Infection and Immunity, November 2000, p. 6378-6383, Vol. 68, No. 11
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany
Received 1 May 2000/Returned for modification 16 June 2000/Accepted 4 August 2000
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ABSTRACT |
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Clostridium novyi alpha-toxin belongs to the family of
large clostridial cytotoxins which act on cells through the
modification of small GTP-binding proteins. We present here an analysis
of the catalytic domain of alpha-toxin. A NH2-terminal
551-amino-acid fragment,
551, was found to contain the full enzyme
activity of the holotoxin, whereas a slightly shortened fragment
encompassing 509 amino acids showed no detectable enzyme activity.
Further characterization of the enzymatically active fragment
551
revealed a substrate specificity for both
UDP-N-acetylglucosamine and UDP-glucose. A
Michaelis-Menten constant of 17 µM was determined for the substrate UDP-N-acetylglucosamine, while that for UDP-glucose was
about 20 times higher, indicating a weaker affinity of the toxin for the latter substrate. Mutation of the aspartic acids of a conserved motif DXD within
551 reduced enzyme activity >700-fold and
inhibited cytotoxicity after microinjection in cells. Inhibition of
enzyme activity of the DXD mutant could be partially overcome by
increased concentrations of manganese ions, suggesting the involvement
of these aspartic acids in Mn2+ binding. By construction of
chimeras of enzymatically active fragments of C. sordellii
lethal toxin and C. novyi alpha-toxin, we located the
region involved in nucleotide-sugar specificity to amino acids 133 through 517.
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INTRODUCTION |
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Type A strains of Clostridium novyi are causative agents of gas gangrene infections in humans and animals (7). Among a variety of different toxins produced by this pathogen is the alpha-toxin, a large 250-kDa protein which exhibits lethal and edematizing activity in vivo (7). In vitro, alpha-toxin is a potent cytotoxin, causing pronounced changes of cell morphology (1, 2). Sharing a considerable sequence homology with the toxins A and B of C. difficile and the lethal toxin from C. sordellii, the alpha-toxin has been grouped into the family of large clostridial cytotoxins (10). All of these toxins are glycosyltransferases, modifying and thereby inactivating different members of the Rho and Ras subfamily of small GTP-binding proteins (14-16). In consequence, several signal transduction pathways are inhibited (19), resulting in the breakdown of cytoskeletal structures (17). While the lethal toxin from C. sordellii modifies Rac, Cdc42, Ras, and the Ras-related GTPases Ral and Rap (11, 14, 18), the other toxins glucosylate Rho, Rac, and Cdc42 (20). Unlike the other members of this toxin family which transfer the glucose moiety from UDP-glucose to their protein substrates, the alpha-toxin uses UDP-N-acetylglucosamine (20).
The large clostridial cytotoxins are thought to consist of three distinct functional domains (21). While repetitive sequences at the C-terminal end of the toxins appear to mediate the receptor binding, a hydrophobic domain in the central region appears to be involved in the translocation from endosomal vesicles into the cytosol (21). Recent publications have shed some light on the enzymatic domain of large clostridial cytotoxins. N-terminal fragments of C. difficile toxin B and C. sordellii lethal toxin exhibit the full glucosyltransferase activity of their respective holotoxins (8, 9). Furthermore, studies with chimeric toxin fragments have delineated the region responsible for the difference in substrate specificity of C. difficile toxin B and C. sordellii lethal toxin within these N-terminal fragments (8). Lastly, a conserved amino acid motif DXD has been shown to be essential for enzyme activity (4). Recent structural data support the notion that this motif is involved in the binding of the nucleotide moiety of the UDP-glucose via coordination of a manganese ion (5).
Here we studied the enzyme domain of C. novyi
-toxin and
present a detailed characterization of its catalytic properties especially with respect to nucleotide-sugar specificity, which separates alpha-toxin from the other members of the family of large
clostridial cytotoxins. Our study localizes the region within the
active domain of C. novyi alpha-toxin, which defines the
difference in nucleotide-sugar specificity.
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MATERIALS AND METHODS |
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Materials. 14C-labeled UDP-hexoses were obtained from DuPont NEN (Dreieich, Germany). PCR primers were from MWG Biotech (Ebersberg, Germany). All other reagents were of analytical grade and were purchased from commercial sources.
Purification of large clostridial cytotoxins. The lethal toxin of C. sordellii 6018 was purified as described for C. difficile toxin B (9). The alpha-toxin of C. novyi was purified as reported previously (20).
PCR amplification. Amplification of lethal toxin fragments and construction of the truncated fragment LT546 were done as described previously (8).
C. novyi type A 19402 (kindly donated by G. Schallehn, Bonn, Germany) was used as a source of chromosomal DNA. Amplification of the N-terminal 900-amino-acid fragment CN1 was performed by the PCR System 2400 (Perkin-Elmer) using the primer pair CN1C-CN1N (5'-AGATCTATGCTTATAACAAGAGAACAA-3' and 5'-GGATCCATCTTTTTCTTTTATTATACT-3'). The reaction was carried out with 300 nmol of primers and 125 ng of chromosomal DNA for 25 cycles (denaturation: 94°C, 10 s; annealing: 48°C, 30 s; and elongation: 68°C, 3 min) in a total volume of 100 µl. The amplified DNA fragment was cloned into pCR2.1 (Invitrogen BV). After mobilization with BamHI, the gene fragment was cloned into pGEX-2T (pGEX-2T-CN1).Cloning of truncated alpha-toxin fragments.
The toxin
fragment CN1 was used for subcloning the truncated fragments
551 and
509.
509 was constructed as follows: pGEX-2T-CN1 was digested
with BstBI, leading to the mobilization of a truncated fragment of 509 amino acids together with a short stretch of the pGEX-2T sequence adjacent to the 5' end of the alpha-toxin fragment. This gene fragment was treated with the Klenow fragment of DNA polymerase I to generate blunt ends, followed by a digestion with BamHI. The resulting fragment was cloned into the pGEX-2T
vector which had been digested with BamHI-SmaI.
551 was constructed using the Seamless Cloning Kit
(Stratagene) as previously described (8). Briefly, toxin fragment CN1 was used as a template for the amplification of the truncated fragment with primers SC9
(5'-GGGGCTCTTCATTCATAATTGAGAGTTCTTCCTATATAAGT-3') and SC10
(5'-GGGGCTCTTCAGAATTCATCGTGACTGACTGACGATC-3'). The amplicon was digested with Eam1104I and relegated.
Site-directed mutagenesis of toxin fragment
551.
The
QuikChange Kit (Stratagene) was used for mutating one nucleotide in
pGEX-2T-
551, according to the manufacturer's instructions. The
primers were constructed as follows: for
551.D284A, primer pair
alphaD284Asen-alphaD284Aanti (5'-GTGGTGTATATTGTGCTTTAGATTTTCTTC-3' and 5'-GAAGAAAATCTAAAGCACAATATACACCAC-3'); and for
551.D286A, primer pair alphaD286Asen-alphaD286Aanti
(5'-TATATTGTGATTTAGCTTTTCTTCCTGGAG-3' and
5'-CTCCAGGAAGAAAAGCTAAATCACAATATA-3').
Construction of alpha-toxin-lethal toxin chimeras. (i)
LT132.
551.
Chimera LT132-
551 was constructed using the
Seamless Cloning technique (Stratagene). Primers SC10
(5'-GGGGCTCTTCAGAATTCATCGTGACTGACTGACGATC-3') and SC14
(5'-GGGGCTCTTCAATCATAAAAAACTTTAACTGTATAATCGCT-3') were employed to amplify a fragment containing the pGEX-2T vector and a gene
fragment consisting of the N-terminal 132 amino acids of lethal toxin.
Lethal toxin fragment LT546 in pGEX-2T served as a template for this
reaction. The alpha-toxin portion of the chimera was amplified using
the primers SC9 (5'-GGGGCTCTTCATTCATAATTGAGAGTTCTTCCTATATAAGT-3') and SC13 (5'-GGGGCTCTTCAGATAAGAATTCATTGCTAGTAAATACATTA-3')
and the fragment
551 in pGEX-2T as a template. Following
Eam1104I digestion, the fragments were ligated to constitute
the chimera. The procedures were carried out according to the
manufacturer's instructions with minor modifications.
551. Chimera LT517.
551 was constructed using the
splicing-by-overlap extension technique as described elsewhere (12). For the generation of the lethal toxin portion,
primers SOE1 (5'-AGATCTATGAACTTAGTTAACAAAGCC-3') and SOE5
(5'-AGCCAACTACTAGTTATTTCCTGTTCTGA-3') were employed in an
amplification using LT546 in pGEX-2T as a template. The alpha-toxin
portion was amplified with the primers SOE6
(5'-CAGGAAATAACTAGTAGTTGGCTTTTAAGA-3') and SOE4
(5'-CCATTGCTGCAGGCATC-3').
Sequencing. Sequencing of CN1 and all its truncated derivatives was done with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer) to check both for correct cloning and mutations due to PCR amplification, sequencing was performed at least twice with overlapping DNA fragments.
Expression of recombinant proteins. The recombinant GTP-binding proteins RhoA, Rac, Cdc42, and Ha-Ras were prepared as previously described (13). The recombinant toxin fragments were expressed and purified as glutathione S-transferase (GST) fusion proteins in accordance with the manufacturer's instructions. GST fusion proteins from the Escherichia coli expression vector pGEX-2T were isolated by affinity chromatography with glutathione-Sepharose (Amersham Pharmacia Biotech), followed by cleavage of the toxin fragment proteins from the GST fusion protein by thrombin treatment (100 µg/ml for 30 min at 22°C). Thrombin was removed via binding to benzamidine-Sepharose.
Glucosylation reaction. Recombinant GTP-binding proteins (50 µg/ml) were incubated with alpha-toxin, recombinant toxin fragments, or chimeric fragments of alpha-toxin and lethal toxin 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 of bovine serum albumin (BSA) per ml, and 10 to 30 µM UDP-[14C]N-acetylglucosamine or UDP-[14C]glucose for 30 min at 37°C. The total volume was 20 µl. Labeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently by phosphorimaging (Molecular Dynamics).
Glucohydrolase reaction. Toxin fragments or mutants were incubated with 20 µM UDP-[14C]N-acetylglucosamine and 100 µM unlabeled UDP-N-acetylglucosamine in a buffer containing 50 mM HEPES (pH 7.5), 100 mM KCl, 2 mM MgCl2, 100 µM BSA, and 1 mM MnCl2. The total volume was 20 µl. In a time course experiment, samples (1.5 µl) were taken at each time point and subjected to thin-layer chromatography with PI-Cellulose plates (Merck), and 0.2 M LiCl was used as the mobile phase to separate hydrolyzed glucose from UDP-glucose. The plates were dried and analyzed by phosphorimaging.
Kinetic experiments. Initial rate data for the glucosyltransferase reaction were determined with regard to UDP-N-acetylglucosamine or UDP-glucose binding by varying the concentration of the respective nucleotide-sugar from about 0.2 to 2 × Km. Experiments were performed at 7.5 µM GST-Rac. Kinetic values were obtained by analysis of Lineweaver-Burk plots of initial velocities from three independent experiments.
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RESULTS |
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N-terminal fragments of large clostridial cytotoxins
contain the full enzyme activity of their respective holotoxins
(8, 9). To obtain functional data for the alpha-toxin from
C. novyi, we constructed fragments of the toxin and
expressed them in E. coli as GST fusion proteins. Figure
1 shows an SDS-PAGE analysis of the
cleaved recombinant proteins
551 and
509, which comprise the
toxin's N-terminal 551 and 509 amino acids, respectively. The
551-amino-acid fragment is of analogous size to the enzymatically active 546-amino-acid fragments of C. difficile toxin B and
C. sordellii lethal toxin described previously (8,
9). These proteins were tested for their enzyme activity in an in
vitro glucosylation assay using 14C-labeled
UDP-N-acetylglucosamine. The large fragment (
551)
catalyzed the modification of the substrate GST-Rac as intensively as
did the holotoxin (Fig. 2). Fragment
509, however, did not express detectable enzyme activity. Fragments
consisting of the central or C-terminal part of the toxin also did not
express enzyme activity (not shown).
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To characterize further the active toxin fragment
551, we determined
its nucleotide-sugar specificity. For this purpose, the toxin fragment
and GST-Rac were incubated at 37°C in the presence of UDP-glucose or
UDP-N-acetylglucosamine, respectively. As shown in Fig.
3, the fragment accepted both
nucleotide-sugars as substrates. However, UDP-glucose resulted in a
weaker labeling than UDP-N-acetylglucosamine. Similar data
were obtained for the holotoxin (not shown). Kinetic experiments
provided further insight into the nucleotide-sugar specificity of
551. The relative specific activities for the glycosyltransferase
reaction (kcat and
kcat/Km) of UDP-glucose and UDP-N-acetylglucosamine, as well as the Michaelis
constants (Km), were compared by varying the
UDP-glucose concentrations from 0.2 to 2 × Km at a fixed GST-Rac concentration of 7.5 µM. The values obtained by Lineweaver-Burk plots are summarized in Table
1. We observed an overall reduced
efficiency of the glycosyltransferase reaction in the presence of
UDP-glucose compared with UDP-N-acetylglucosamine, which
was caused both by an increase in Km, and a
decrease in kcat for the reaction involving
UDP-glucose as a substrate.
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C. novyi alpha-toxin is the only member of the large
clostridial cytotoxin family to accept
UDP-N-acetylglucosamine as a nucleotide-sugar substrate,
the other toxins exclusively use UDP-glucose. To localize the region
defining nucleotide-sugar specificity, chimeric toxin fragments of
alpha-toxin and C. sordellii lethal toxin were tested for
their enzyme activity. A chimera consisting of the
NH2-terminal 132 amino acids of lethal toxin and amino
acids 133 through 551 of alpha-toxin possessed the protein substrate
and the nucleotide-sugar specificity of the alpha-toxin fragment
551. On the other hand, a chimera comprising the first 517 amino
acids of lethal toxin and amino acids 518 through 551 of alpha-toxin
possessed the same substrate and cosubstrate specificity as lethal
toxin (Fig. 3 and 4).
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Mutation of the conserved aspartic acids of the amino acid motif DXD in
the large clostridial cytotoxins C. difficile toxin B and
C. sordellii lethal toxin drastically reduces enzyme
activity (4). To probe the significance of the DXD motif in
alpha-toxin, the aspartic acids of this motif in
551 were replaced
individually by alanine.
551.D284A was about 1,000-fold less
efficient for modification of GST-Rac protein relative to the wild type
(Fig. 5). Similar results were obtained
with
551.D286A (not shown).
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Given the impact of manganese on enzyme activity of lethal toxin DXD
mutants (4), we measured the manganese ion dependency of the
glucosyltransferase activity of
551.D286A. As shown in Fig.
6, wild-type fragment activity was only
moderately enhanced by increasing concentrations of manganese ions,
e.g., no increase in labeling was observed by increasing the
Mn2+ concentration from 0.2 to 2 mM. In contrast, the
activity of
551.D286A was dramatically increased with the addition
of Mn2+ from 0.2 to 2 mM.
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Finally, we tested the toxin fragment
551 and
551.D286A for
cellular activity by microinjection into HeLa cells. The wild-type fragment
551 elicited the characteristic morphological changes observed with the holotoxin. The cells rounded up about 30 min after
microinjection. In contrast,
551.D286A had no effect on the
morphology of HeLa cells (Fig. 7).
Moreover, the truncated fragment
509 was inactive when introduced
into cells (not shown).
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DISCUSSION |
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In this study, we present an analysis of the catalytic domain of C. novyi alpha-toxin. The data were obtained by investigating the enzymatic properties of recombinant alpha-toxin fragments. As observed for other clostridial cytotoxins, an N-terminal fragment of about 550 amino acids was necessary and sufficient for full enzyme activity, whereas a truncated fragment consisting of the N-terminal 509 amino acids possessed no detectable glucosyltransferase activity (Fig. 2). Both UDP-N-acetylglucosamine and UDP-glucose served as substrates for alpha-toxin, a finding that had not been observed in earlier publications. This discrepancy might be explained by improvement of conditions to purify and store the toxins. Kinetic data clearly show that UDP-N-acetylglucosamine is a preferred substrate compared to UDP-glucose. Alpha-toxin exhibits both an increased affinity (ca. 20-fold) and an increased turnover rate (ca. 10-fold) with UDP-N-acetylglucosamine resulting in a 180-fold increase in transferase efficiency with UDP-N-acetylglucosamine relative to UDP-glucose. The data could be explained by the fact that the two nucleotide-sugars share the same general structure and similar electronic properties apart from the bulky side chain of UDP-N-acetylglucosamine. Therefore, UDP-glucose might be bound in the cleft for UDP-N-acetylglucosamine, but without forming the interactions mediated by the side chain with the enzyme which might result in the decreased affinity.
Alpha-toxin is the only large clostridial cytotoxin to use UDP-N-acetylglucosamine as a substrate. To elucidate the molecular background of this special feature, chimeric toxin fragments were constructed to locate the region involved in nucleotide-sugar recognition. The size of the regions swapped between the toxin fragments was chosen in analogy to the chimeric proteins of C. difficile toxin B and C. sordellii lethal toxin described previously (8). From the data obtained by the characterization of two lethal toxin-alpha-toxin fragment chimeras, we propose amino acids 133 through 517 contain the region determining the different nucleotide-sugar specificity of the two toxins. Because the nucleotide moieties of UDP-glucose and UDP-N-acetylglucosamine are unchanged, this region is most likely involved in the binding of the sugar moiety of the substrate.
A tryptophan conserved at position 102 within all the clostridial cytotoxins has been recently shown to be important for the nucleotide-sugar affinity of lethal toxin fragments (3). Located outside of the above-mentioned region, this residue should interact with the nucleotide moiety of the substrates. This conclusion is consistent with data obtained from recently published crystal structures of two glycosyltransferases (5, 6). In both of these studies, an aromatic amino acid residue was proposed to be involved in a stacking interaction with the uracil ring of the nucleotide. This residue is conserved throughout the members of the respective glycosyltransferase families. Likewise, a tryptophan 102 analogue is conserved among all proteins related to the catalytic subunit of large clostridial cytotoxins (3).
Another characteristic feature of a variety of glycosyltransferases is the amino acid motif DXD, which has been shown to coordinate a manganese ion in one of the above-mentioned crystal structures (6). Site-directed mutagenesis of the alpha-toxin fragment's DXD motif resulted in mutants exhibiting a drastically reduced enzyme activity in glycosyltransferase assays and a lack of intracellular activity (Fig. 5 and 7). In the presence of high concentrations of manganese ions, the activity of the mutants was considerably increased, a finding which is consistent with the view that the DXD motif is involved in the coordination of a manganese ion. However, we cannot exclude that manganese ions increase enzyme activity independently of the DXD motif and that the observation of the enhancement was possible because the basal activity was low. The increase in activity of the wild-type fragment by low amounts of manganese ions can be explained by the general dependency of large clostridial cytotoxins on manganese ions (Fig. 6).
Investigating chimeric toxin fragments of C. difficile toxin
B and C. sordellii lethal toxin, we have shown recently that a region of amino acids 271 through 516 defines the substrate specificity of the cytotoxins (8). Corroborating these data, amino acids 133 through 551 were found to be sufficient to define the
substrate specificity of
-toxin as shown by the lethal
toxin-alpha-toxin chimera LT132.
551 (Fig. 4). In addition to this
active chimera, we constructed several fusion proteins of alpha-toxin
and lethal toxin (LT270.
551, LT132.
408.LT546, and LT408.
551).
However, all of these chimeric proteins were inactive. On the other
hand, the addition of amino acid residues 518 to 551 of alpha-toxin to
the inactive fragment (residues 1 to 517) of lethal toxin restored the
activity of lethal toxin. Thus far it is not clear whether amino acids
518 to 551 harbor catalytic important residues or have a
structure-stabilizing effect on the transferase.
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ACKNOWLEDGMENTS |
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We thank K. Thoma for excellent technical assistance.
This study was financially supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany. Phone: 0761-2035301. Fax: 0761-2035311. E-mail: aktories{at}ruf.uni-freiburg.de.
Editor: D. L. Burns
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