From the Institut für Experimentelle und
Klinische Pharmakologie und Toxikologie der Universität
Freiburg, Hermann-Herder-Strasse 5, D-79104 Freiburg, the
§ GBF (German Research Center for Biotechnology),
Mascheroder Weg 1, D-38124 Braunschweig, and the ¶ Institut
für Toxikologie der Medizinischen Hochschule Hannover,
Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany
Received for publication, December 7, 2000
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ABSTRACT |
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Clostridium botulinum C3 is the
prototype of the family of the C3-like transferases that ADP-ribosylate
exclusively RhoA, -B and -C. The ADP-ribose at Asn-41 results in
functional inactivation of Rho reflected by disaggregation of the actin
cytoskeleton. We report on a new C3-like transferase produced by a
pathogenic Staphylococcus aureus strain. The transferase
designated C3Stau was cloned from the genomic DNA. At the
amino acid level, C3Stau revealed an identity of 35% to C3
from C. botulinum and Clostridium limosum
exoenzyme, respectively, and of 78% to EDIN from S. aureus. In addition to RhoA, which is the target of the other
C3-like transferases, C3Stau modified RhoE and Rnd3. RhoE
was ADP-ribosylated at Asn-44, which is equivalent to Asn-41 of RhoA.
RhoE and Rnd3 are members of the Rho subfamily, which are deficient in
intrinsic GTPase activity and possess a RhoA antagonistic cell
function. The protein substrate specificity found with recombinant Rho
proteins was corroborated by expression of RhoE in Xenopus
laevis oocytes showing that RhoE was also modified in
vivo by C3Stau but not by C3 from C. botulinum. The poor cell accessibility of C3Stau was
overcome by generation of a chimeric toxin recruiting the cell entry
machinery of C. botulinum C2 toxin. The chimeric
C3Stau caused the same morphological and cytoskeletal
changes as the chimeric C. botulinum C3. C3Stau
is a new member of the family of the C3-like transferases but is also
the prototype of a subfamily of RhoE/Rnd modifying transferases.
Various bacterial protein toxins interfere with eukaryotic cell
functions by catalyzing a posttranslational modification of essential
cellular regulator proteins such as ADP-ribosylation of the Rho
proteins by C3-like transferases. Clostridium botulinum exoenzyme C3 is the prototype of a family encompassing exoenzymes from
Clostridium limosum and Bacillus cereus and from
Staphylococcus aureus
(EDIN)1 (1-4). The members
of this family are similar in structure and homologous to each other.
They are single-chained ADP-ribosyltransferases with a molecular mass
of ~25 kDa. The C3-like transferases are in fact mere exoenzymes
devoid of the cell entry apparatus harbored by other toxins, and they
are thought to enter cells by nonspecific pinocytosis. C3 catalyzes
ADP-ribosylation of the RhoA, -B and -C subtypes but not of other
members of Rho and Ras subfamilies (3-5). Only in the presence of the
detergent sodium dodecyl sulfate, the Rac protein is a poor substrate
(4). The ADP-ribose moiety is transferred from NAD+ to the
acceptor amino acid Asn-41 and is linked N-glycosidically to
the amide group of the carboxylate side chain of Asn-41 (6).
The Rho proteins belong to the Ras superfamily of low molecular mass
GTPases, which are the major regulators of the actin cytoskeleton, but
they are also involved in cell cycle progression, transcriptional
activity, and in cooperation with Ras in cell transformation (7-9).
Because ADP-ribosylation of Rho in intact cells results in
disaggregation of the actin cytoskeleton, ADP-ribosylation has been
classified as an inactivating modification. The molecular basis of the
inactivation is the decreased interaction of Rho with the exchange
factors, which promote activation (10), and the sequestration by
the negative regulator guanine nucleotide exchange
factor.2 Although C3 induces
the breakdown of the actin cytoskeleton, an effect that can be easily
monitored, it is now clear that C3-catalyzed ADP-ribosylation
inactivates all Rho functions (for reviews see Refs. 12-14).
Because of its confined protein substrate specificity, C3 has been
advanced to a widely used tool in cell biology to selectively turn off
cellular Rho functions. The poor cell accessibility of C3 has been
overcome by the creation of chimeric C3 toxins using the cell entry
apparatus of other toxins (15, 16). We report here on a novel C3-like
transferase produced by a pathogenic S. aureus strain that
ADP-ribosylates RhoE/Rnd3 subtype proteins in addition to RhoA, -B and
-C.
Materials and Chemicals--
Culture supernatant was subjected
to purification using the FPLC System, an anion exchange MonoQ column,
and a cation exchange MonoS column from Amersham Pharmacia Biotech.
Oligonucleotides were obtained from Mannfred Weichselgartner
(Ebersberg, Germany), PCR was carried out using the Gene Amp 2400 System from PerkinElmer Life Sciences, and DNA sequencing was
carried out with the Big Dye Terminator Cycle Sequencing Ready Reaction
Kit from PerkinElmer Life Sciences. The Topo-TA-vector system
was from Invitrogen (Groningen, The Netherlands), the pGEX2T
vector system from Amersham Pharmacia Biotech, and the
Quickchange Kit was from Stratagene (Heidelberg, Germany).
Restriction enzymes and T4-DNA ligase were from New England
Biolabs. C. botulinum C3 exoenzyme was purified as
described (17). All other chemicals were from commercial sources.
RhoE, as described by the group of Settleman and co-workers (18), RhoA,
Rac1, CDC42, and TC10 were purified as glutathione S-transferase fusion
proteins, and RhoD was expressed as maltose-binding protein in
Escherichia coli. Rnd3 was cloned using the RhoE
construct from Settleman and co-workers (18) and an extension
consisting of the published nucleotide sequence encoding for an
N-terminal 15-amino acid fragment from Rnd3 (22). In some cases,
the GST portion was cleaved off with thrombin (100 µg/ml), which was
removed by precipitation with benzamidine-Sepharose beads (Amersham
Pharmacia Biotech).
Purification--
S. aureus strain HMI6 was a
clinical isolate from a patient with postoperative infection. The
identity of this isolate was confirmed in the laboratory by routine
tests. For purification of the ADP-ribosyltransferase, bacteria were
grown in LB medium at 37 °C overnight. After centrifugation at
4 °C, ammonium sulfate was added to a concentration of 70% to
precipitate proteins from the culture supernatant. The pellet was
resuspended in 50 mM HEPES, pH 7.0, and dialyzed against
the same buffer overnight to remove the salt. The solution was added
onto the MonoQ column using a 10-ml super loop, and the flow through
was collected, dialyzed against a buffer containing 50 mM
HEPES, pH 6.0, and loaded onto the MonoS column. The transferase was
eluted with a linear gradient at 0.3 M NaCl in 50 mM HEPES, pH 6.0.
ADP-ribosylation Reaction--
Recombinant RhoA, RhoE/Rnd3, and
other GTPases (2 µM) were ADP-ribosylated in a buffer
containing 50 mM HEPES, pH 7.3, 2 mM MgCl2, 20 µM
[adenylate-32P]NAD, and 100 µg/ml bovine
serum albumin for up to 4 h at 37 °C. The total volume was 25 µl. The recombinant toxin was applied in a concentration of 1 nM or as indicated.
SDS-PAGE--
SDS-polyacrylamide gel electrophoresis was
performed according to the methods of Laemmli (19). Gels were stained
with Coomassie Brilliant Blue R-250, dried, and further analyzed by the
PhosphorImager SI from Molecular Dynamics.
Amino Acid Sequencing Analysis--
The eluted fraction from
MonoS column containing the transferase was separated by SDS-PAGE,
transferred onto polyvinylidene difluoride membrane (Amersham Pharmacia
Biotech), and visualized with Amido Black. N-terminal amino acid
sequencing was performed on an excised band using an Applied Biosystems
447A pulse-liquid protein sequencer.
Amplification from Genomic DNA--
Genomic DNA from
strain S. aureus HMI6 was prepared by standard methods.
Primers for amplification of the HMI6 gene were designed according to the determined N-terminal amino acid sequence and in
consideration of the staphylococci codon usage. For the 3' end, the
primer was designed according to a region 100 base pairs downstream of
the in frame stop codon in the published EDIN sequence (2). PCR was
performed under the following conditions: 500 ng of template, 2.5 mM of each dideoxynucleotide, 5 µl of 10-fold concentrated Mg2+-free buffer, different
concentrations of MgCl2, and variant units of
Taq polymerase (New England Biolabs). The primers were:
HMI6-N, 5' AGA TCT GCC GAG ACT AAA AAT TTT ACA G 3'; and HMI6-C, 5' GGA TCC TAA GTT TAA AGC GTA TTT TTA G 3'. PCR products were separated by
gel electrophoresis, purified, ligated into the TOPO-TA vector, and
sequenced. For mobilization of the C3Stau gene, the
HMI6-5' end primer and a primer corresponding to the 3' end of
C3Stau were used. The primers were flanked by a 5'
BglI and a 3' BamHI site, which were used for the
ligation of the C3Stau gene into the pGEX vector. The amino
sequence of C3Stau is available in the
GenBankTM/EBI data base under accession number
AJ277173.
Expression of Recombinant C3Stau
Transferase--
For expression of the transferase, bacteria were
grown overnight in LB media in the presence of 100 µg/ml of
ampicillin, followed by inoculation in fresh media. At an
A600 of 0.8, isopropyl-1-thio- Preparation of Oocyte Lysates Overexpressing Human
RhoE--
Human RhoE cRNA was transcribed in vitro using
the RhoE-pNK2 as template and injected into defolliculated
Xenopus laevis oocytes. After 24 h at 19 °C, lysates
were prepared as described from cRNA-infected cells and noninjected
cells (20).
Construction of the C2IN-C3Stau Fusion
Toxin--
The fusion toxin containing the N-terminal 225 amino acids
from C. botulinum C2 toxin and the full-length
C3Stau was constructed using the 5' BglI
site and the 3' BamHI site flanking the toxin gene.
Construction, expression, and purification of GST fusion protein
were carried out as described (16).
Cytotoxic Assay--
NIH3T3 and KB cells were grown to
subconfluency at 37 °C in Dulbecco's minimum essential medium
containing 10% fetal calf serum, 2 mM
L-glutamate, 100 units penicillin/ml, and 100 µg/ml streptomycin at 5% CO2. For cytotoxic assays, subconfluent
cells in 24-well plates containing coverslips were treated for 3 h
with 200 ng/ml activated C2II toxin, C2IN-C3Stau alone (100 ng/ml), or with 200 ng/ml activated C2II and C2IN-C3Stau.
As a control, cells were treated with 200 ng/ml activated C2II and 100 ng/ml C2IN-C3 (limosum) for the same time. Cells growing on coverslips
were washed twice with phosphate-buffered saline and fixed with 4%
paraformaldehyde and 0.1% Triton X-100 in phosphate-buffered saline
for 30 min. Actin was stained with phalloidin-rhodamine (600 ng/ml) and
the coverslips were mounted in Moviol.
Several clinical isolates of S. aureus were screened
for C3-like activity. An isolate designated HMI6 was identified to
possess ADP-ribosyltransferase activity, which modified recombinant
RhoA. The transferase was purified from the culture supernatant
by applying ammonium sulfate precipitation and ion exchange
chromatography. The partially purified protein was excised from
SDS-polyacrylamide gel and N-terminally sequenced. Twenty-three amino
acids of the N terminus were identified revealing an identity of 78%
(18 of 23 amino acids) with the EDIN transferase from S. aureus (Fig. 1). The
ADP-ribosyltransferase from S. aureus was designated
C3Stau to make a distinction from C3bot
(C. botulinum), C3lim (C. limosum),
and C3cer (B. cereus).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside was added to a
final concentration of 0.2 mM. After incubation for 6 h at 37 °C, bacteria were pelleted, resuspended in lysis buffer
containing 50 mM HEPES, pH 7.3, 2 mM
MgCl2 and 1 mM phenylmethylsulfonyl fluoride,
and broken with a French Press (SLM Aminco). Debris was removed
by centrifugation for 15 min at 15,000 × g at 4 °C, and GST fusion protein was purified from supernatant with
glutathione-Sepharose beads. The GST carrier was cleaved with thrombin
followed by its removal with benzamidine beads.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of the N-terminal amino acids of
the ADP-ribosyltransferase C3Stau from S. aureus
strain HMI6 with EDIN from S. aureus strain
E1. Partially purified C3Stau was N-terminally
sequenced. The amino acids marked in white are those
different in C3Stau.
The gene coding for C3Stau was amplified from the genomic
DNA by PCR. The 5' primers were deduced from the identified N-terminal amino acid sequence, and the basis for the 3' primers was the noncoding
region EDIN, the sequence of which is known. Indeed, one major PCR
product was obtained. From this product, the C3Stau gene
was further cloned. The determined sequence is presented in Fig.
2, and the alignment with the sequences
of EDIN, C3bot, and C3lim is given in Fig.
3. The molecular mass of
C3Stau was calculated as 23,640 Da, and the theoretical
isoelectric point was 9.4. The sequence comparison revealed an identity
of 78% with EDIN and 35% with C3bot and
C3lim. The C3Stau transferase gene was cloned
into the pGEX-2T expression vector. GST-C3Stau was nicely
expressed in E. coli, and the removal of the GST part by
thrombin treatment resulted in a stable protein with a molecular mass
of 24 kDa (Fig. 4). The recombinant
C3Stau was used to compare its properties with that of
C3bot exoenzyme, the prototype of C3-like transferases.
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To test the protein substrate specificity, recombinant Rho subfamily
proteins (RhoA, Rac1, Cdc42, RhoD, RhoE, TC10, and RhoN) were incubated
with C3Stau in the presence of [32P]NAD. As
shown in Fig. 5A, RhoA was
[32P]ADP-ribosylated by C3bot and
C3Stau. Surprisingly, RhoE was modified by
C3Stau but not by C3bot. The new substrate
specificity of C3Stau was corroborated by showing that Rnd3
was also a substrate (Fig. 5A). Rnd3 is an isoform of RhoE
possessing an N-terminal 15-amino acid extension.
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The time course revealed that RhoE/Rnd3 was ADP-ribosylated more slowly by C3Stau compared with RhoA, and the linear phase of ADP-ribosylation lasted for hours. C3bot, in contrast, did not catalyze any incorporation of ADP-ribose into RhoE/Rnd3 (Fig. 5B). RhoE and Rnd3 have been identified recently as GTPase-deficient low molecular mass GTP-binding protein, which has preferentially bound GTP (18, 21, 22). Because RhoA in the GDP-bound but not in the GTP-bound form is the preferred substrate for C3bot (23), RhoE was artificially loaded with GDP and tested for ADP-ribosylation. However, the nucleotide occupancy did not change the kinetics of modification (data not shown). Thus, for unknown reasons, RhoE/Rnd3 in contrast to RhoA was ADP-ribosylated with slow velocity.
Sequential ADP-ribosylation of recombinant RhoA, i.e. first with C3bot followed by C3Stau (and vice versa), indicated that both transferases linked the ADP-ribose to Asn-41 of RhoA (data not shown). This finding was confirmed by using the mutant RhoAsn41Ile, which was not a substrate for C3Stau and C3bot (Fig. 5C). Asn-41 is equivalent to Asn-44 in RhoE. Its exchange to Ile (RhoEAsn44Ile) completely prevented ADP-ribosylation by C3Stau, indicating that Asn-44 is the acceptor amino acid in RhoE (Fig. 5C).
To test whether cellular RhoE/Rnd3 was a substrate for
C3Stau, cell lysates and cellular subfractions were
[32P]ADP-ribosylated with C3bot and
C3Stau. However, no radioactive band in addition to RhoA
was detected (data not shown). It is conceivable that RhoE/Rnd3 is
poorly expressed in the cell lines tested compared with RhoA and that
radioactively labeled RhoA masked the traces of labeled RhoE/Rnd3.
Therefore, a sequential ADP-ribosylation was performed, i.e.
nonradioactive ADP-ribosylation by C3bot was followed by
C3Stau-catalyzed [32P]ADP-ribosylation.
Preincubation with C3bot followed by
C3Stau-catalyzed [32P]ADP-ribosylation
resulted in a radioactive labeling, whereas the opposite way showed no
labeling, indicating a protein substrate that is modified by
C3Stau but not by C3bot (Fig.
6A).
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Another approach to check whether RhoE was an in vivo substrate for C3Stau was the expression of RhoE in oocytes from X. laevis. In lysates from noninjected control oocyte, C3Stau and C3bot [32P]ADP-ribosylated only RhoA (one band) (Fig. 6B). However, in lysates from oocytes expressing the RhoE protein, C3Stau ADP-ribosylated two polypetides (double band), whereas C3bot modified only one single band (Fig. 6B). Thus, cellular RhoE was a substrate for C3Stau, corroborating the findings with recombinant Rho GTPases.
Exoenzymes C3bot and C3lim are cytotoxic to
cultured cell lines to induce disaggregation of actin filaments but
only when applied at micromolar concentrations (13). The same property
was true for C3Stau (data not shown). To overcome this
limitation, a chimeric C3Stau was constructed analogous to
that construct of C3lim with C. botulinum C2
toxin (16). C3Stau was fused to the enzymatically deficient
C2I component, and the cell entry was mediated by the receptor binding
component C2II from C. botulinum. C2IN-C3Stau
was as nontoxic to cells as C3Stau was, but in the presence
of C2II C2IN-C3Stau induced the typical C3-like morphology
and cytoskeletal changes (Fig.
7A). The differential
ADP-ribosylation of lysates from intoxicated cells clearly demonstrated
the in vivo ADP-ribosylation of cellular RhoA (Fig.
7B).
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DISCUSSION |
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The first exoenzyme that was identified to ADP-ribosylate the Rho protein was exoenzyme C3 from C. botulinum. Because several isoforms of C3, all produced by C. botulinum types C and D, were identified, it was initially thought that C3 was associated with or even part of the botulinum neurotoxins. However, the later identification of homologues of C3 produced by C. limosum, B. cereus, and S. aureus, which definitely do not harbor genes for neurotoxins, proved that C3 and C3-like exoenzymes are independent from neurotoxins (24). All the C3 homologues are single chain proteins that are released from the bacteria and are, therefore, to be classified as exoenzymes. Compared with bacterial protein toxins such as pertussis toxin, cholera toxin, or Pseudomonas exotoxin A, the C3 homologues are devoid of a cell entry apparatus and seem to enter cells by nonspecific pinocytosis (25). Their function as virulence factors is unclear, but there is a report that the C3 homologue EDIN from S. aureus inhibits differentiation and induces hyperplasia of epidermis (26). The C3 homologue C3Stau, presented in this study, is also produced by a pathogenic S. aureus strain. C3Stau shows 78% identity and 90% homology to EDIN but only 35% identity to C3 isoforms and the exoenzyme from C. limosum.
The identification of C3 exoenzyme is tightly linked to the elucidation of cellular Rho functions (13, 24). The reason for this is the remarkable substrate specificity of C3bot, which ADP-ribosylates only the subtypes RhoA, -B, and -C but not other target proteins. Surprisingly, C3Stau modifies, in addition to RhoA, -B, and -C, the recently identified Rho subfamily members, RhoE/Rnd3. The acceptor amino acid in RhoE is Asn-44, the equivalent to the acceptor residue in RhoA, Asn-41. However, compared with RhoA, RhoE/Rnd3 is slowly modified; the linear phase lasts about 4 hours but eventually results in complete modification. The reason for this different property is unknown but may be based on its lack of GTP hydrolyzing activity. The basis for this is the exchange of three conserved amino acids in RhoE/Rnd3, which is canonically involved in the GTPase activity. GTPase activity of RhoE can be restored by replacement with the conserved amino acids (18). It is, therefore, conceivable that RhoE/Rnd3 possesses a conformation that resembles the active GTP-bound form independent of nucleotide occupancy. This notion explains the finding that loading of RhoE with GDP does not change the kinetics of ADP-ribosylation, whereas RhoA-GDP is modified about five times faster than RhoA-GTP (23). However, it is also conceivable that RhoE/Rnd3 needs interaction with other proteins or binding to membranes to be rapidly modified.
RhoE/Rnd3 belongs to a new branch of the Rho subfamily, the Rnd proteins (22). The members Rnd1, Rnd2, and Rnd3, the last one being nearly identical with RhoE, are about 50% identical with RhoA, but they show a remarkable functional difference to RhoA. They are deficient in intrinsic and GTPase acticating protein-stimulated GTPase activity (18, 21, 22). Furthermore, RhoE/Rnd3 has the opposite function of RhoA and can be classified as a functional RhoA antagonist (21, 22). How RhoE/Rnd3 is regulated is so far unclear, possibly by expression or by subcellular translocation.
The initial step of RhoA activation, the interaction with guanine nucleotide exchange factor Lbc, is blocked by ADP-ribosylation (10). Based on this finding it is conceivable that RhoE/Rnd3 acts by sequestering exchange factors, thereby preventing activation of RhoA. ADP-ribosylation might inhibit binding of RhoE/Rnd3 to the exchange factors, thereby allowing the normal activation cascade of RhoA.
In the case of C3bot and C3lim, inactivation of RhoA by ADP-ribosylation allows RhoE/Rnd3 to act, thereby inducing disaggregation of the actin cytoskeleton. C3Stau, however, inactivates both RhoA and its antagonist RhoE/Rnd3. Thus, one expects fewer morphological effects for C3Stau than for C3bot. However, the experimental data do not support this notion. The major reason for this might be the low expression of RhoE/Rnd3 in all tissues tested, which does not allow a direct functional antagonism to RhoA (22).
C3Stau belongs to the family of C3-like transferases
because of its homology and its ability to ADP-ribosylate RhoA.
However, it is also the prototype of a novel subfamily of the C3-like
transferases because of its extended protein substrate specificity,
modifying RhoE and Rnd3 in addition to RhoA. C3Stau and
EDIN are produced by pathogenic S. aureus strains. S. aureus bacteria are reported to escape endosomes after
phagocytosis by endothelial cells and to exist freely in the cytoplasm
(11, 27). Based on this scenario, C3Stau as well as EDIN
are released inside the cytoplasm and can immediately reach their
targets RhoA, RhoE, and Rnd3. Under these conditions they in fact do
not need any cell entry machinery.
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ACKNOWLEDGEMENTS |
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We thank Jürgen Dumbach for expert technical assistance, Dr. J. Settleman (Massachusetts General Hospital Cancer Center and Harvard Medical School) for providing the RhoE construct, and Dr. H. Takeshima (Tokyo) for the RhoN gene.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 388 and Project Ak6/10.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.
To whom correspondence should be addressed: Tel.:
49-511-532-2812; Fax: 49-511-532-2879; E-mail:
just.ingo@mh-hannover.de.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M011035200
2 H. Genth, M. Schmidt, H. Barth, K. Aktories, and I. Just, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: EDIN, epidermal differentiation inhibitor from S. aureus strain E1; PCR, polymerase chain reaction; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; C3bot, C. botulinum exoenzyme C3; C3lim, C. limosum ADP-ribosyltransferase; C3Stau, ADP-ribosyltransferase from S. aureus strain HMI6.
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