(Received for publication, July 24, 1995; and in revised form, December 18, 1995)
From the
We have previously isolated a fibroblast mutant cell with high
resistance to the two Rho-modifying glucosyltransferase toxins A and B
of Clostridium difficile. We demonstrate here a low level of
UDP-glucose in the mutant, which explains its toxin resistance since:
(i) to obtain a detectable toxin B-mediated Rho modification in lysates
of mutant cells, addition of UDP-glucose was required, and it promoted
the Rho modification dose-dependently; (ii) high pressure liquid
chromatography analysis of nucleotide extracts of cells indicated that
the level of UDP-glucose in the mutant (0.8 nmol/10 cells)
was lower than in the wild type (3.7 nmol/10
cells); and
(iii) sensitivity to toxin B was restored upon microinjection of
UDP-glucose. Using the mutant as indicator cell we also found that the
related Clostridium sordellii lethal toxin is a
glucosyltransferase which requires UDP-glucose as a cofactor. Like
toxin B it glucosylated 21-23-kDa proteins in cell lysates, but
Rho was not a substrate for lethal toxin.
Antibiotic-associated diarrhea and its potentially fatal form
pseudomembranous colitis are human diseases caused by the anaerobic
intestinal pathogen Clostridium difficile(1) . Only
strains producing the enterotoxin (ToxA) ()and the cytotoxin
(ToxB) are pathogenic(1) . Both toxins disrupt the actin
cytoskeleton, causing rounding of cultured cells (2, 3) and inhibition of cytokinesis(4) . The
cytoskeletal collapse is mediated via a modification of the small
GTPases Rho, Rac, and Cdc42, members of the Rho subfamily of
Ras-related proteins(5, 6) . Recently the molecular
mechanism for this modification has been
resolved(7, 8) . A glucose moiety is covalently
coupled to amino acid Thr-37 (determined for RhoA), located in the
putative effector domain of the GTPase. As a cofactor for this
enzymatic reaction UDP-glucose (UDP-Glc) is required. Thus both the C. difficile toxins were defined as
glucosyltransferases(7, 8) .
The C. difficile toxins together with Clostridium sordellii hemorrhagic
toxin and lethal toxin (LT) as well as Clostridium novyi -toxin belong to the family of large clostridial
cytotoxins(9) . Antibodies to LT cross-react with ToxB, but the
cytopathogenic effects (CPE) induced by these toxins differ
morphologically(10) . The molecular mode of action of LT is
unknown.
No cell type with a natural resistance against the C.
difficile toxins is known. Only after chemical mutagenesis of
Chinese hamster fibroblasts (Don cells) were we able to isolate a
mutant cell which is highly resistant to both the C. difficile toxins(11) . However, extreme doses of the toxins still
induce rounding of mutant cells. The difference in sensitivity as
compared to the wild type cell is 10 for ToxB and 10
for ToxA(11) . The mutant cell is resistant also to
microinjected ToxB whereas wild type cells are sensitive. (
)Thus, the ToxB resistance of the mutant cell cannot be
explained by either a mutation in the cell surface receptor or in the
endocytic (12) internalization process.
The present study was undertaken to elucidate the molecular basis for the toxin resistance of this cell. We report that the level of UDP-Glc is lowered in the mutant cell and that sensitivity to ToxB can be restored by microinjection of UDP-Glc. Using the mutant cell as indicator we also found that LT from C. sordellii acts as a glucosyltransferase which requires UDP-Glc as a cofactor.
Alternatively, the difference in amount of UDP-Glc between wild type
and mutant cells was estimated by enzymatic conversion of endogenous
UDP-Glc to UDP-GlcUA (15) followed by determination of the area
of the UDP-GlcUA peak generated by ion exchange HPLC(16) .
Briefly, protein-free extracts of cells were prepared by addition of 2
volumes of 10% trichloroacetic acid to pelleted cells. After incubation
(30 min, 4 °C) and centrifugation, the trichloroacetic acid was
extracted twice with ether. 30 µl of the preparation were added to
70 µl of reaction buffer (0.27 M glycine, 1.6 mM NAD, 4.3 mM EDTA; pH 8.7) and the reaction initiated by
addition of UDP-Glc:dehydrogenase (2 µl, 65 milliunits/ml). After
incubation for 1 h at 22 °C when the reaction was completed
(monitored by the formation of NADH at 340 nm), the whole mixture (100
µl) was loaded onto an anion exchange column (QMA MemSep,
Millipore) and eluted for 50 min with a linear gradient of
KHPO
(0.1-0.5 M; pH 4.5). The
eluting nucleotides were detected at 254 nm and the corresponding areas
calculated.
Figure 1:
Modification of Rho in wild type and
mutant cells treated with ToxB. Wild type (lane 2) and mutant
cells (lanes 4 and 6) were treated at 37 °C with
ToxB (200 ng/ml) for 1 h (lanes 2 and 4) or 16 h (lane 6). Controls without toxin: wild type (lane 1)
and mutant, 1 and 16 h (lanes 3 and 5). All wild type
and mutant cells were completely rounded after 1- and 16-h toxin
treatment, respectively (lanes 2 and 6), whereas the
mutant cells treated for 1 h were morphologically unaltered (lane
4). Cell lysates were prepared and Rho was labeled by C3-catalyzed
[P]ADP-ribosylation as described under
``Experimental Procedures.'' Proteins were separated by 12.5%
SDS-PAGE, and radiolabeled bands detected by PhosphorImager
analysis.
Figure 2:
ToxB-induced modification of Rho in wild
type and mutant cell lysates in the absence and presence of UDP-Glc.
Lysates of wild type (lane 2) and mutant (lane 4)
cells were treated with ToxB (2 µg/ml) for 1 h at 37 °C.
Controls without toxin: wild type (lane 1) and mutant (lane 3). Rho in the cell lysates was radiolabeled by
C3-catalyzed [P]ADP-ribosylation as described
under ``Experimental Procedures.'' Proteins were separated by
12.5% SDS-PAGE, and radiolabeled bands detected by PhosphorImager
analysis. Panel A, without UDP-Glc; panel B, with 100
µM UDP-Glc in the lysis
buffer.
Figure 3:
Influence of UDP-Glc on ToxB-induced
modification of Rho in wild type and mutant cell lysates; dose response
curves. Lysates of wild type and mutant cells were treated for 1 h at
37 °C with ToxB (2 µg/ml) in lysis buffer with increasing
concentrations of UDP-Glc. Rho in the cell lysates was radiolabeled by
C3-catalyzed [P]ADP-ribosylation as described
under ``Experimental Procedures.'' Proteins were separated by
12.5% SDS-PAGE, and radiolabeled bands detected by PhosphorImager
analysis. Panel A, lanes from left to right: 0, 0.009, 0.018,
0.037, 0.075, 0.150, 0.30, 1.5, 3.0, 6.0, 12.5, 25, and 100 µM UDP-Glc; C, controls without toxin. Panel B, the
bands shown in panel A were quantified using Image Quant
software and percent modification was calculated as described under
``Experimental Procedures.''
These results suggested that the mutant
cell may have less UDP-Glc available for glucosylation than the wild
type. However, the data are also consistent with the possibility that
the mutant cell could contain components acting as competitive
inhibitors of either Rho or UDP-Glc. The first possibility was assessed
by titrating the toxin. At each toxin dose the Rho modification was
identical in lysates from both cell types (Fig. 4A).
This excludes competitive interference with Rho or with the toxin by
some factor present only in the mutant. The second possibility was
assessed by directly labeling the substrates with decreasing amounts of
UDP-[C]Glc. If a competitive inhibitor of
UDP-Glc was present in mutant cell lysates, the substrates should be
less labeled than substrates in the wild type at low concentrations of
UDP-[
C]Glc. In contrast they were more labeled,
ruling out this possibility (Fig. 4B). On the other
hand the observed result could be expected if the mutant has a lower
level of UDP-Glc, since this would imply less competition from
endogenous UDP-Glc with the added UDP-[
C]Glc.
Figure 4:
Modification of ToxB substrates at
decreasing amounts of toxin and UDP-[C]Glc. Panel A, lysates of wild type and mutant cells were treated in
the presence of 100 µM UDP-Glc for 1 h at 37 °C with
different amounts of toxin B (inset, from left to right: 2, 8,
24, 74, 222, 666, and 2000 ng/ml). Rho in the cell lysates was then
radiolabeled by C3-catalyzed [
P]ADP-ribosylation
as described under ``Experimental Procedures.'' Proteins were
separated by 12.5% SDS-PAGE, and radiolabeled bands detected and
quantified by PhosphorImager analysis. Percent ToxB-induced
modification was calculated as described under ``Experimental
Procedures.'' Panel B, lysates of wild type and mutant
cells were treated for 1 h at 37 °C with ToxB (6 µg/ml) in the
presence of different amounts of UDP-[
C]Glc (inset, from left to right: 0.75, 1.5, 3, 6, 12, 25, 50, and
100 µM). Proteins were separated and analyzed as for panel A. Percent labeling was calculated in relation to the
labeling intensity registered at 100 µM UDP-[
C]Glc.
Figure 5: Quantification of UDP-Glc by HPLC analysis. Panels A and B, nucleotides were extracted from wild type (panel A) and mutant (panel B) cell monolayers and separated by reverse phase HPLC (see ``Experimental Procedures''). The relevant peak (No. 2, retention time 7.8 min) was identified by addition of 100 µM UDP-Glc as an internal standard (accounting for 40% of the peak area in the wild type). Amounts of UDP-Glc in the extracts were calculated from the integrated peak areas as described under ``Experimental Procedures.'' All other major peaks (numbered 1 and 3-5 to facilitate comparison) are in close agreement between mutant and wild type. Panels C and D, nucleotides were extracted from wild type (panel C) and mutant (panel D) cell pellets, the UDP-Glc was enzymatically converted to UDP-GlcUA, and the latter subsequently separated and quantified by ion exchange HPLC (see ``Experimental Procedures''). After subtraction of the endogenous levels of UDP-GlcUA (which were identical in wild type and mutant cells, accounting for 20% of the area in the wild type) the areas of the enzymatically generated UDP-GlcUA were compared. The position of UDP-GlcUA was determined using a purified standard which eluted at 27.6 min.
Figure 6: Effect of microinjection of UDP-Glc into mutant cells treated with ToxB. Panel A, semiconfluent mutant cells were incubated at 37 °C with 12 ng/ml ToxB. After 1 h, when similarly treated wild type cells had developed a complete CPE, the mutant cells were microinjected with 100 mM UDP-Glc (1 and 2) or 100 mM UDP-GlcUA (3 and 4) in buffer containing 2% FITC-dextran to localize microinjected cells. The cells were further incubated at 37 °C for 30 min, then fixed and photographed as described under ``Experimental Procedures.'' Panel B, mutant cells were treated with ToxB as described above and then microinjected with 100, 50, or 10 mM UDP-Glc in buffer containing 2% FITC-dextran. Approximately 100 cells were microinjected per experiment. The percentage of affected cells was calculated as described under ``Experimental Procedures.'' Mean values S.D. of three experiments are presented.
The normal
intracellular concentration of UDP-Glc is reported to be approximately
100 µM(18) , and dilution of microinjected
compounds is calculated to be 1/100. Thus microinjection of 50 mM UDP-Glc should restore the cellular level to the order of
magnitude reported to be physiological. This amount (the
ED) was only 5 times higher than an amount which had no
effect (10 mM), and half the amount which gave almost full
effect, showing that the concentrations used were in the physiological
range of cellular UDP-Glc concentration. This experiment also
demonstrated that a 5 times difference in UDP-Glc concentration (from
10 to 50 mM) could in fact be a limiting factor for toxin
B-mediated CPE. In conclusion, the ToxB-resistance of the mutant cell
was abolished by raising its cytosolic level of UDP-Glc.
Figure 7: Effect of LT on wild type and mutant cells. Panel A, semiconfluent cultures of wild type and mutant cells growing in 96-well microtiter plates were treated at 37 °C with LT (625 ng/ml), and the percentage of rounded cells after the indicated time periods was calculated. Panel B, wild type (2) and mutant cells (4) incubated for 8 h with LT (625 ng/ml). Controls without toxin: wild type (1) and mutant (3). The cells were fixed and stained with FITC-phalloidin as described under ``Experimental Procedures.''
As these findings
indicated a role for UDP-Glc also in intoxication by LT, its ability to
transfer glucose to cellular proteins was examined. At least two
21-23-kDa proteins were labeled in cell lysates treated with LT
in the presence of UDP-[C]Glc (Fig. 8A, lane 3). The labeled bands migrated roughly
as those labeled by ToxB (Fig. 8A, lane 1). To decide
whether also LT uses Rho as a substrate, lysates were first exposed to
C3 in the presence of non-labeled NAD in order to ADP-ribosylate Rho.
They were then treated with ToxB or LT in the presence of
UDP-[
C]Glc. The upper ToxB-glucosylated band (Fig. 8A, lane 2) was identified as Rho because of the
shift in the molecular weight due to ADP-ribosylation. In contrast,
there was no shift in any of the LT-glucosylated bands after the same
treatment (Fig. 8A, lane 4), suggesting that Rho is not
a substrate for LT. This notion was verified with recombinant Rho which
served as a substrate for ToxB but not for LT (Fig. 8B). We conclude that LT acts as a
glucosyltransferase. The substrates for LT were subsequently identified
as Ras, Rac, and Rap(30) .
Figure 8:
Glucosylation of 21-23-kDa proteins
in cell lysates and recombinant Rho-glutathione S-transferase
fusion protein by ToxB and LT. Panel A, lysates from wild type
cells were incubated without (lanes 1 and 3) or with (lanes 2 and 4) 10 µg/ml C3 and 2 mM NAD
for 1 h at 37 °C. The lysates were then treated with 6 µg/ml
ToxB (lanes 1 and 2) or 5 µg/ml LT (lanes 3 and 4) in the presence of
UDP-[C]Glc (100 µM) at 37 °C
for 1 h. Proteins were separated by 12.5% SDS-PAGE, and radiolabeled
bands detected by PhosphorImager analysis. Panel B, recombinant Rho-glutathione S-transferse fusion protein
(1 µg) was treated with 6 µg/ml ToxB (lanes 1 and 3) or 5 µg/ml LT (lanes 2 and 4) in the
presence of UDP-[
C]Glc (100 µM) at
37 °C for 1 h. Proteins were separated on 12.5% SDS-PAGE, and
radiolabeled bands detected by PhosphorImager analysis (lanes 1 and 2). Lanes 3 and 4, corresponding
Coomassie Blue-stained gel.