1 Groupe de Recherche sur les Antimicrobiens et les Micro-organismes (UPRES EA 2656, IFR 23), Université de Rouen, UFR Médecine-Pharmacie, 22 Boulevard Gambetta, F-76183 Rouen Cedex, France
2 INSERM U 614 (IFR 23), Université de Rouen, UFR Médecine-Pharmacie, 76183 Rouen Cedex, France
3 Unité de Biochimie et Structure des Protéines, INRA, 78352 Jouy-en-Josas Cedex, France
Correspondence
Jean-Louis Pons
Jean-Louis.Pons{at}univ-rouen.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the acd sequence reported in this paper is AY775569.
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INTRODUCTION |
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Autolytic systems of several Gram-positive low G+C bacteria have been studied (Oshida et al., 1995; Smith et al., 2000
; Tomasz, 2000
). Bacillus subtilis is considered to be a model system for investigating the roles of autolysins in the cell-wall metabolism of endospore-forming Gram-positive bacteria (Smith et al., 2000
). Members of the genus Clostridium, which belong to anaerobic microflora of humans and are a potential cause of human infections, belong to this Gram-positive bacteria low G+C phylum. A few PGHs are described in members of this genus, such as an amidase (SleC) and a muramidase (SleM) in Clostridium perfringens (Chen et al., 1997
; Miyata et al., 1995
). However, to our knowledge, the autolytic system of C. difficile, which is recognized as a major nosocomial enteric pathogen causing pseudomembranous colitis and many cases of antibiotic-associated diarrhoea (George, 1984
), has not been investigated.
The aim of this study was the search for a putative PGH gene in the available C. difficile 630 genome, and the molecular characterization and expression of Acd, an autolysin of C. difficile with N-acetylglucosaminidase activity.
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METHODS |
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Escherichia coli strain M15 harbouring pREP4, which constitutively expresses the Lac repressor protein encoded by the lacI gene (QiaExpress System; Qiagen), was used as a recipient. LuriaBertani (LB) broth (Difco) was used for cultivation of E. coli cells at 37 °C with shaking (200 r.p.m.). When required, the antibiotics ampicillin (100 µg ml1) and kanamycin (25 µg ml1) (Sigma), and IPTG (1 mM) (Sigma) were added, according to the manufacturer's instructions.
B. subtilis 168 HR (Foster, 1991) was cultivated in LB broth (Difco) at 37 °C with shaking (200 r.p.m.) and was used (i) as a substrate in renaturing SDS-PAGE and (ii) to prepare cell-wall extracts for the study of hydrolytic bond specificity.
General DNA techniques, PCR and DNA sequencing.
Chromosomal DNA extraction from C. difficile colonies was performed using the InstaGene Matrix kit (Bio-Rad). DNA fragments used in the cloning procedures and PCR products were isolated from agarose gels with the Qiaquick gel extraction kit (Qiagen), according to the manufacturer's instructions. Plasmid DNA from E. coli was isolated and purified with the Qiagen Plasmid midi kit. Primers (MWG-Biotech) acd F and acd R (Table 1) were used for the amplification of a DNA fragment encoding Acd devoid of its signal peptide sequence and for the construction of hexa-His fusion protein. A 1790 bp DNA fragment was amplified with these primers from C. difficile 630 total DNA. PCR was performed on a GeneAmp System 2700 thermal cycler (Applied Biosystems) in a final volume of 50 µl containing 0·5 µM each primer, 200 µM each deoxynucleoside triphosphate and 1 U PfuTurbo DNA polymerase (Stratagene) in a 1x cloned Pfu DNA polymerase reaction buffer [20 mM Tris/HCl, pH 8·8, 10 mM KCl, 2 mM MgSO4, 10 mM (NH4)2SO4]. The PCR mixtures were denatured (2 min at 95 °C), then the amplification procedure followed, consisting of 30 s at 95 °C, annealing for 2 min at 60 °C and ending with an extension step at 68 °C for 4 min. A total of 40 cycles were performed. DNA sequences were determined with an Applied Biosystems 310 automated DNA sequencer using an ABI-PRISM Big Dye Terminator Sequencing kit (Perkin Elmer). Primers (MWG-Biotech) used are listed in Table 1
.
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Detection of cell-wall lytic enzymes by SDS-PAGE and renaturing SDS-PAGE.
Cell-wall lytic enzymes were extracted by the following two procedures. LiCl extractions were carried out as described by Groicher et al. (2000). Briefly, a pellet of 200 ml anaerobic C. difficile 630 cell culture (overnight at 37 °C) was resuspended in 5 ml 5 M LiCl solution and shaken at 200 r.p.m. at 4 °C for 20 min. After centrifugation (20 000 g, 20 min, 4 °C), the supernatant was dialysed overnight at 4 °C against 0·01 M potassium phosphate buffer (pH 7·0) and concentrated 20-fold in a Centricon-3 concentrator (Amicon). SDS extractions were carried out as described by Leclerc & Asselin (1989)
. Briefly, a pellet of 200 ml anaerobic C. difficile 630 cell culture (overnight at 37 °C) was resuspended in 75 ml 4 % (w/v) SDS. The suspension was shaken at 150 r.p.m. for 90 min at room temperature and twice sonicated on ice for 1 min. The extract was heated at 90 °C for 15 min and centrifuged at 12 000 g for 15 min at room temperature. The supernatants from LiCl and SDS extractions were stored at 80 °C.
SDS-PAGE was performed as described by Laemmli (1970) with 15 % polyacrylamide separating gels. Lytic activity was detected by using SDS-polyacrylamide gels (Leclerc & Asselin, 1989
) containing 0·2 % (w/v) Micrococcus lysodeikticus ATCC 4698 (Sigma), B. subtilis 168 HR (Foster, 1991
) and C. difficile 630 lyophilized cells. To allow for protein renaturation after electrophoresis, the gels were gently shaken at 37 °C for 16 h in 250 ml 25 mM Tris/HCl (pH 8·0) containing 1 % (v/v) Triton X-100. Bands of lytic activity were visualized by staining with 1 % (w/v) methylene blue (Sigma) in 0·01 % (w/v) KOH and subsequent destaining with distilled water.
Preparation of cell-wall peptidoglycan.
Peptidoglycan from B. subtilis 168 HR vegetative cells was prepared as described previously (Huard et al., 2003) with some modifications. Briefly, pelleted cells were resuspended in 10 % (w/v) SDS and boiled for 25 min. Insoluble material was recovered by centrifugation (20 000 g, 10 min, 20 °C) and boiled again in 4 % (w/v) SDS for 15 min after resuspension. The resulting insoluble wall preparation was then washed with hot distilled water (60 °C) six times to remove SDS. The covalently attached proteins were removed by treatment with Pronase (2 mg ml1) for 90 min at 60 °C, then by trypsin (200 µg ml1) for 16 h at 37 °C. The walls were then recovered by centrifugation (20 000 g, 10 min, 20 °C), washed once in distilled water and resuspended in hydrofluoric acid (HF) (48 %, v/v, solution); the mixture was incubated at 4 °C for 24 h. The insoluble material was collected by centrifugation (20 000 g, 10 min, 20 °C) and washed repeatedly by centrifugation and resuspension twice with Tris/HCl buffer (250 mM, pH 8·0) and four times with distilled water until the pH reached 5·0. The material was lyophilized and then stored at 20 °C.
Determination of hydrolytic bond specificity.
B. subtilis 168 HR cell walls (4 mg) were incubated overnight at 37 °C with purified Acd-His recombinant protein (370 µg) in a final volume of 500 µl sodium phosphate buffer (pH 8·0). Samples were boiled for 3 min to stop the reaction. The insoluble material was removed by centrifugation at 14 000 g for 15 min. Half of the soluble muropeptide fraction was further digested with mutanolysin (2500 U ml1) (Sigma). The soluble muropeptides obtained after digestion were reduced with sodium borohydride at a final concentration of 8 mg ml1 (Atrih et al., 1999). The reduced muropeptides were then separated by RP-HPLC with an LC Module I system (Waters) and a Hypersyl PEP100 C18 column (250x4·6 mm, particle size 5 µm) (Thermo Finnigan) as described by Atrih et al. (1999)
. Muropeptides were analysed by MALDI-TOF MS using a Voyager-DE STR mass spectrometer (Perseptive Biosystems) as reported previously (Huard et al., 2003
).
RT-PCR.
Total RNA extraction was performed at various times of broth culture of C. difficile 630 by using RNAprotect Bacteria Reagent and an RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. First, for RNA stabilization, 1 ml BHI cell culture was added to 2 ml RNAprotect Bacteria Reagent and the mixture was incubated for 5 min at room temperature. The pelleted cells were resuspended in 100 µl TE (10 mM Tris/HCl, 1 mM EDTA, pH 8·0) containing lysozyme (50 mg ml1), incubated for 10 min at room temperature and then treated with 350 µl RLT buffer (Qiagen) for lysis. After addition of 250 µl ethanol to the lysate, RNase-free DNase I (Qiagen) treatment was performed for DNA removal. The RNA was purified in succeeding steps with spin columns and finally eluted with 50 µl RNase-free water. The RNA was quantified spectrophotometrically and stored at 70 °C. RT-PCR was performed using the OneStep RT-PCR kit (Qiagen). Amplification of a constant amount of total RNA (20 ng) was performed with primers designated in Table 1 and in a final volume of 25 µl, according to the manufacturer's instructions, including 0·5 µM each RT 16S primer or 1 µM each RT acd (this study) and tpi (Lemée et al., 2004b
) primer. A reverse transcription reaction for 30 min at 50 °C was followed by an initial heating step of PCR amplification at 95 °C for 15 min to activate the DNA polymerase as well as to inactivate the reverse transcriptase. Then a touch-down procedure followed, consisting of 30 s at 95 °C, annealing for 30 s at temperatures decreasing from 55 to 45 °C during the first 11 cycles (with 1 °C incremental steps in cycles 1 to 11) and ending with an extension step at 72 °C for 30 s. A total of 40 cycles were performed. The analysis of acd expression at various stages of growth was performed in two experiments. Simultaneously, PCR was performed on RNA samples with the same oligonucleotides to exclude false-positive amplification from residual DNA.
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RESULTS |
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Determination of Acd hydrolytic bond specificity
Sequence homology analysis suggested that acd could encode an N-acetylglucosaminidase. To determine Acd hydrolytic specificity, the recombinant enzyme was used to digest cell walls from B. subtilis 168 HR. Mutanolysin, an autolysin with muramidase activity, was also used as a digestion control. As expected, the soluble muropeptides released from mutanolysin digestion were identical to those described by Atrih et al. (1999) (data not shown), but were completely different from soluble muropeptides released from Acd digestion, as revealed by RP-HPLC analysis (Fig. 4
a). We could thus conclude that Acd does not possess a muramidase activity. Half of the Acd-soluble muropeptide fraction was further analysed by RP-HPLC, which revealed three major peaks, named peaks 1, 2 and 3 (Fig. 4a
). MALDI-TOF MS generated molecular ions with m/z values of 892·26, 1815·74 and 1814·74 for these three peaks, respectively (Table 2
). According to previously described data (Atrih et al., 1999
; Huard et al., 2003
), these m/z values correspond to a disaccharide tripeptide muropeptide with one amidation for peak 1, and to a disaccharide tripeptide disaccharide tetrapeptide with one or two amidations for peaks 2 and 3, respectively (Table 2
, Fig. 5
). The other half of the Acd-soluble muropeptide fraction was incubated with mutanolysin. The RP-HPLC digested muropeptides and pattern revealed new peaks (4, 5, 6, 7 and 8) (Fig. 4b
). MALDI-TOF analysis of these peaks generated molecular ions with m/z values of 689·23, 1409·58, 1612·82, 1408·64 and 1611·91, respectively (Table 2
). These values correspond to a disaccharide tripeptide with one amidation and missing one N-acetylglucosamine for peak 4, to a disaccharide tripeptide disaccharide tetrapeptide with one amidation and missing two or one N-acetylglucosamines for peaks 5 and 6, respectively, and to a disaccharide tripeptide disaccharide tetrapeptide with two amidations and missing two or one N-acetylglucosamines for peaks 7 and 8, respectively (Table 2
, Fig. 5
). These results reveal that the muropeptides generated by Acd hydrolysis could be further cleaved by a muramidase (mutanolysin), indicating that N-acetylglucosamine is present on the reducing end of the disaccharide of these muropeptides. Finally, these results demonstrate that Acd has an N-acetylglucosaminidase activity, as initially suggested by the sequence homology data.
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DISCUSSION |
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Like most of the previously described bacterial PGHs (Heilmann et al., 1997; Hell et al., 1998
; Milohanic et al., 2001
; Oshida et al., 1995
), Acd has a modular structure with two main domains. The first 22 aa of the N-terminal domain of the deduced Acd sequence might constitute a putative signal peptide, or a retention signal with a transmembrane domain as described for a few proteins like cell-wall hydrolases in B. subtilis (Tjalsma et al., 2004
). Most known PGHs exhibit repeated sequences that could be involved in cell-wall binding. Although LysM domains involved in cell-wall targeting and peptidoglycan binding (Bateman & Bycroft, 2000
) have been described in the major autolysin of Lactococcus lactis (AcmA) (Buist et al., 1995
) and in a muramidase (MurA) of Listeria monocytogenes (Carroll et al., 2003
), such repeated sequences were not found in Acd. The PGH of C. difficile is also devoid of LPXTG or other LPXTG-like motifs enabling covalent binding to peptidoglycan (Comfort & Clubb, 2004
; Fischetti et al., 1990
). However, the N-terminal domain exhibits repeated sequences probably involved in cell-wall interactions (Wren, 1991
). These repeated sequences contain four putative GW modules, which constitute another motif for cell-surface anchoring. GW modules might interact with cell-wall polymers such as teichoic or lipoteichoic acids and are present in many surface proteins produced by Gram-positive bacteria, including L. monocytogenes InlB and Ami (Braun et al., 1997
; Cabanes et al., 2002
; Milohanic et al., 2001
) and the four staphylococcal surface autolysins, S. aureus Atl (Oshida et al., 1995
), S. caprae AtlC (Allignet et al., 2001
), S. epidermidis AtlE (Heilmann et al., 1997
) and S. saprophyticus Aas (Hell et al., 1998
).
The C-terminal domain (residues 415607) of Acd exhibits significant homology with the catalytic domain of LytD, a glucosaminidase from B. subtilis (39 % identity, 57·2 % similarity) and the C-terminal glucosaminidase domain of S. aureus Atl (40 % identity, 53·6 % similarity). Recent results with B. subtilis LytG (Horsburgh et al., 2003) or L. lactis AcmB (Huard et al., 2003
) indicate that PGHs presenting sequence homology may have different activities and that it is necessary to determine their hydrolytic bond specificity experimentally by analysis of the produced muropeptides. Since the purified recombinant Acd protein was found to hydrolyse B. subtilis vegetative cell walls in renaturing SDS-PAGE experiments, the hydrolytic bond specificity of Acd was further investigated in B. subtilis vegetative peptidoglycan, whose molecular structure has been previously studied in detail (Atrih et al., 1999
). RP-HPLC and MALDI-TOF MS analysis of muropeptides generated by Acd hydrolysis clearly confirmed the N-acetylglucosaminidase activity that was suggested through sequence homology analysis.
Regarding the deduced Acd amino acid sequences from 12 C. difficile strains, Acd is highly conserved, particularly in the C-terminal catalytic domain which includes the enzymic site of Acd. This supports the hypothesis that Acd has an important function in the physiology of C. difficile. The variation of acd expression during C. difficile vegetative growth was studied by RT-PCR, with 16S rRNA and tpi genes chosen as transcriptional controls owing to their housekeeping function. The present results indicate that acd is mostly transcribed during vegetative growth and suggest that the acd gene is involved in cellular physiological functions like peptidoglycan turnover or cell separation. Further investigation of the putative role of Acd in vegetative growth would require targeted disruption of the acd gene. However, the inability to transform this species, possibly due to the activity of endogenous restriction systems, hampers this type of investigation. Several genetic tools have been tentatively used to introduce heterologous DNA in C. difficile: (i) electroporation gene transfer (Ackermann et al., 2001), the efficiency of which was contested (Purdy et al., 2002
); (ii) use of conjugative transposons (Mullany et al., 1994
) or plasmids (Liyanage et al., 2001
), which showed low efficiency; (iii) removal or methylation of restriction sites in vectors derived from C. difficile plasmids, although this had not yet been used for the disruption of genes (Purdy et al., 2002
). An alternative antisense RNA approach was also elaborated, but was found to be insufficient to decrease the expression of the corresponding protein (Roberts et al., 2003
). Therefore, further improvements are required in genetic manipulation to study the function of targeted genes in C. difficile.
Although PGHs are implicated in several physiological functions, previous studies also suggested that autolysins may play an indirect role in the pathogenesis of some bacteria. It has been suggested that autolysins of Streptococcus pneumoniae facilitate the release of potent pro-inflammatory agents such as cell-wall components or pneumolysin, the main pneumococcal toxin (Canvin et al., 1995; Diaz et al., 1992
). Another study proposed that proteins lacking an N-terminal signal sequence could be exported from the cytoplasm by cell lysis or via the flagellar export machinery, the holin systems or other unidentified export systems (Tjalsma et al., 2004
). The pathogenesis of C. difficile is mainly due to toxin A (enterotoxin) and toxin B (cytotoxin) (Lyerly et al., 1988
), which are synthesized without any N-terminal signal peptide. The release of these toxins from the cytoplasm could be possible (i) by using a holin-like protein, TcdE, whose gene is located between the toxin A and B genes in the C. difficile pathogenicity locus (Tan et al., 2001
), or (ii) by cell lysis mediated by one or several PGHs. Thus the potential role of Acd in the pathology of C. difficile should be further investigated.
In conclusion, we report here the first molecular characterization of an autolysin of C. difficile, named Acd. This PGH displays N-acetylglucosaminidase activity and could thus play an important role in the physiology of this organism.
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ACKNOWLEDGEMENTS |
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Received 10 January 2005;
accepted 31 March 2005.
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