(Received for publication, December 20, 1995; and in revised form, February 12, 1996)
From the
The lethal toxin (LT) from Clostridium sordellii belongs to the family of large clostridial cytotoxins causing morphological alterations in cultured cell lines accompanied by destruction of the actin cytoskeleton. C. sordellii LT exhibits 90% homology to Clostridium difficile toxin B, which has been recently identified as a monoglucosyltransferase (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K.(1995) Nature 375, 500-503). We report here that LT too is a glucosyltransferase, which uses UDP-glucose as cosubstrate to modify low molecular mass GTPases. LT selectively modifies Rac and Ras, whereas the substrate specificity of toxin B is confined to the Rho subfamily proteins Rho, Rac, and Cdc42, which participate in the regulation of the actin cytoskeleton. In Rac, both toxin B and LT share the same acceptor amino acid, threonine 35. Glucosylation of Ras by LT results in inhibition of the epidermal growth factor-stimulated p42/p44 MAP-kinase signal pathway. LT is the first bacterial toxin to inactivate Ras in intact cells.
Clostridium sordellii produces two major virulence
factors, the hemorrhagic toxin and the lethal toxin (LT), ()which are causally involved in diarrhea and enterotoxemia
in domestic animals and in gas gangrene in man(1, 2) .
The hemorrhagic toxin exhibits hemorrhagic activity, whereas LT causes
severe edemas. In addition both toxins are lethal and cytotoxic. The
cytotoxic effects on cell monolayers are characterized by
redistribution of the actin
cytoskeleton(3, 4, 5) . In respect to
physicochemical and immunological properties LT resembles toxin B
(ToxB) from Clostridium difficile, whereas hemorrhagic toxin
is more similar to toxin A (ToxA) from C.
difficile(5, 6, 7) . Recently, LT has
been cloned and sequenced(8) . As deduced from these data, LT
has a molecular mass of 270,614 Da and shows 76% identity (90%
homology) with C. difficile ToxB and 47% identity (73%
homology) with C. difficile ToxA, respectively. In common with
ToxA and ToxB, LT possesses C-terminal-located repetitive peptides that
are probably involved in cell receptor binding(9) .
Furthermore, Clostridium novyi
-toxin, which has been
recently sequenced, exhibits the same structural features(10) .
Although the primary structure of LT is closer to ToxB than ToxB is to
ToxA, LT-induced cytotoxic effects are clearly distinct from those
induced by ToxB. Whereas ToxB causes cell shrinkage and formation of
neurite-like extensions, LT induces more pronounced rounding of cells,
which become grouped in small cell
clusters(3, 5, 11) . ToxB-induced destruction
of actin filaments results in accumulation of the breakdown products in
the perinuclear space, whereas LT leads to a diffuse
distribution(3, 11) .
Recently, we reported that ToxA and ToxB from C. difficile are monoglucosyltransferases that selectively modify the low molecular mass GTP-binding proteins of the Rho subfamily(12, 13) , whereas other members of the Ras superfamily are not substrates. The cosubstrate UDP-glucose is cleaved by ToxA/ToxB, and the glucose moiety is transferred to amino acid threonine 37 of Rho, which is located in the effector domain of the Rho proteins(12, 13, 14) . The substrate proteins of ToxA and ToxB are Rho, Rac, and Cdc42, which are involved in the regulation of the actin cytoskeleton. Whereas Rho governs the formation of focal adhesions and stress fibers(15) , Rac is involved in membrane ruffling(16) , and Cdc42 is involved in the formation of filopodia(17, 18) . Glucosylation renders Rho functionally inactive, eventually resulting in a redistribution of the microfilament system(12) .
We report here the identification of C. sordellii lethal toxin as a glucosyltransferase that modifies the low molecular mass GTP-binding proteins Rac and Ras.
Before cell
lysis, the cells were rinsed with ice-cold phosphate-buffered saline
(pH 7.2) and were then disrupted mechanically by sonication (5 times on
ice) in the presence of lysis buffer (2 mM MgCl,
40 µg/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride,
20 µg/ml leupeptin, 80 µg/ml benzamidine, 50 mM HEPES,
pH 7.4), followed by centrifugation for 10 min at 2,000
g. The supernatant was used for the glucosylation reaction.
For the MAP kinase assay, the cells were rinsed with ice-cold
phosphate-buffered saline and were then disrupted mechanically by
sonication (5 times on ice) in the presence of lysis buffer (150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM orthovanadate, 40 µg/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 10 mM HEPES, pH 7.4), followed by centrifugation for 15 min at 14,000
g. The supernatant was used for the MAP kinase assay.
Lysates from NIH 3T3 cells were incubated with C.
sordellii LT in the presence of
UDP-[C]glucose,
UDP-[
C]galactose, or
UDP-[
C]N-acetylglucosamine (Fig. 1A). Proteins in the molecular mass range of
20-22 kDa were only labeled in the presence of
UDP-[
C]glucose, indicating incorporation of
[
C]glucose into these cellular proteins. When
the concentration of LT was increased (up to 40 µg/ml) and the
incubation time was prolonged (up to 180 min) no additional proteins
were labeled (data not shown). To study the protein substrate
specificity of LT in more detail, recombinant low molecular mass
GTP-binding proteins of the Ras superfamily were tested. Under
conditions that were sufficient to elicit full transferase activity of C. difficile ToxA and ToxB(12, 13) , LT
catalyzed only minimal incorporation of glucose into the recombinant
GTP-binding proteins. Therefore, we tested whether the transferase
activity of LT might require a factor that is present in cell lysates.
Several cellular subfractions were analyzed to enhance glucosylation of
recombinant Rho subfamily proteins. A cytosolic factor that was
heat-stable and had a molecular mass below 500 Da (fraction F500) was
preliminarily characterized to stimulate LT-catalyzed glucosylation (Fig. 1C).
Figure 1:
Glucosyltransferase activity of C.
sordellii LT. A, cosubstrate specificity. NIH 3T3 cell
lysates were incubated with LT (4 µg/ml) in the presence of 10
µM of UDP-[C]hexoses (17 nCi) for
60 min at 37 °C. Lane 1, UDP-
[
C]galactose; lane 2,
UDP-[
C]glucose; lane 3,
UDP-N-acetyl[
C]glucosamine. B,
protein substrate specificity. Recombinant low molecular mass
GTP-binding proteins (each 50 µg/ml) (dissolved in 2 mM MgCl
, 1 mM MnCl
, 100 µg/ml
bovine serum albumin, 50 mM HEPES, pH 7.4) were incubated with
LT (4 µg/ml) and 10 µM UDP-[
C]glucose (17 nCi) for 30 min at 37
°C. C, effect of EDTA on LT-catalyzed glucosylation. Rac1
(50 µg/ml dissolved in 2 mM MgCl
, 100
µg/ml bovine serum albumin, 50 mM HEPES, pH 7.4) was
[
C]glucosylated by LT (lanes 1-6)
or ToxB (lanes 7 and 8) for 30 min at 37 °C. Lanes 1, 3, 5, and 7, without EDTA; lanes 2, 4, 6, 8, in the presence
of 2 mM EDTA; lane 1, control; lane 2, plus
2 mM EDTA; lane 3, addition of fraction F500; lane 4, F500 plus 2 mM EDTA; lane 5,
addition of 1 mM MnCl
; lane 6, 1 mM MnCl
plus 2 mM EDTA; lane 7,
glucosylation by ToxB; lane 8, ToxB plus 2 mM EDTA.
(F500 was prepared by incubating the cytosolic fraction of rat brain
homogenate for 15 min at 95 °C followed by filtration through a
membrane with a 500-Da cut-off.) D, guanyl nucleotide
dependence of the glucosylation reaction. Rac1 and Ha-Ras (each 50
µg/ml) were incubated with 100 µM of either GTP (lanes 1 and 3) or GDP (lanes 2 and 4) for 15 min at 37 °C followed by LT-catalyzed
glucosylation for 30 min as described under ``Experimental
Procedures.'' PhosphorImager data of the 12.5% SDS-PAGE are
shown.
As shown in Fig. 1B, Rac was the exclusive substrate of the Rho subfamily. Neither Rho nor Cdc42 was glucosylated. Surprisingly, Ras was modified by LT, whereas other members of the Ras superfamily such as Rab and Arf were not substrates. Denaturation of either Rac/Ras or LT completely inhibited glucosylation, indicating that the native structure of both substrate protein and transferase are essential for the glucosylation reaction (data not shown).
Since the activities of various eukaryotic
glycosyltransferases are stimulated by Mn(24, 25) several metal ions were tested.
LT-catalyzed glucosylation was only stimulated in the presence of
Mn
with maximal incorporation of glucose at 1 mM of Mn
. Combination of Mn
with
fraction F500 caused no additional increase in glucosylation,
indicating that the stimulating factor in the fraction F500 is most
likely Mn
. In contrast, glucosyltransferase activity
of the related ToxA/B was inhibited rather than stimulated by
Mn
(data not shown). Chelation of Mn
by EDTA (2 mM) or the addition of EDTA (2 mM)
to fraction F500 completely blocked glucosylation of Rac by LT but not
by ToxB (Fig. 1C). Increasing concentrations of
MgCl
up to 10 mM caused a decrease in LT-catalyzed
glucosylation but did not change ToxB-catalyzed modification of Rac.
All these findings indicate that Mn
is required for
LT transferase activity but does not interfere with the Mg
dependent GTP-binding to Rac protein.
To test whether the
guanyl nucleotide-bound state of Rac affects the incorporation of
glucose, Rac was incubated with either GDP or GTP prior to
glucosylation. Rac was a better substrate for LT in the GDP-bound form
than in the GTP-bound form (Fig. 1D). Identical effects
of GDP/GTP were observed for Ras (Fig. 1D). The guanyl
nucleotide dependence of glucosylation of the Rho subfamily proteins by
ToxA/B is based on the location of the acceptor amino acid threonine 37
in the GTP-binding and hydrolysis domain(12, 26) . To
determine whether LT uses the same acceptor site as ToxA/B, sequential
glucosylation by either transferase was performed. Rac was modified by
LT (or ToxB) in the presence of unlabeled UDP-glucose, followed by a
second glucosylation performed in the presence of
UDP-[C]glucose and ToxB (or LT) (Fig. 2A). Previous glucosylation completely inhibited
the second glucosylation with either toxin, indicating that LT most
likely catalyzes incorporation of glucose at the same acceptor amino
acid of Rac as does ToxB. As expected, treatment of RhoA with LT did
not inhibit subsequent
C glucosylation by ToxB (Fig. 2A). To confirm these data by a different
approach we made use of the deglucosylation reaction, which might be
even more stereospecific than the sequential glucosylation. Instead of
recombinant Rac we used membrane fractions of NIH 3T3 cells because
they can be washed to remove UDP-glucose, which blocks deglucosylation.
In the presence of a surplus of UDP (30 mM), LT was able to
cleave the previously incorporated glucose moiety and to again form
UDP-glucose (Fig. 2B). The reversal of LT-catalyzed
glucosylation of Rac (lower band) was blocked after the removal of LT.
However, when ToxB was added, deglucosylation of Rac was observed
again. The upper band ( Fig. 1and Fig. 2B),
which is not a substrate protein for ToxB, was not deglucosylated by
ToxB. These experiments indicate that LT and ToxB share the same
acceptor amino acid in the Rac protein.
Figure 2:
Acceptor amino acid of LT-catalyzed
glucosylation. A, sequential glucosylation. Rac1 (50
µg/ml) was C-glucosylated with either LT (4 µg/ml
plus 1 mM MnCl
) (lane 1) or ToxB (1
µg/ml) (lane 2). For sequential glucosylation Rac1 was
glucosylated with LT (lane 3) in the presence of unlabeled
UDP-glucose (10 µM) for 45 min at 37 °C followed by a
second glucosylation in the presence of
UDP-[
C]glucose (10 µM, 17 nCi) and
ToxB. Lane 4, first glucosylation with ToxB followed by
C glucosylation with LT. For control, RhoA (50 µg/ml)
was incubated with LT plus 1 mM MnCl
(lane
5) or ToxB (lane 6) in the presence of
UDP-[
C]glucose. For sequential glucosylation
RhoA was incubated with unlabeled UDP-glucose and LT followed by a
second
C glucosylation with ToxB (lane 7). B, deglucosylation of Rac by either LT or ToxB. Membrane
fractions from NIH 3T3 cells were
C-glucosylated by LT as
described under ``Experimental Procedures.'' The membranes
were washed 3 times and resuspended in 2 mM MgCl
/50 mM HEPES, pH 7.4. Deglucosylation was
initiated by the addition of UDP (30 mM) and either LT (4
µg/ml plus 1 mM MnCl
) or ToxB (1 µg/ml).
After 90 min at 37 °C the reaction was analyzed by 12.5% SDS-PAGE
followed by evaluation using the PhosphorImager. Lane 1, LT
only; lane 2, UDP only; lane 3, LT plus UDP; lane
4, ToxB only; lane 5, ToxB plus UDP. Upper band,
unidentified substrate protein; lower band,
Rac.
LT catalyzed C
glucosylation of three substrate proteins (Fig. 1). The middle
band of the triplet was identified as Rac by immunoblot of
two-dimensionally separated cell lysates
C-glucosylated by
LT (not shown). The faint lower band corresponds to Ras (see below).
The identity of the upper one is still unclear, but glucosylation is
GDP-dependent, indicating that this substrate is a low molecular mass
GTP-binding protein. To study whether cellular Ras is actually a
substrate of LT, lysates from rat brain homogenates were glucosylated
by either LT or ToxB in the presence of
UDP-[
C]glucose followed by immunoprecipitation
of Ras by anti-Ras antibody immobilized to Sepharose beads. As
illustrated in Fig. 3A,
C-glucosylated Ras
was precipitated only from lysates treated with LT but not from
ToxB-treated lysates. The precipitated Ras comigrates in SDS-PAGE with
the faint lower band. Immunoblot analysis of the precipitated Ras
protein showed that both samples had the same amount of protein (data
not shown). These results were reproduced with lysates from NIH 3T3
cells, but the amount of precipitated Ras was lower than from rat brain
lysate (data not shown). These precipitation experiments indicate that
cellular Ras is a substrate for LT. To test whether Ras is glucosylated
in the intact cell, differential glucosylation was applied. If LT
catalyzes glucosylation of Ras in the intact cell, subsequent
C glucosylation of the lysate should show decreased
incorporation of [
C]glucose. Because of the low
concentration of Ras in NIH 3T3 cells, Ras was enriched by
immunoprecipitation with anti-Ras after the
C
glucosylation. As shown in Fig. 3B, treatment of intact
cells with LT completely blocked subsequent glucosylation of the
lysate, indicating previous modification in the intact cell.
Figure 3:
Immunoprecipitation of glucosylated Ras. A, lysates from rat brain homogenate were incubated with LT (4
µg/ml) or ToxB (1 µg/ml) in the presence of
UDP-[C]glucose (40 µM for LT and 10
µM for ToxB) for 45 min at 37 °C. Before the addition
of anti-Ras beads, samples (lanes 1 and 3) were
applied directly to SDS-PAGE. The beads were incubated with the lysates
for 2 h at 4 °C by agitating head-over-head. Thereafter, beads were
washed 3 times with radioimmune precipitation buffer and boiled with
sample buffer. The proteins were separated by 12.5% SDS-PAGE and
C incorporation was evaluated with the PhosphorImager. Lane 1, lysate glucosylated with LT; lane 2,
precipitation of Ras from lysate glucosylated with LT; lane 3,
lysate glucosylated with ToxB; lane 4, precipitation of Ras
from lysate glucosylated with ToxB. B, NIH 3T3 cells were
incubated without and with LT (40 ng/ml) for 9 h. Thereafter, cell
lysates were prepared and
C-glucosylated with LT. Ras was
enriched by immunoprecipitation as described above and separated by
12.5% SDS-PAGE followed by evaluation of
C incorporation
with the PhosphorImager. Lane 1, Ras from control cells; lane 2, Ras from cells intoxicated with
LT.
To test whether glucosylation induced inactivation of Ras resulting in decreased MAP kinase activity, EGF-stimulated p42/p44 MAP kinase activity was determined. Serum-starved NIH 3T3 cells were treated with LT and ToxB, respectively, until the cells showed the typical morphology. The Ras-regulated MAP kinase signal pathway was stimulated with EGF for 10 min. In cell lysates phosphorylation of specific substrates of p42/p44 MAP kinases were determined in the linear phase of the phosphorylation reaction. As depicted in Fig. 4, EGF stimulation increased basal p42/p44-induced phosphorylation by factor of about 2.5. The same was true for lysates from ToxB-treated cells, which showed no difference in basal and stimulated phosphorylation compared with control. In contrast, LT treatment completely inhibited EGF-stimulated kinase activity without altering basal MAP kinase activity. Even after prolonged incubation times, stimulated phosphorylation was not different from basal values, indicating complete inactivation of Ras by LT. The same results were obtained with stimulation by 10% fetal calf serum (not shown).
Figure 4: Effect of C. sordellii LT on the p42/p44 MAP kinase. Serum-starved NIH 3T3 cells were treated with LT (40 ng/ml) or ToxB (0.3 ng/ml) for 9 h followed by stimulation with EGF (2 ng/ml) for 10 min. Thereafter, cell lysates were prepared, and the p42/p44 MAP kinase-induced phosphorylation of substrate peptides was determined in a filter assay as described under ``Experimental Procedures.'' EGF-stimulated phosphorylation of control cells was set at 100%. The data are given as mean values (±S.E., n = 6).
C. sordellii lethal toxin belongs to the family of
large clostridial cytotoxins, which is characterized by a single-chain
structure and a molecular mass of 250-300 kDa(11) . This
family, which comprises C. difficile ToxA and ToxB, C.
novyi -toxin, C. sordellii lethal toxin (LT), and
hemorrhagic toxin, exhibits cytotoxicity by affecting predominantly the
microfilament cytoskeleton. ToxA and ToxB, which are coexpressed by
pathogenic C. difficile strains, show an identity of 49% (63%
homology) at the amino acid level(27) . LT from C.
sordellii has 76% identity (90% homology) with ToxB and 47% with
ToxA. Thus, LT is more closely related to ToxB than ToxB is to
ToxA(8) . This high degree of identity prompted us to study
whether ToxB and LT share the same molecular mode of action. Here we
present evidence that LT is a glucosyltransferase that selectively uses
UDP-glucose as a cosubstrate as was shown previously for ToxA and ToxB.
UDP-galactose or UDP-N-acetylglucosamine are not used. Whereas
ToxA and ToxB glucosylate the Rho subfamily proteins Rho, Rac and
Cdc42(12, 13) , LT modifies only one member of this
subfamily, namely Rac. Moreover, Ras, which is the prototype of a
separate subfamily of the large superfamily of low molecular mass
GTPases, is a substrate for LT but not for the C. difficile toxins. The Rho subfamily has been shown to be involved in the
regulation of the actin cytoskeleton. Rho participates in the formation
of focal adhesions and stress fibers, whereas Rac regulates membrane
ruffling and Cdc42 regulates the formation of microspikes and
filopodia(15, 16, 17, 18, 28) .
In addition, Rac exhibits a cell type-specific function in neutrophil
granulocytes, where it regulates superoxide anion production (29) . Recently, the Rho proteins have been identified as being
involved in the activation of transcription factors via the Ras pathway (30) and a Ras-independent signal
cascade(31, 32, 33) . Thus, LT as well as
ToxA/B could act in two ways. First, they inactivate the regulatory
proteins of the actin cytoskeleton. Second, they may interfere with
gene expression by inhibiting the signal cascade in which Rho and Ras
lead to activation of transcription factors.
The morphological alterations of cultured cells induced by LT are different from those caused by ToxB. Whereas ToxB causes cell shrinkage, resulting in cell adhesion points resembling neurite-like extensions, LT induces merely rounding of cells(3, 5, 11) . However, despite the different morphology both toxins predominantly cause destruction of the actin filament system. The different features in morphology and the recent finding that overexpression of the isoforms RhoA, B, and C increases resistance to ToxA and ToxB but not to LT (34) are consistent with the differences in the target proteins for these toxins.
In cell lysates LT exhibits glucosyltransferase
activity leading to the modification of cellular proteins.
Glucosylation of the recombinant target proteins, however, depends on
the presence of a cellular factor, which has been identified as the
cation Mn. Combination of the cytosolic subfraction
F500 with Mn
did not result in further stimulation,
and the bivalent cation chelator EDTA blocked the stimulatory effect of
the F500 fraction as well as that of Mn
, indicating
that Mn
is actually the cellular factor required for
LT enzymic activity. Stimulation by Mn
is also
observed with eukaryotic glycosyltransferases (24, 35) . In contrast, Mn
does not
enhance transferase activity of ToxA or ToxB. This finding suggests
that Mn
does not compete with Mg
binding at Rac but most likely acts directly on LT. Since EDTA
has no effect on ToxA/B transferase activity, the possibility of
binding of Mn
to ToxB can be excluded. Therefore, the
Mn
dependence of LT activity may reflect structural
differences between LT and ToxB, although both toxins show 90%
homology. As observed with ToxA/B, LT glucosylates recombinant
proteins, indicating that isoprenylation, the functionally relevant
posttranslational modification of low molecular mass GTPases, is not
essential for glucosylation.
The acceptor amino acid of both ToxB-
and ToxA-glucosylated RhoA has been determined as
Thr-37(12, 13) . Thr-37, which corresponds to Thr-35
in Rac, is located in the effector domain and is crucial for GTP
binding. As can be deduced from the crystal structure of Ras, the
hydroxy group of threonine is ligand for Mg, which
coordinates the
- and
-phosphates of
GTP(26, 36) . In the GDP-bound form, the hydroxy group
is exposed to surface of the molecule and is accessible for
glucosylation. Consistent with this concept is the finding that
GDP-bound Rac and Ras, respectively, are glucosylated to a greater
extent than the GTP-bound form.
The sequential glucosylation of Rac by LT followed by ToxB, and vice versa indicates that both toxins share the same acceptor amino acid in Rac. This finding was verified by the technique of deglucosylation. The deglucosylation of LT-modified Rac by LT and ToxB, respectively, gives evidence that ToxB and LT glucosylate Rac at the same acceptor amino acid, namely Thr-35.
Applying immunoprecipitation we showed that Ras from cell lysates is a substrate for LT. Furthermore, using the method of differential glucosylation we present evidence that Ras is actually a substrate in the intact cell. Inhibition of EGF- and fetal calf serum-stimulated p42/p44 MAP kinases (extracellular regulated kinase 1/extracellular regulated kinase 2) in LT-treated cells indicates that glucosylation renders Ras inactive, resulting in a blocked signal pathway. Inhibition of the MAP kinase pathway is due to glucosylation of Ras but not of Rac, because ToxB that glucosylates Rac does not alter basal or EGF-stimulated p42/p44 MAP kinase activity. Furthermore, the possibility of indirect effects of the destroyed cytoskeleton on MAP kinases can be excluded because LT causes comparable depolymerizing of the actin filaments, as does ToxB.
It is likely that glucosylation of Rac, which is reportedly involved in the regulation of the cortical actin cytoskeleton and which acts upstream of Rho(16, 28) , contributes to the cytotoxic effects of LT. Recently, Hall and co-workers (16) presented evidence that oncogenic Ras acts upstream of the Rac protein, which itself is upstream of Rho subtype proteins. There is some evidence that oncogenic Ras mediates malignant phenotype by interfering with Rho subfamily proteins(37) . However, at the moment it is not clear whether inactivation of Ras by glucosylation does contribute to the morphological effects of LT or whether inactivation of Ras results only in blockade of cell proliferation, which may be effective at conditions of long term intoxication.
Ras is also a substrate of Pseudomonas aeruginosa exoenzyme S, which catalyzes ADP-ribosylation. However, Ras belongs to a group of heterogenous substrate proteins including Ral, Rap1A, Rap2, Rab3, Rab4, and even the intermediate filament protein vimentin(38, 39) . Furthermore, the substrate specificity depends on exoenzyme S concentration(38) . In contrast, the substrate specificity of LT does not vary with concentration of LT. The most important difference, however, is that LT modifies Ras in intact cells, whereas exoenzyme S acts on Ras in cell lysates only.
In conclusion, C. sordellii lethal toxin (LT) has been identified as a glucosyltransferase that uses UDP-glucose as cosubstrate to modify Ras and Rac proteins. LT belongs to the novel family of clostridial glucosyltransferases that glucosylate the crucial threonine residue in the effector domain of low molecular mass GTP-binding proteins. Despite the high homology of C. sordellii lethal toxin to C. difficile ToxB, lethal toxin differs in its substrate specificity and the cofactor dependence. LT renders Ras inactive by glucosylation, resulting in inhibition of the EGF-stimulated MAP kinase pathway. LT is the first bacterial toxin that causes inactivation of Ras in intact cells.