From the Institut für Pharmakologie und
Toxikologie der Albert-Ludwigs-Universität Freiburg,
Hermann-Herder-Str. 5, D-79104 Freiburg, Germany, ¶ Medical
Research Council Laboratory of Molecular Biology, Hills Road, Cambridge
CB2 2QH, United Kingdom, and
Biochemie-Zentrum Heidelberg, INF
328, D-69120 Heidelberg, Germany
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ABSTRACT |
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A fragment of the N-terminal 546 amino acid residues of Clostridium sordellii lethal toxin possesses full enzyme activity and glucosylates Rho and Ras GTPases in vitro. Here we identified several amino acid residues in C. sordellii lethal toxin that are essential for the enzyme activity of the active toxin fragment. Exchange of aspartic acid at position 286 or 288 with alanine or asparagine decreased glucosyltransferase activity by about 5000-fold and completely blocked glucohydrolase activity. No enzyme activity was detected with the double mutant D286A/D288A. Whereas the wild-type fragment of C. sordellii lethal toxin was labeled by azido-UDP-glucose after UV irradiation, mutation of the DXD motif prevented radiolabeling. At high concentrations (10 mM) of manganese ions, the transferase activities of the D286A and D288A mutants but not that of wild-type fragment were increased by about 20-fold. The exchange of Asp270 and Arg273 reduced glucosyltransferase activity by about 200-fold and blocked glucohydrolase activity. The data indicate that the DXD motif, which is highly conserved in all large clostridial cytotoxins and also in a large number of glycosyltransferases, is functionally essential for the enzyme activity of the toxins and may participate in coordination of the divalent cation and/or in the binding of UDP-glucose.
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INTRODUCTION |
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The family of large clostridial cytotoxins comprises
Clostridium difficile toxins A and B, the lethal toxin
(LT)1 and hemorrhagic toxin
of Clostridium sordellii, and the -toxin from
Clostridium novyi (1, 2). Whereas C. difficile
toxins A and B are the major virulence factors of the
antibiotic-associated diarrhea and pseudomembranous colitis (3-6), the
toxins from C. sordellii and C. novyi are
implicated in gas gangrene syndrome (7, 8). All these toxins possess
glycosyltransferase activity and modify small GTPases of the Rho family
(2, 9-12). The toxins differ in cosubstrate and protein substrate
specificity. Whereas
-toxin from C. novyi catalyzes the
N-acetylglucosaminylation by using UDP-GlcNAc as a
cosubstrate (13), the other toxins use UDP-glucose and cause
glucosylation of the protein targets. All Rho subfamily proteins
including Rho, Rac, and Cdc42 are modified by C. difficile
toxins A and B, the hemorrhagic toxin of C. sordellii, and
the C. novyi
-toxin (9, 10, 13, 14). By contrast, the
lethal toxin from C. sordellii glucosylates Rac and Cdc42 but not (or much less) Rho. In addition, Ras proteins (Ras, Rap, Ral)
are modified by C. sordellii lethal toxin (11).
Toxin-induced glucosylation of low molecular mass GTPases occurs at Thr37 of Rho (Thr35 of all other GTPases) (10). This amino acid residue is conserved in all low molecular mass GTPases and involved in divalent cation and nucleotide binding. Moreover, Thr35/Thr37 is located in the effector region of the GTPases, and glucosylation appears to block the interaction of the GTPases with their effectors (15, 16). Thus, glucosylation of Rho proteins by large clostridial cytotoxins renders the GTPases biologically inactive (17).
The large clostridial cytotoxins possess molecular masses between 250 and 308 kDa and are, therefore, the biggest bacterial toxins known. It has been suggested that these toxins are constructed of three functional parts (18). The C terminus, which consists of a large region of repetitive oligopeptides, is assumed to be important for receptor binding to the eukaryotic target (19). A hydrophobic region almost in the middle of the toxins is believed to participate in membrane translocation, and finally, the N terminus harbors the enzyme activity. Deletion analysis performed with C. difficile toxin B, and with C. sordellii lethal toxin revealed that a fragment consisting of the N-terminal 546 amino acid residues possesses full glucosyltransferase activity and is able to induce the typical cytotoxic effects after microinjection (20, 21).
Recently, it has been reported that various families of glycosyltransferases, which exhibit high sequence homology within the same family but no overt similarity between different families, share a common small motif consisting of DXD, which appears to be essential for enzyme activity (26). Sequence alignments of large clostridial cytotoxins with these glycosyltransferases revealed that the same motif is also conserved in the bacterial toxins. Therefore, we studied whether this short motif has any functional relevance and is involved in enzyme activity of the toxins. Here we report that the exchange of aspartic acid residues 286 and 288, which form the DXD motif in C. sordellii lethal toxin, decreases the glucosyltransferase activity several thousand-fold and blocks the glucohydrolase activity, indicating an essential role in catalysis. Moreover, we identified additional amino acid residues located in close vicinity of the DXD motif, which are of functional importance and conserved in all large clostridial cytotoxins.
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EXPERIMENTAL PROCEDURES |
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Materials-- 14C-labeled UDP-hexoses were obtained from DuPont NEN Life Science Products (Dreieich, Germany). Polymerase chain reaction primers were from MWG Biotech (Ebersberg, Germany). All other reagents were of analytical grade and purchased from commercial sources.
Polymerase Chain Reaction Amplification-- Amplification of the C. difficile toxin B fragment CDB1 and C. sordellii CS1 and construction of C-terminal truncated fragments B546 and LT546 were performed as described previously (20, 21).
Site-directed Mutagenesis of Toxin Fragments LT546 and B546-- The QuikChange KitTM (Stratagene) was used for mutating one or two nucleotides in the pGEX2T-LT546 construct or in the pGEX2T-B546 construct, respectively. Procedures were carried out according to the manufacturer's instructions. Primers were constructed as follows: D270ALT546, primer pair S1D270Asen/anti (5'-CTGCTGCTTCTG CTATATTACGAATATC-3'/5'-GATATTCGTAATATAGCAGAAGCAGCAG-3'); R273ALT546, primer pair S1R273Asen/anti (5'-CTTCTGATATATTAGCAATATCTATGTTAAAAG-3'/5'-CTTTTAACATAGATATTGCTAATATATCAGAAG-3'); K278ALT546, primer pair S1K278Asen/ anti (5'-CGAATATCTATGTTAGCAGAAGATGGTGG-3'/5'-CCACCATCTTCTGCTAACATAGATATTCG-3'); G281ALT546, primer pair S1G281Asen/anti (5'-GTTAAAAGAAGATGCTGGTGTATATTTAG-3'/5'-CTAAATATACACCAGCATCTTCTTTTAAC-3'); G282ALT546, primer pair S1G282Asen/anti (5'-GTTAAAAGAAGATGGTGCTGTATATTTAGATG-3'/5'-CATCTAAATATACAGCACCATCTTCTTTTAAC-3'); Y284ALT546, primer pair S1Y284Asen/anti (5'-GATGGTGGTGTAGCTTTAGATGTTGAC-3'/5'-GTCAACATCTAAAGCTACACCACCATC-3'); D286ALT546, primer pair S1D286Asen/anti (5'-GTGGTGTATATTTAGCTGTTGACATCTTAC-3'/5'-GTAAGATGTCAACAGCTAAATATACACCAC-3'); D288ALT546, primer pair S1D288Asen/anti (5'-GTATATTTAGATGTTGCCATCTTACCAGG-3'/5'-CCTGGTAAGATGGCAACATCTAAATATAC-3'); D286NLT546, primer pair S1D286Nsen/anti (5'-GGTGGTGTATATTTAAATGTTGACATCTTAC-3'/5'-GTAAGATGTCAACATTTAAATATACACCACC-3'); D288NLT546, primer pair S1D288Nsen/anti (5'-GTATATTTAGATGTTAACATCTTACCAGGTA-3'/5'-TACCTGGTAAGATGTTAACATCTAAATATAC-3'); D286A/D288ALT546, primer pair S1D286AD288Asen/anti (5'-GTGTATATTTAG CTGTTGCCATCTTACCAG-3'/5'-CTGGTAAGATGGCAACAGCTAAATATACAC-3`); P291ALT546, Primer pair S1G291Asen/anti (5'-GATGTTGACATCTTAGCAGGTATACAACC-3'/5'-GGTTGTATACCTGCTAAGATGTCAACATC-3'); D286AB546, primer pair B1D286sen/ anti (5'-GTGGTATGTATTTAGCTGTTGATATGTTAC-3'/5'-GTAACATATCAACAGCTAAATACATACCAC-3'); D288AB546, primer pair B1D288sen/anti (5'-GTATTTAGATGTTGCTATGTTACCAGGAAT -3'/5'-ATTCCTGGTAACATAGCAACATCTAAATAC-3').
Sequencing-- Sequencing of CS1 and CDB1, their truncated derivatives LT546 and B546, and the mutated clones was done with the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer) to check both for correct cloning and mutations due to polymerase chain reaction amplification. Sequencing was performed at least twice with overlapping DNA fragments.
Expression of Recombinant Proteins-- The recombinant GTP-binding proteins RhoA, Rac1, Cdc42, Ha-Ras, and Ral were prepared from their fusion proteins as described (10, 11). The recombinant toxin fragments were expressed and purified as glutathione S-transferase fusion proteins in accordance with the manufacturer's instructions (see Fig. 2). Glutathione S-transferase fusion proteins from the Escherichia coli expression vector pGEX2T were isolated by affinity chromatography with glutathione-Sepharose (Amersham Pharmacia Biotech, Germany) followed by removing glutathione S-transferase fusion proteins with glutathione elution buffer (10 mM glutathione in 50 mM Tris-HCl, pH 8.0). Recombinant Rap1a was a gift of Dr. A. Wittinghofer (MPJ Dortmund, Germany).
Glucosylation Assay-- Recombinant GTP-binding proteins (50 µg/ml) were incubated with recombinant toxin fragment LT546 or mutated fragments of LT at the indicated concentrations in a buffer containing 50 mM Hepes (pH 7.5), 100 mM KCl, 2 mM MgCl2, 1 mM MnCl2, 100 µg/ml bovine serum albumin, and 10 µM 14C-UDP-glucose for 30 min at 37 °C or for the indicated periods. The total volume was 20 µl. Labeled proteins were analyzed by SDS-PAGE and subsequently by PhosphorImager analysis (Molecular Dynamics, Inc.). Quantitative data are given as arbitrary units as means ± S.E. (n = 3).
Glucohydrolase Assay-- LT546 and the mutated fragments (at the indicated concentrations) were incubated with 20 µM 14C-labeled UDP-glucose and 100 µM unlabeled UDP-glucose in a buffer containing 50 mM Hepes (pH 7.5), 100 mM KCl, 2 mM MgCl2, 100 µM bovine serum albumin, 100 µM MnCl2. The total volume was 10 µl.
Samples of 1.5 µl were taken out at each time point and subjected to thin layer chromatography with PI-cellulose plates (Merck, catalog number 1.05579, Darmstadt, Germany) and 0.2 M LiCl as mobile phase in order to separate hydrolyzed glucose from UDP-glucose. The plates were dried and analyzed by PhosphorImager analysis. Quantitative data are given as pmol per time indicated as means ± S.E. (n = 3).Photoaffinity Labeling--
Indicated proteins (2 µg) were
incubated on ice, in the presence of 50 µM
[-32P]5N3-UDPGlc, reactions were allowed
to equilibrate for 30 s, followed by UV irradiation with a
hand-held UV lamp (254/366 nm) with the glass face removed at a
distance of 3 cm for 3 min. For competition experiments, 1 mM of unlabeled UDP-glucose was added to the reaction
mixture just before the addition of
[
-32P]5N3-UDPGlc. Reactions were
terminated by the addition of 5 µl of sample buffer. Thereafter,
radiolabeled proteins were analyzed by SDS-PAGE and PhosphorImager
analysis.
Microinjection Studies-- For microinjection studies, HeLa cells were grown for 24 h in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 4 mM glutamine/penicillin/streptomycin and plated on Cellocate (Eppendorf, Germany) coverslips at about 105 cells/dish at 37 °C and 5% CO2. Microinjection was performed with the Microinjector 5242 and Micromanipulator 5171 from Eppendorf.
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RESULTS |
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Effects of Alteration of the DXD Motif of C. sordellii Lethal Toxin on Glucosyltransferase and Glucohydrolase Activities-- It has been reported recently that the amino acid motif DXD is crucial for the activity of the yeast mannosyltransferase Mnn1p (26).2 This motif is also conserved among the large clostridial cytotoxins in a region of high homology of the toxins (Fig. 1). To elucidate the significance of this motif for the enzymatic activity of the toxins, we constructed various mutants of the enzymatically active N-terminal fragment LT546 of C. sordellii lethal toxin and expressed them in E. coli as glutathione S-transferase fusion proteins. The conserved amino acid residues were changed to alanine or asparagine, respectively, and Fig. 2A shows the SDS-PAGE analysis of the proteins.
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Photoaffinity Labeling of DXD Mutants--
To test whether the
DXD motif mutant proteins, which exhibited blocked or
largely reduced enzyme activity, were still able to interact with the
cosubstrate UDP-glucose, we performed photoaffinity labeling
experiments with azido-UDP-glucose
([-32P]5N3-UDP-glucose). Fig.
5 shows that the wild-type toxin fragment was labeled by [
-32P]5N3-UDP-glucose after
UV irradiation. The addition of unlabeled UDP-glucose blocked the
radiolabeling. By contrast, the single mutants D286ALT546 and
D288ALT546 and the double mutant D286A/D288ALT546 were not labeled,
suggesting that the respective aspartic acid residues are important for
UDP-glucose binding or for photoaffinity labeling with
azido-UDP-glucose.
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Effects of Manganese Ions on the Activity of DXD Mutants of C. sordellii Lethal Toxin-- Like various eukaryotic glycosyltransferases (22, 23) the glucosyltransferase activity of C. sordellii lethal toxin depends on manganese ions (11). Therefore, we studied whether alteration of the DXD motif affects the Mn2+ dependence of the glucosyltransferase activity of LT. As reported recently for the holotoxin, the active wild-type fragment of LT exhibited maximal activity at 1 mM Mn2+ (Fig. 6). The maximal increase in activity by the addition of manganese was by about 40% under the conditions used. Further increase in the concentration of the divalent cation > 1 mM reduced the enzyme activity. The single exchange of the aspartic acid residues of the DXD motif of LT changed the Mn2+ dependence. As shown in Fig. 6, the enzyme activity of the D288ALT546 mutant was affected at higher concentrations of Mn2+ than the wild-type fragment. Whereas at 10 mM Mn2+ the enzyme activity of the wild-type LT fragment was decreased, the enzyme activity of the mutant was increased by about 20-fold. Similar findings were obtained with the D286ALT546 mutant (not shown). By contrast, the double mutant was not affected by Mn2+, not even at a high concentration of the divalent cation (not shown).
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Microinjection Studies with DXD Mutants of C. sordellii Lethal Toxin-- In order to further characterize the mutant fragments, we tested their biological activity by microinjection studies. For this purpose, the wild-type toxin fragment or the mutant toxins were injected into HeLa cells. As reported recently for C. difficile toxin B, microinjection of the wild-type fragment LT546 induced the typical morphological changes observed after treatment of cells with the holotoxin (Fig. 7A). Changes occurred 30 min after treatment of cells. In contrast to these findings, no change in cell morphology was observed, even 4 h after microinjection, when the single mutant D288ALT546 (Fig. 7C) or D286ALT546 (not shown) or the double mutant protein D286AD288ALT546 (Fig. 7D) was applied.
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Effects of Mutations in the Surroundings of the DXD Motif--
We
also mutated various other amino acid residues that are conserved in
all clostridial toxins and are located in the region of the
DXD motif (Fig. 1). We tested the mutants K278ALT546,
G281ALT546, G282ALT546, and P291ALT546 for transferase activity with
Ras as a substrate. As can be seen in Fig.
8, all of these mutants exhibited similar
properties with respect to both glucosyltransferase and glucohydrolase
activity as compared with the wild-type toxin fragment, indicating no
essential roles in catalysis. Moreover, these mutants were fully active
after microinjection into culture cells (shown for K278ALT546; Fig.
7B) and could be labeled by
[-32P]5N3-UDP-glucose (shown for
G282ALT546; Fig. 5). By contrast, exchange of amino acid residues
Tyr284, Asp270, or Arg273 to
alanine decreased the enzyme activity, whereas the transferase and
glycohydrolase activity of the Tyr284 mutant was reduced by
40- and 50-fold, respectively (Fig. 9). The glucosyltransferase activities of the D270ALT546 and R273ALT546 mutants were reduced by at least 200-fold (Fig. 9), and no
glucohydrolase activity was detected (Fig. 9C). Whereas
microinjection of the Y284ALT546 mutant induced the typical
cytotoxicity, the D270ALT546 and R273ALT546 mutants exhibited variable
results. Thus, we cannot exclude the possibility that these mutant
proteins possess a small cytotoxic activity dependent on the amount of
toxin microinjected.
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DISCUSSION |
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Recently, we and others reported that the N terminus of C. difficile toxin B and of the lethal toxin from C. sordellii harbors the glucosyltransferase activity of the toxins
(20, 21, 24). Here we continued the structure-function analysis of this
family of large clostridial cytotoxins and report the demonstration
that a small motif formed by two aspartic acid residues
(DXD) appears to be essential for enzyme activity of the
toxins. Changes of the aspartic acid residues at positions 286 and 288 of C. sordellii lethal toxin to alanine or asparagine
reduced the enzyme activity by at least 5000-fold. Microinjection
studies corroborated that these mutant proteins had lost their
cytotoxic activity. Because the amino acid exchanges blocked
transferase and glycohydrolase activity but did not cause major changes
in the sensitivity toward trypsin digestion, we suggest that these
aspartic acid residues are essentially involved in the enzyme activity
of the toxin. The finding that also in C. difficile toxin B
changes of the DXD motif caused inhibition of the enzyme
activity supports their essential role. In fact, these aspartic acid
residues appear to form a highly conserved motif (DXD),
which is present not only in all large clostridial cytotoxins but also
in a large number of different eukaryotic transferases (26). Recently,
it has been shown that the DXD motif is essential for the
Golgi -1,3-mannosyltransferase Mnn1p of S. cerevisiae
(26) (see Fig. 1). The same motif, which is frequently surrounded by a
hydrophobic region, was recognized in several related transferases such
as in
-1,4-galactosyltransferases,
-1,3-galactosyltransferases,
glucuronyltransferases, fucosyltransferases, glycogenins, and others
(26). Moreover, this motif is observed also in various bacterial
glycosyltransferases like LPS synthases. Of all of the
glycosyltransferases containing the DXD motif, those showing
most similarity to the clostridial toxins in the region surrounding the
motif were members of the family related to the Och1p
-1,6-mannosyltransferase (Fig. 1).
It has been suggested that the DXD motif participates in the
coordination of the divalent cation by glycosyltransferases (26). It
has been shown that the activity of C. sordellii lethal
toxin depends on the presence of Mn2+ ions (11). Moreover,
recent studies in our laboratory indicate that the activities of all
large clostridial cytotoxins depend on Mn2+
ions.2 Divalent cations
(mostly manganese ions) are suggested to be important for the binding
of the nucleotide-sugar that serves in all glycosyltransferases as the
cosubstrate (25). The notion that the DXD motif is somehow
involved in the UDP-glucose binding of LT is supported by the
photoaffinity studies with azido-UDP-glucose. The wild-type fragment of
LT was radiolabeled in the presence of
[-32P]5N3-UDP-glucose after UV
irradiation. The incorporation of azido-UDP-glucose was specific by the
criterion of competitive inhibition induced by excess unlabeled
UDP-glucose. By contrast, mutants of the DXD motif were not
photoaffinity-labeled in the presence of the azido-UDP-glucose derivative. Interestingly, at high concentrations of Mn2+
ions, the activity of the single DXD mutants was largely
increased (about 20-fold), whereas the double mutants exhibited no
activity even in the presence of 10 mM Mn2+. If
the DXD motif is indeed involved in divalent cation binding, it is feasible that the presence of one aspartic acid residue of the
DXD motif allows binding of Mn2+ and
UDP-glucose, albeit with lower affinity, explaining the increase in
activity with high concentrations of manganese ions. However, it should
be noted that, even at high concentrations of Mn2+, the
activities of the single DXD mutants were reduced by several hundred-fold as compared with the wild-type LT fragment. Moreover, we
did not observe photoaffinity labeling of 5-azido-UDP-glucose with the
single DXD mutants even at high Mn2+
concentrations (not shown).
In addition to the DXD motif, we changed several other amino acid residues that are located in the same region and are highly conserved within the family of large clostridial cytotoxins. Exchange of the glycine doublet (Gly281/Gly282), which was conserved not only in the clostridial glucosyltransferases but also in the yeast Och1p family of mannosyltransferases, did not change the enzyme activity of the mutant toxin. Unaltered activity was also observed with the mutant K278ALT546. However, we observed a 50-fold reduction with the mutant Y284ALT546 and an about 200-fold reduction in activity with the D270ALT546 and R273ALT546 mutants. However, all of these mutations did not show as large a decrease in activity as that observed with the DXD motif mutants, suggesting that these amino acid residues play not a direct role in catalysis. Nevertheless, those residues that showed the most effect after exchange apart from the DXD motif are also conserved in the Och1p family.
The full enzyme activity and cytotoxicity (after microinjection) of
C. difficile toxin B and of C. sordellii lethal
toxin depend on the presence of the N-terminal 546 amino acid residues of the holotoxins. The amino acid sequences of these fragments are
about 75% identical, showing no major changes in the similarity of
amino acid sequences in the area around the DXD motif. Most distantly related in their primary structure are C. novyi
-toxin and C. sordellii lethal toxin with only 35% amino
acid sequence identity at the N terminus (amino acids 1-546 of LT;
amino acids 1-551 of
-toxin). However, a stretch of 30 amino acid
residues between amino acid residues 264 and 294 is about 73%
identical. Part of this highly conserved region is the DXD
motif. Thus, from the data reported in this paper, we conclude that
this region is essential for enzyme activity and most likely part of or
near the active site of the glucosylating toxins.
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ACKNOWLEDGEMENT |
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The excellent technical assistance by K. Thoma is gratefully acknowledged.
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FOOTNOTES |
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* This study was supported by the Deutsche Forschungsgemeinschaft AK6/11-1 and by the Fonds der Chemischen Industrie.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.
§ These two authors contributed equally to this work.
** To whom correspondence should be addressed: Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 5, 79104 Freiburg, Germany. Tel.: 0761-2035301; Fax: 0761-2035311; E-mail: aktories{at}ruf.uni-freiburg.de.
1 The abbreviations used are: LT, C. sordellii lethal toxin; CS1, N-terminal C. sordellii lethal toxin fragment of amino acid residues 1-900; LT546, N-terminal C. sordellii lethal toxin fragment of amino acid residues 1-546; CDB1, N-terminal C. difficile toxin B fragment of amino acid residues 1-900; B546, N-terminal C. difficile toxin B fragment of amino acid residues 1-546; PAGE, polyacrylamide gel electrophoresis; 5N3-UDPGlc, 5-azido-UDP-glucose.
2 H. Genth, K. Aktories, F. Hofmann, C. Busch, and I. Just, manuscript in preparation.
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REFERENCES |
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