin cytoskeleton induced by the group of
RhoA-modifying toxins (4, 20). The molecular consequences of this
modification in terms of RhoA interaction with downstream effectors and
with RhoA controlling proteins has been partially elucidated (21, 22).
However, the sequence of events leading to induction of the early
recognized neurite-like CPE (23), which is produced also by other
RhoA-modifying toxins (including C3 from Clostridium
botulinum), is barely understood (24). Presently, the relevant
substrate for the induction of a CPE by toxins that do not modify RhoA
is unknown.
In this study we have explored the molecular events leading to the CPE
by toxin B variants from toxin A-negative strains. We demonstrate (i)
that glucosylation of R-Ras induces cell detachment and rounding of the
cell body by inactivation of integrins, and (ii) an imbalance in the
activation state of RhoA via the inactivation of Rac1. The biological
relevance of the two different types of CPE is discussed in the context
of the pathology produced by C. difficile.
Materials--
C. difficile toxins TcdB-10463,
TcdB-1470, TcdB-8864, TcdA-10463, and C. sordellii toxin
(TcsL) were kindly provided by Dr. Christoph von Eichel-Streiber
(Johannes Gutenberg-Universitãt, Mainz, Germany).
Escherichia coli cytotoxic necrotizing factor (CNF) 1 was
prepared as described previously (25). Eucaryotic expression vectors
containing myc-tagged small GTPase mutants were
kindly provided by Dr. Alan Hall (University College London, London,
England) and Dr. Patrice Boquet, (INSERM, Nice, France). Lipofectin was from Invitrogen. Fluorescein
isothiocyanate-phalloidin and epidermal growth factor (EGF) were from
Sigma. Anti-phospho ERK antibodies were from Cell Signaling, and
anti-ERK antibodies were from Upstate Biotechnologies. The GST-R-Ras
fusion protein construct was a generous gift from Adrienne Cox
(University of North Carolina). The GST-tagged rhotekin RhoA-binding
domain (RBD) was expressed from plasmid pGEX-2T-RBD (kindly
provided by Dr. Martin Alexander Schwartz, Scripps Research Institute,
La Jolla, CA). The GST-tagged GTPase-binding domain of p21-activated
kinase 1 (PBD) was expressed from a derivative pGEX-2T plasmid (kindly provided by Dr. Gary Bokoch, Scripps Research Institute, La Jolla, CA).
All plasmids were transformed into the E. coli BL-21 strain, and expression of fusion proteins was induced with
isopropyl-1-thio- Glucosyltransferase Activity--
5 µl of
UDP-[14C]glucose dissolved in ethanol were vacuum-dried,
and 10 µl of reaction 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) containing
recombinant GST-R-Ras (2 µg) were added. The mixture was incubated
for 1 h at 37 °C with 5 µg/ml of TcdB-10463, TcdB-1470, or
TcdB-8864 or 100 µg/ml of TcdA-10463. The reaction was stopped by
heating at 95 °C in Laemmli sample buffer. Proteins were separated
by 12.5% SDS-PAGE. Radiolabeled R-Ras was detected by
PhosphorImager analysis (Molecular Dynamics).
Cell Culture, Plasmids, and Transfection--
Mouse BalbC 3T3
fibroblasts (ATCC CCL-163) were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum, 5 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml). Human epithelial HeLa cells (ATCC CCL-2) were cultured in Eagle's minimal essential medium supplemented as
indicated above. Both cell lines were incubated at 37 °C in a humid
atmosphere containing 5% CO2. Transfection of cells with plasmids encoding myc-tagged V12Rac1, V38R-Ras, V12Rap1, or
V14RhoA was performed with Lipofectin according to the manufacturer's instructions. In brief, 50000 cells were seeded on 13-mm glass coverslips and cultivated for 24 h. Lipofectin was mixed with 1 µg of plasmid in Eagle's minimal essential medium without fetal bovine serum and antibiotics, and the complex was allowed to form for
15 min at room temperature. The mixture was then laid onto subconfluent
HeLa cells, and the cells were incubated for 6 h at 37 °C. The
original medium was changed to fully supplemented medium. Cells were
intoxicated 48 h after this treatment at the conditions indicated
in the figure legends.
Immunofluorescence--
Cells cultivated on 13-mm glass
coverslips and transfected as described above were intoxicated with
clostridial toxins as indicated in the figure legends. Cells were then
fixed with 3.7% paraformaldehyde in phosphate-buffered saline (PBS)
and permeabilized with 0.5% Triton X-100 in PBS. Cells were further
incubated with anti-Myc 9E10 monoclonal antibody (Santa Cruz
Biotechnology) for 30 min, followed by secondary TRITC anti-mouse
antibody for an additional 30 min. For staining of the actin
cytoskeleton, cells were incubated for 30 min with 0.5 µg/ml
fluorescein isothiocyanate-phalloidin (Sigma). Fluorescence and phase
contrast were visualized using an Olympus BH-2 microscope, and images
were captured using a digital camera (MagnaFire). The percentage of
cells protected from clostridial toxins was calculated as the fraction
of cells showing no CPE as viewed by phase contrast among 300 cells
positive for GTPase expression as determined by immunofluorescence
using a monoclonal anti-Myc antibody.
Scanning Electron Microscopy--
Subconfluent cells grown in
13-mm glass slides, intoxicated as indicated in the figure legends,
were chemically immobilized with 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 4 °C for 2 h and
post-fixed in 1% osmium tetroxide in phosphate buffer for 30 min.
Fixed cells were dehydrated in increasing concentrations of ethanol and
frozen in tert-butyl alcohol. Cells were freeze-dried by
sublimation and coated with 20 nm of gold-palladium (10 mAmp, 2 min). Cells were observed and photographed with an S-570 (Hitachi) scanning electron microscope operating at 15 kV.
Tyrosine Phosphorylation of ERKs--
BalbC 3T3 cells growing in
24-well plates were serum-starved overnight and treated with
clostridial toxins or left untreated as indicated in the figure
legends. Cells were then stimulated with 100 ng/ml EGF for 5 min,
washed twice with ice cold PBS, and lysed in Laemmli sample buffer.
Protein concentration was determined by the Bio-Rad DC method
according to the manufacturer's instructions, and equal amounts of
protein (20 µg) were loaded onto a 12.5% SDS-polyacrylamide gel.
Separated proteins were transferred to a polyvinylidene difluoride
(PVDF) membrane and probed with anti-phospho-ERK or anti-ERK
antibodies. Membranes were further incubated with peroxidase-conjugated
anti-mouse antibodies, and the detected bands were visualized with a
chemiluminescence Western blotting kit (Pierce Super Signal WestDura).
Adhesion Assays--
Cells cultivated on 24-well plates to 90%
confluence were intoxicated or left untreated as indicated in the
figure legends. After treatment, cells were washed three times with
PBS, the remaining cells were lysed with SDS-PAGE sample buffer, and
the protein concentration was quantified using bovine serum albumin as standard.
Quantification of GTP-RhoA, GTP-Rac1, and GTP-Cdc42--
For
precipitation steps, GTP-RhoA was quantified with GST-tagged RBD (26),
and GTP-Rac1 and GTP-Cdc42 were quantified with GST-PBD (27). HeLa
cells cultured in 6-well plates were intoxicated at the conditions
indicated in the figure legends. After treatment, cells were washed
with ice-cold PBS and lysed with 500 µl of ice-cold precipitation
buffer (1% Triton X-100, 0.1% SDS, 0.3% Nonidet P-40, 500 mM NaCl, 10 mM MgCl2, and 50 mM Tris, pH 7.2). Lysates were clarified by centrifugation
at 14000 rpm for 1 min. Twenty µl of each lysate were saved as
control for total GTPase content. GTP-loaded RhoA GTPases were
precipitated with Sepharose beads coupled to either GST-PBD or GST-RBD
protein. Samples were incubated for 30 min at 4 °C with shaking,
washed with precipitation buffer, and resuspended in 25 µl of Laemmli
sample buffer for analysis by SDS-polyacrylamide gel electrophoresis.
Samples transferred to a PVDF membrane were probed with either rabbit
antibodies against RhoA or Cdc42 (Santa Cruz Biotechnology) or with
anti-Rac1 monoclonal antibody (Transduction Laboratories). Probing and
developing were performed with peroxidase-labeled secondary antibodies
and the chemiluminescence Western blotting kit (Pierce Super Signal
WestDura), respectively. GTP-Cdc42, GTP-RhoA, and GTP-Rac1 levels were
calculated using the Scion Image software for Windows and compared with
control total levels of Cdc42, RhoA, and Rac1.
RhoA- and R-Ras-modifying LCTs Induce Distinguishable
CPEs--
The CPE induced by TcdA-10463, TcdB-10463, TcdB-1470, and
TcdB-8864 was studied in 3T3 fibroblasts and epithelial HeLa cells by
scanning electron microscopy. After intoxication with TcdA-10463 or
TcdB-10463, 3T3 fibroblasts showed a neurite-like CPE characterized by
the presence of long protrusions attached to the substratum (Fig.
1). HeLa cells treated with the same
toxins developed a more rounded phenotype with few protrusions (Fig.
1); these protrusions are barely seen under light microscopy (data not
shown). After intoxication with TcdB-1470 or TcdB-8864, 3T3 fibroblasts
presented a rounded phenotype. The cells appeared to be detaching from
the substratum, and some presented blebs on their surface. A rounded CPE was also evident in HeLa cells, and filopodia-like structures emanating from the cell body were observed (Fig. 1). RhoA glucosylation is essential for the CPE induced by TcdB-10463 (4, 20); however, the
relevant in vivo substrate for LCTs not affecting RhoA is not known. We tested the ability of TcdA-10463 and TcdB-8864 to glucosylate recombinant R-Ras in vitro. Because of its low
enzymatic activity (2, 3) TcdA-10463 was used 100 times more
concentrated than the negative control TcdB-10463. Under these
conditions we did not detect any radioactive signal from R-Ras after
TcdA-10463 treatment but detected a radioactive band after TcdB-8864
treatment, similar to the positive control, TcdB-1470 (Fig.
2A). This indicates that R-Ras
is a substrate also for this toxin B variant. Thus, among LCTs there
exists a strict correlation between RhoA glucosylation and induction of
a neurite-like CPE and between R-Ras glucosylation and induction of a
rounding CPE, suggesting R-Ras to be a crucial in vivo
substrate for the latter group of LCTs. To test this hypothesis HeLa
cells transiently transfected with constitutively active V38R-Ras were
intoxicated with TcdB-1470. At conditions inducing 100% CPE by
TcdB-1470 in control non-transfected cells, V38R-Ras-transfected cells
showed no sign of CPE (Fig. 2B). On the other hand, V38R-Ras expression did not protect cells against TcdB-10463-induced CPE (Fig.
2B). The degree of protection obtained by transfection of activated GTPases was quantified. Almost no cells expressing V38R-Ras showed a detectable CPE after TcdB-1470 treatment whereas V12Rac1 and
V12Rap1 expression conferred a lower degree of protection (Fig.
2C). Only ~10% of cells expressing V14RhoA (negative
control) were protected against the TcdB-1470-induced CPE. Similar
results were obtained with 3T3 fibroblasts (data not shown). Altogether these results indicate that R-Ras is a major in vivo
substrate determining the CPE of one group of LCTs.
R-Ras-modifying Toxins Induce Cell Detachment--
Cells treated
with the R-Ras-modifying toxins TcdB-1470 or TcdB-8864 rounded up in
less than 1 h and seemed loosely attached to the substratum,
whereas TcdB-10463-treated cells still remained attached 24 h
after intoxication (data not shown). These observations, together with
the reported role of R-Ras in controlling the activation state of
integrins (18), prompted us to investigate whether TcdB-1470 treatment
induces cell detachment. 3T3 fibroblasts were intoxicated for 1 h
with the different toxins, and after several washing steps, the
remaining cells were quantified. TcdB-10463-treated cells remained
attached to the substratum to the same extent as control
non-intoxicated cells whereas TcdB-1470 induced a massive detachment of
cells (Fig. 3A). The same
result was obtained with TcdB-8864 and with HeLa epithelial cells (data
not shown). Integrin engagement has been proven to be essential for
EGF-mediated activation of ERK kinases (28); thus we tested whether
TcdB-1470 treated cells were able to support this signaling pathway.
Serum-starved cells intoxicated for 4 h with TcdB-10463,
TcdB-1470, or TcdB-8864 were treated with EGF, and the tyrosine
phosphorylation status of ERK kinases was investigated. ERK kinases
were phosphorylated in cells intoxicated with TcdB-10463 to the same
extent as in non-treated control cells upon stimulation with EGF. On
the other hand, the EGF-induced phosphorylation of ERK kinases in
TcdB-1470- or TcdB-8864-treated cells was significantly impaired when
compared with the control cells (Fig. 3B). This result
indicates that TcdB-10463-intoxicated cells have a biologically
relevant adhesive phenotype whereas focal contacts are affected by
intoxication with TcdB-1470 or TcdB-8864.
Dynamics in the Activation State of RhoA-GTPases after LCTs
Intoxication--
TcdB-8864 and TcdB-1470 (Fig. 1) induce
filopodia-like structures in HeLa cells. These structures appear before
the cells are rounded (~30 min), peak at 60 min, and then disappear
after 3 h of intoxication. C. sordellii TcsL induces a
similar phenomenon (14). We used the RhoA GTPase activator CNF1 from
E. coli to assess the role of these GTPases in the induction
of filopodia after TcdB-1470 intoxication. When HeLa cells were
pre-treated with CNF1 for 2 h and further incubated with
TcdB-1470, a strong promotion in both the number and length of
filopodia was observed (Fig. 4). This
result suggested an active involvement of some member(s) of the RhoA
GTPase subfamily in the filopodia formation after toxin treatment.
Thus, we monitored the activation state of individual RhoA GTPases
after intoxication with TcdB-1470 and TcdB-10463 by pull-down assays
(26, 29). A decrease in the level of Rac1-GTP by TcdB-1470 was evident
as early as 30 min post-incubation (Fig.
5A). This decrease was
followed at 60 min by disappearance of Rac1 from total cell lysates.
Although this finding might suggest proteolytic degradation of Rac1
after glucosylation, this interpretation is not likely to be correct,
because the anti-Rac1 antibody used in this study has been shown
recently not to recognize glucosylated
Rac1.2 The same
anti-Rac1 antibody recognized two isoforms of this protein whereas the
PBD-Sepharose beads preferentially pulled down only one isoform (Fig.
5A). The level of RhoA-GTP exhibited a significant and
consistent increase at 60 min post-intoxication (Fig. 5B), i.e. at the time when filopodia formation by TcdB-1470 was
maximal (Fig. 4). After 120 min of incubation, RhoA-GTP returned to
initial levels (Fig. 5B). The level of Cdc42-GTP remained
essentially unaltered throughout intoxication (Fig. 5C).
These results are consistent with the reported in vitro
substrates for TcdB-1470 (10). When the same set of experiments was
repeated with TcdB-10463, a decrease in Rac1-GTP, RhoA-GTP, and
Cdc42-GTP levels was evident (Fig. 6).
The inactivation kinetics of all three proteins followed a similar
pattern with a significant decrease detected at 30 min. GTP-bound
proteins reached undetectable levels at 60 min post-incubation. The
Rac1 signal disappeared from total lysates 60 min after TcdB-10463 addition, mirroring the TcdB-1470-induced effect on this protein (Fig.
6A).
Altogether, these results suggest that glucosylation of other GTPases,
probably Rac1, leads to transient RhoA activation, a reciprocal balance
demonstrated previously on epithelial cells and fibroblasts (29, 30).
To evaluate this hypothesis we studied the opposite scenario through
the use of CNF1. The levels of RhoA-GTP, Rac1-GTP, and Cdc42-GTP
increased after 30 min of incubation with CNF1 and peaked at 1 h.
However, whereas Rac1-GTP and Cdc42-GTP levels remained augmented even
after 24 h of incubation, the RhoA-GTP level showed a decrease at
4 h, and at 24 h had returned to basal levels (Fig.
7C). Thus, the reciprocal
balance between Rac1 and RhoA is also observed upon toxin-induced
activation of Rac1. To further explore this point, cells treated with
CNF1 for 24 h were intoxicated with TcdB-1470, and the levels of
RhoA-GTP were monitored. Under these conditions, an increase in the
RhoA-GTP level was clearly detectable 60 min after TcdB-1470 addition,
peaked at 120 min, and returned to basal level after 180 min (Fig.
8A). A parallel experiment
determined that Rac1-GTP levels start to decline 60 min after TcdB-1470
addition to CNF-intoxicated cells strengthening the correlation between
Rac1 glucosylation and transient activation of RhoA.
In this report we characterized in molecular terms the CPE induced
by toxin B variants (TcdB-1470 and TcdB-8864) produced by toxin
A-negative strains of C. difficile and compared it with the
CPE induced by classic toxin B from strain VPI-10463.
R-Ras Modification Plays a Crucial Role for the Induction of a
Rounding CPE by Toxin B Variants--
We demonstrate that modification
of R-Ras is a crucial event for induction of the CPE elicited by
non-RhoA-modifying LCTs. This conclusion is based on several
considerations. First, all R-Ras-modifying toxins tested elicit a
complete rounding of the cell body accompanied by induction of
filopodia-like structures in epithelial cells, whereas toxins that do
not modify this GTPase induce a neurite-like CPE. R-Ras glucosylation
seems to be dominant over RhoA modification in determining the
resulting CPE, because (i) simultaneous treatment with TcdB-10463 and
TcdB-1470 results in a rounding phenotype (data not shown), and (ii) a
variant toxin from C. difficile that has been reported
recently to glucosylate both RhoA and R-Ras induces a rounding
phenotype (31). Second, analysis of in vitro modified
substrates indicates that R-Ras, Rac1, Rap, and Ral are four common
substrates for non-RhoA-modifying toxins. Of those, Ral is not detected
in lysates from the cells used in this study (10); it is rather a more
abundant protein in neuronal tissue. Rac1 and Rap are not likely
candidates, because they are also substrates for toxins inducing a
neurite-like CPE (2, 4, 5). Thus, R-Ras remains the most likely
candidate. We confirmed this hypothesis, because transfection with
constitutively activated R-Ras protected nearly 100% of cells against
TcdB-1470-induced CPE whereas under the same conditions no protection
was conferred against TcdB-10463. Rac1 and Rap1 transfection conferred
a lower degree of protection, which can be attributed to substrate
competition (Rac1) or to the reported role (Rap1) in the control of
integrin-mediated cell adhesion (32, 33). Finally, the phenotype of
TcdB-1470-intoxicated cells correlates with the reported role of R-Ras
in the control of cell adhesiveness through modulation of integrin
avidity (18). Although TcdB-10463-intoxicated cells did not detach from
the substratum, TcdB-1470 induced a massive detachment of cells,
indicating that glucosylation of R-Ras, but not RhoA, alters the
activation state of integrins. Furthermore, EGF-mediated activation of
ERK kinases, an event requiring proper adhesion of the stimulated cells
(28), was inhibited by 4 h of treatment with TcdB-1470. We have
shown previously (10) that 1 h of treatment with this toxin does
not interfere with this signaling route even if under those conditions
the intracellular substrates are completely modified. Thus, the
interference we observe by prolonging the treatment depends on the
detachment of cells and not on the modification of some GTPase(s)
essential for this pathway such as previously shown for the C. sordellii TcsL (14), which glucosylates Ras.
What is the sequence of events occurring at the level of focal
adhesions during cell intoxication by C. difficile
cytotoxins? Based on the present results and the facts that (i) RhoA
has a role in the induction of focal adhesions and stress fibers (34), and (ii) vinculin disappears rapidly from focal adhesions upon TcdB-10463 intoxication (10), we propose the following model: RhoA
inactivation by LCTs leads to disaggregation of adaptor proteins located at the cytoplasmic side of the focal adhesion, such as vinculin; this causes disassembly and in turn release of the anchored stress fiber from the complex. There is not necessarily a
depolymerization of the F-actin into monomeric G-actin, because the
remaining protrusions still stain with phalloidin, which labels F-actin
exclusively. Because TcdB-10463-intoxicated cells retain their ability
to adhere to extracellular matrix proteins, we deduce that the integrin avidity to the extracellular matrix is not altered by RhoA
glucosylation. R-Ras modification by toxin variants, on the other hand,
induces integrin inactivation that, in turn, will induce its release
from the extracellular matrix initiating a complete disassembly of focal adhesions, including the stress fibers. These events will therefore culminate in a CPE differing from the one induced by RhoA-modifying toxins.
Toxin Variants Induce a Misbalance in the Activation State of Rho
GTPases--
Intoxication of HeLa cells with TcdB-1470 and TcdB-8864
induced the formation of filopodia-like structures closely resembling those induced by TcsL, which were shown previously (14) to contain fimbrin. Because filopodia formation is an active process modulated by
RhoA GTPases (7) we analyzed their involvement in this particular feature induced by toxin variants. Pre-activation of RhoA GTPases by
CNF1 treatment synergized with toxin variants to induce the formation
of giant filopodia, suggesting an active role of some GTPase(s) in this
phenomenon. Because some of the RhoA GTPases are substrates, at least
in vitro, for the toxin variants, we analyzed the activation
state of RhoA GTPases during early intoxication with toxin variants
compared with the classic toxin TcdB-10463. As predicted from in
vitro glucosylation assays with recombinant substrates (4),
TcdB-10463 rapidly reduced the activity of RhoA, Rac1, and Cdc42. On
the other hand, the TcdB-1470 treatment induced a rapid decrease in the
activity of Rac1 and did not affect the activation state of Cdc42,
again as predicted from in vitro studies (10).
Interestingly, RhoA activity was transiently elicited, peaking at the
same time as filopodia formation peaks after TcdB-1470 intoxication.
This observation is in agreement with recent reports indicating the
existence of a network of negative signaling between RhoA GTPases. It
was observed that in different cell lines Rac1 modulates the activity
of RhoA by inhibiting it (29, 30). In this context, it can be
postulated that Rac1 inactivation by TcdB-1470, and in general toxin
variants, releases the negative control on RhoA allowing its activity
to increase. This of course does not occur upon TcdB-10463
intoxication, because RhoA itself is a substrate for this toxin (4). We
could confirm this hypothesis by creating the opposite scenario through
the use of CNF1, an activator of RhoA GTPases. Upon CNF1 intoxication
the activity of RhoA, Rac1, and Cdc42 increased, but although the two
latter remained permanently activated, RhoA-GTP rapidly returned to a basal level, even though it remained covalently modified as determined by the shift in molecular weight. Furthermore, we could release the
CNF1-induced negative control of Rac1 on RhoA by TcdB-1470 treatment.
These data indicate that Rac1 overactivation exerts a negative control
over RhoA even though RhoA has lost its ability to interact with GTPase
activating proteins because of CNF1 modification (35). The molecular
details for this negative control of Rac1 on RhoA are currently
unknown, but bacterial toxins like TcdB-1470 and CNF1 seem to be
promising tools for its further elucidation. In conclusion, we have
shown that treatment with C. difficile toxin variants
induces a transient activation of RhoA, probably because of Rac1
inactivation, and resulting in the formation of filopodia-like
structures characteristic of the CPE induced by these toxins.
Besides solving some molecular details behind the CPE induced by
C. difficile toxin variants, this report contributes with significant findings concerning RhoA GTPase-modifying toxins. We
determined that Rac1 is no longer detected in total cell lysates by
Western blotting upon glucosylation by LCTs. Thus, the monoclonal antibody used in this study will become an important tool in the field
of clostridial toxins to follow the dynamics of intoxication in intact
cells avoiding previously used radioactive-dependent approaches. This report also illustrates the in vivo
kinetics of inactivation of Rho GTPases upon treatment with LCTs and
indicates a full agreement between the reported in vitro
substrates for these toxins (4, 10) and their behavior in
vivo with regard to activity state upon modification. Our results
with CNF1 also agree with the reported in vitro activation
of the Rho GTPases and are consistent with recent reports concerning
in vivo activation of Rho GTPases (36, 37). We did not
observe any degradation in vivo of the CNF1-modified GTPases
in the HeLa cells used in this study, contrasting the finding by Doye
et al. (37) in a rat bladder carcinoma cell line and in
primary endothelial HUVEC cells. These authors, however, report a
decreased ubiquitinylation of Rac1 upon CNF1 modification in
transformed epithelial cell lines consistent with our observations in
HeLa cells (37). Furthermore, all GTPases, whether modified by LCTs or
CNF1, showed similar kinetics in their activity responses, indicating
that all the substrates are modified with the same efficiency by these
bacterial toxins.
The long term aim of this kind of studies is to determine how toxin B
variants from C. difficile TcdA-negative strains are able to
induce clinical entities of their own, a finding contradicting the
dogma that TcdA is responsible for the pathophysiological effects
induced by this bacterium. One possible explanation is that R-Ras
modification leads to more deleterious effects than RhoA modification
via the induction of cell detachment, which for adherent cells is a
potent inductor of apoptosis. Additionally, transient activation of
RhoA could induce the release of chemical mediators by epithelial cells
favoring the development of diarrhea. Thus, it is possible that
R-Ras-modifying variants of toxin B are able to take over the
diarrheagenic functions of TcdA in C. difficile disease
whereas this does not seem possible for the RhoA-modifying TcdB-10463.
Evaluation of this hypothesis by in vivo experiments will
provide a deeper understanding of the relevant clinical entities such
as antibiotic associated diarrhea and pseudomembranous colitis, which
in turn would imply better treatment options for these diseases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside. The recombinant
proteins were purified with glutathione-Sepharose 4B (Amersham
Biosciences). UDP-[14C]glucose (specific activity,
318 mCi/mmol) was from PerkinElmer Life Sciences. All other
reagents were of analytical grade and obtained from local commercial sources.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (90K):
[in a new window]
Fig. 1.
Morphological effect induced by LCTs in
epithelial cells and fibroblasts. Epithelial HeLa cells
(A, C, E, G, and
I) and BalbC 3T3 fibroblasts (B, D,
F, H, and J) were treated with
TcdA-10463 (C and D), TcdB-10463 (E
and F), TcdB-1470 (G and H) or
TcdB-8864 (I and J) until a CPE was achieved in
100% of the cells. Control cells were left untreated (A and
B). Cells were intoxicated for 1 h with all toxins at a
concentration of 50 ng/ml except for the TcdA-10463 treatment, which
was for 4 h at a concentration of 500 ng/ml. Cells were observed
by scanning electron microscopy as described under "Experimental
Procedures." Bar, 10 µm.
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Fig. 2.
Relevance of R-Ras as substrate for
TcdB-1470. A, purified recombinant R-Ras (2 µg) in
GST fusion form was incubated with TcdB-10463 (5 µg/ml), TcdA-10463
(100 µg/ml), TcdB-8864 (5 µg/ml), or TcdB-1470 (5 µg/ml) in the
presence of UDP-[14C]glucose (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.
The Coomassie-stained gel and PhosphorImager analysis of the same gel
are shown. B, HeLa cells transfected with
myc-tagged constitutively active V38R-Ras were intoxicated
with TcdB-1470 (A and B) or TcdB-10463
(C and D) for 2 h. R-Ras expressing cells
were visualized by immunofluorescence using anti-Myc antibodies
(B and D); the same fields were observed by
phase-contrast microscopy (A and C).
C, HeLa cells transfected with constitutively active
myc-tagged V12Rac1, V12Rap1, V38R-Ras, or V14RhoA were
intoxicated with TcdB-1470 for 2 h. The percentage of 300 successfully transfected cells (visualized as in B)
protected from CPE is shown. Mean of triplicate transfections and S.D.
from one representative experiment of three is shown.
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Fig. 3.
Effect of TcdB-1470 and TcdB-10463 on cell
adhesion. A, monolayers of BalbC 3T3 cells in 24-well
plates were treated 1 h with TcdB-10463 or TcdB-1470 or left
untreated (Control). After treatment the cultures were
extensively washed with PBS, and cells remaining attached to the
substrate were lysed by addition of SDS-containing buffer. Protein
concentration ([Protein]), corresponding to adhered cells,
was determined in lysates. Mean of triplicate samples and S.D. from one
representative experiment of three is shown. B, BalbC 3T3
cells were serum-starved for 16 h. Cells were then intoxicated for
4 h with TcdB-10463, TcdB-1470, or TcdB-8864 or left untreated.
After intoxication cells were stimulated with EGF for 5 min, washed
with ice-cold PBS, and lysed in Laemmli sample buffer. Twenty µg of
protein of each lysate were loaded on 12.5% SDS-polyacrylamide gels,
proteins blotted on PVDF, and probed with antibodies against
phospho-ERK (upper panel) or total ERK (lower
panel) proteins. Immune complexes were visualized by the
chemiluminescence reaction. ERK 1 (p44) and ERK 2 (p42) isoforms are shown.
View larger version (49K):
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Fig. 4.
CNF1 pretreatment enhances filopodia-like
structures induced by TcdB-1470. HeLa cells were treated with CNF1
for 2 h (B) or left untreated (A). Then
cells were intoxicated with TcdB-1470 (A and B)
for an additional hour. Cells were visualized by phase-contrast
microscopy.
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Fig. 5.
Effect of TcdB-1470 on the activation state
of RhoA GTPases. Confluent monolayers of HeLa cells in 6-well
plates were intoxicated with TcdB-1470 for the indicated times. After
treatment, cells were lysed, and 20 µl were separated as control for
total amount of GTPases. Lysates were then incubated with RBD-GST
(B, upper panel) or PBD-GST (A and
C, upper panel)-Sepharose beads for 30 min.
Active proteins were pulled down by centrifugation, resolved in 10%
SDS-PAGE, and transferred to PVDF membranes. Small GTPases were
detected using anti-Rac1 (A), anti-RhoA (B), or
anti-Cdc42 (C) antibodies. D, the intensity of
the bands corresponding to Rac1-GTP (open circles), RhoA-GTP
(open triangles), and Cdc42-GTP (closed circles)
was quantified and corrected according to the amount of total GTPase in
the respective internal control (A, B, and
C, lower panel). Each time point was
expressed in relation to the amount of active GTPase found at 0 min of
incubation with the toxin. A representative experiment of three is
shown.
View larger version (29K):
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Fig. 6.
Effect of TcdB-10463 on the activation state
of RhoA GTPases. Confluent monolayers of HeLa cells in 6-well
plates were intoxicated with TcdB-10463 for the indicated times. After
treatment, cells were lysed, and 20 µl were separated as control for
total amount of GTPases. Lysates were then incubated with RBD-GST
(B, upper panel)- or PBD-GST (A and
C, upper panel)-Sepharose beads for 30 min.
Active proteins were pulled down by centrifugation, resolved in 10%
SDS-PAGE, and transferred to PVDF membranes. Small GTPases were
detected using anti-Rac1 (A), anti-RhoA (B), or
anti-Cdc42 (C) antibodies. D, the intensity of
the bands corresponding to Rac1-GTP (open circles), RhoA-GTP
(open triangles), and Cdc42-GTP (closed circles)
was quantified and corrected according to the amount of total GTPase in
the respective internal control (A, B, and
C, lower panel). Each time point was
expressed in relation to the amount of active GTPase found at 0 min of
incubation with the toxin. A representative experiment of three is
shown.
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[in a new window]
Fig. 7.
Effect of CNF1 on the activation state of
RhoA GTPases. Confluent monolayers of HeLa cells in 6-well plates
were intoxicated with CNF1 for the indicated times. After treatment,
cells were lysed, and 20 µl were separated as control for total
amount of GTPases. Lysates were then incubated with RBD-GST
(B, upper panel)- or PBD-GST (A and
C, upper panel)-Sepharose beads for 30 min.
Active proteins were pulled down by centrifugation, resolved in 10%
SDS-PAGE, and transferred to PVDF membranes. Small GTPases were
detected using anti-Rac1 (A), anti-RhoA (B), or
anti-Cdc42 (C) antibodies. D, the intensity of
the bands corresponding to Rac1-GTP (open circles), RhoA-GTP
(open triangles), and Cdc42-GTP (closed circles)
was quantified and corrected according to the amount of total GTPase in
the respective internal control (A, B, and
C, lower panel). Each time point was expressed in
relation to the amount of active GTPase found at 0 min of incubation
with the toxin. A representative experiment from three is shown.
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[in a new window]
Fig. 8.
TcdB-1470 treatment induces RhoA reactivation
in CNF1-treated cells. A, confluent monolayers of HeLa
cells in 6-well plates were treated with CNF1 for 3 or 24 h or
left untreated. Cells treated with CNF1 for 24 h were further
incubated with TcdB-1470 for the indicated times. Cells were then
lysed, and 20 µl were separated as control for total amount of RhoA
(lower panel). Lysates were incubated with RBD-GST-Sepharose
beads for 30 min. RhoA-GTP (upper panel) was pulled down by
centrifugation, resolved in 10% SDS-PAGE, transferred to PVDF
membranes, and detected using anti-RhoA antibodies. B,
confluent monolayers of HeLa cells in 6-well plates were treated with
CNF1 for 24 h or left untreated. Cells were further incubated with
TcdB-1470 for the indicated times. Cells were then lysed, and lysates
were incubated with PBD-GST Sepharose beads for 30 min. Rac1-GTP was
pulled down by centrifugation, resolved in 10% SDS-PAGE, transferred
to PVDF membranes, and detected using anti-Rac1 antibodies.
C, the intensity of the bands corresponding to RhoA-GTP on
cells treated with CNF1 for 24 h in A (upper
panel) was quantified and corrected according to the amount of
total GTPase in their corresponding internal control (lower
panel). Each time point was expressed in relation to
the amount of active GTPase found in non-CNF1 non-TcdB-1470-treated
cells. A representative experiment of three is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by Swedish Medical Research Council Grant 05969, Research Contract ICA4-CT-1999-10001 from the European Community, Research and Technological Development Project NOVELTARGETVACCINES, and International Foundation for Science Grant B/3222-1 (to E. C. O.) and by Ministerio de Ciencia y Tecnología/Consejo Nacional de Ciencia y Tecnología, Costa Rica and Vicerrectoría de Investigación, Universidad de Costa Rica.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 part of the Central
American/Karolinska International Research Training Program. To whom
correspondence should be addressed. Tel.: 506-2380761; Fax:
506-2381298; E-mail: echaves@cariari.ucr.ac.cr.
Recipient of a grant from the Swedish International Development
Agency (Sida/SAREC), as part of the Central American/Karolinska International Research Training Program.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M209244200
2 I. Just, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: TcdA, C. difficile toxin A; TcdB, C. difficile toxin B; TcsL, C. sordellii lethal toxin; CNF, cytotoxic necrotizing factor from E. coli; LCT, large clostridial cytotoxin; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; RBD, rhotekin RhoA-binding domain; PBD, GTPase-binding domain of p21 activated kinase 1; TRITC, tetramethylrhodamine isothiocyanate; CPE, cytopathic effect; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride.
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