Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 5 rue JB Clément, F-92296 Châtenay-Malabry cedex, France1
Author for correspondence: Tuomo Karjalainen. Tel: +33 1 46 83 55 49. Fax: +33 1 46 83 13 03. e-mail: tuomo.karjalainen{at}cep.u-psud.fr
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ABSTRACT |
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Keywords: Clostridium difficile, adherence, pathogenesis, GroEL, heat shock
Abbreviations: GST, glutathione S-transferase; HSP, heat shock protein
The nucleotide sequence of the groESL locus of strain 79-685 was assigned GenBank accession number AF093568; that of strain ATCC 53603 was assigned GenBank accession number AF159449. The nucleotide sequence of the 16S rDNA of strain 79-685 was assigned GenBank number AF072474.
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INTRODUCTION |
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One aspect of C. difficile pathogenesis that has been studied by us is its interaction with target cells and identification of the bacterial factors involved in this process (Eveillard et al., 1993 ; Karjalainen et al., 1994
). Adhesion and colonization of animal tissue by bacteria is an important step in establishing infection. It is probable that without attachment, C. difficile cannot colonize and will be quickly removed by non-specific host defence mechanisms. It is likely that after destruction of normal flora by antibiotic treatment, C. difficile finds itself in a stressful environment that could serve as a stimulus for cell attachment and subsequent colonization. We have previously observed that C. difficile cell adherence is enhanced by heat, osmotic and acid shock as well as iron insufficiency (Waligora et al., 1999
). Furthermore, attachment was partially inhibited by an antiserum raised to GroEL of Mycobacterium leprae. This evoked a potential role for heat shock proteins (HSPs) in adherence. Therefore, we were interested in finding out whether GroEL, a member of the Hsp60 family of chaperonins, plays a role in C. difficile colonization.
In this study we undertook the isolation of the C. difficile groELS operon from two strains, and characterization and expression of the hsp60 gene. PCR amplification of the groEL gene coupled with RFLP analysis was used in an attempt to differentiate between clinical isolates. The role of Hsp60 in cell adhesion was examined by inhibition assays using antiserum and the purified Hsp60 protein, and by investigating the localization of the protein in various cell compartments.
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METHODS |
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The entire operon from the translation-initiation codon of GroES until the stop codon of GroEL was subsequently obtained by PCR using primers designed on basis of alignment of the 5' ends of the groES gene of Clostridium thermocellum (P48212 and P48223), C. perfringens (P26821) and Clostridium acetinobutylicum (M74572), and the 3' ends of the groEL gene of the same bacteria (primers 5'-ATGARWATYARACCAYTWGGWGACAFRG-3' and 5'-TTARTACATTCCGYCCATKCCCCRTWCC-3'). Finally, sequences upstream of the operon were obtained by amplification with groEL-specific internal primer (5'-CAATAACAGCTAATACATCAC-3') and a promoter-specific primer (5'-TGCTTCTGCAGGTACAGCTAAT-3'), and sequences downstream were obtained with the primer pair 5'-GGAATAGTAGCTGGTGGAGG-3' and 5'-TACTGATTATCTAAATATGTG-3'. These primers were designed on the basis of the genome sequence of C. difficile strain 630 that recently became available on the Internet (http://www.sanger.ac.uk).
Amplified products were purified by the Wizard Gel Extraction Kit (Promega) and the nucleotide sequences of both strands of the amplified products were obtained by using the Taq DyeDeoxy and Big Dye Terminator Cycle sequencing kits purchased from Perkin Elmer. The labelled extension products were analysed with an ABI PRISM 310 Genetic Analyser (Perkin Elmer). Additional primers were designed to obtain additional internal sequence.
Southern blotting.
DNA of C. difficile strains was prepared as described above. DNA (2 µg) was digested with HindIII and AccI according to instructions provided by the supplier (Pharmacia), the fragments separated through a 0·8% agarose gel and electrically transferred to a nylon membrane (Roche). A 340 bp amplified PCR product of the groEL gene of C. difficile strain 79-685 was used as a probe, labelled in a standard PCR reaction (see above) with 25 µl of the following nucleotide mix: 25 µl 1 mM digoxigenin-11-dUTP (Roche), 4·6 µl 10 mM dTTP, 7·1 µl 10 mM dATP, 7·1 µl 10 mM dGTP, 7·1 µl 10 mM dCTP (Promega) and 2·5 µl sterile water. Hybridization was carried out overnight at 42 °C with 1 µg probe (ml DIG Easy Hyb)-1 (Roche). The subsequent washing steps and detection with CSPD (disodium 3-{4-methoxys p iro[1,2-dioxetane-3,2'-(5'-chloro) tricyclo(3.3.1.13,7)decan]-4-yl} phenyl phosphate; Roche) were carried out as recommended by the manufacturer.
RFLP analysis.
A 1448 bp amplicon encompassing groEL was obtained by PCR as described above on genomic DNA from 12 strains using primers 5'-ATGARWATYARACCAYTWGGWGACAFRG-3' and 5'-TACAACAGCTACTCCTCCAGC-3'. The DNA was digested with eight restriction enzymes under the conditions recommended by the supplier (New England Biolabs). These digests were then subjected to electrophoresis in a 1% agarose gel.
Cloning of C. difficile 79-685 groEL into an expression vector.
Two oligonucleotide primers, PGEX1 (5'-ATTGAATTCGAGGGGTTTAAAATGGCTA-3') and PGEX2 (5'-TTAGTACATTCCGTCCATGCCCGTTCCT-3'), incorporating an EcoRI site in PGEX1 (shown in bold; the translation-initiation codon of GroEL is underlined), were synthesized and used to amplify by PCR the full-length coding region of groEL of strain 79-685 in a standard amplification reaction as described above. The resulting 1·6 kb DNA product was blunt-ended by Klenow [1 U in 30 µl One-Phor-All buffer (Pharmacia) for 2 h at 37 °C] and subsequently digested with EcoRI. The fragment was ligated (1 U T4 ligase; Life Technologies) into the EcoRI and SmaI sites of pGEX-6P-1 (Pharmacia) and transformed into E. coli XLOLR (Stratagene). A transformant was identified carrying a plasmid which, after nucleotide sequencing of the junction between vector and insert, was found to be capable of producing an in-frame fusion protein between glutathione S-transferase (GST) and GroEL. This plasmid was transformed into E. coli BL21.
Expression and purification of the fusion protein.
An overnight culture of E. coli BL21 containing pGEX-6P-1groEL grown in LB broth (Difco) containing carbenicillin (50 µg ml-1) was diluted 1:100 into 4 l LB medium containing carbenicillin and the culture grown to OD600 0·6 at 37 °C. The expression of the fusion protein was induced by addition of 1 mM IPTG and the culture was continued for 12 h at room temperature. Bacteria were collected by centrifugation and resuspended in 200 ml 20 mM Tris/HCl, pH 7·5. The bacteria were lysed by incubation with freshly prepared lysozyme at 100 µg ml-1 (Sigma) for 15 min at 30 °C, followed by sonication at intervals of 5 s for 1 h at 80% power (Bioblock Scientific 72442 Vibra Cell) and three successive freezethaw cycles. Insoluble material was removed by centrifugation at 14000 g for 10 min, and the fusion protein was purified from the supernatant by a single-step affinity chromatography using glutathioneSepharose-4B and protocols from Pharmacia. A 2 ml bed volume was used for each 200 ml sonicate; the column was washed three times with 20 ml PBS, followed by cleavage of the GroEL moiety bound to glutathioneSepharose with 80 U Prescission protease (Amersham Pharmacia Biotech) per 1 ml bed volume.
Antibodies.
A rabbit polyclonal, monospecific GroEL antiserum was prepared. The band corresponding to the purified C. difficile GroEL protein was cut out of the PAGE gel and was lyophilized. Purified protein (200 µg) was injected with Freunds complete adjuvant into New Zealand White rabbits (AGROBIO), followed by four boosters with 100 µg protein in Freunds incomplete adjuvant at day 14, 28 and 42. The rabbits were killed and bled 21 d after the last injection. Antibodies were purified on protein ASepharose (Pharmacia-Biotech) according to the suppliers recommendations and used at a 1:2000 dilution in immunoblots.
Antiserum against C. difficile was produced as described previously (Karjalainen et al., 1994 ). Monoclonal antibodies raised against the 65 kDa HSP of M. leprae were obtained from UNPD/World Bank/WHO Special Program for Research and Training in Tropical Diseases, Atlanta, GA.
N-terminal protein sequence.
The purified C. difficile GroEL protein was electroblotted onto a PVDF membrane (Roche) with 50 mM Tris/HCl, 50 mM boric acid buffer and stained with 0·1% amido black (Sigma). The band was excised and N-terminal sequencing was performed by the Laboratory of Microsequencing of Proteins, Pasteur Institute, Paris.
Immunoelectron microscopy.
After adsorption of a bacterial preparation to Formvar-coated grids (Sigma) for 5 min, the grids were floated on PBS with 1% BSA for 30 min, then incubated with GroEL antibodies diluted 1:10 for 1 h and washed three times in PBS. They were incubated with a 1:20 dilution of protein A conjugated with 10 nm diameter colloidal gold particles (Sigma) for 1 h, washed three times with PBS and fixed with 3% glutaraldehyde. After three washings the grids were stained with phosphotungstic acid before observation by transmission electron microscopy.
Immunofluorescence.
Bacteria were cultured for 24 h at 37 °C in TGY and shocked at 43 °C for 20 min. A drop of the PBS-washed culture was deposited on a microscope slide and dried. The slide was immersed in PBS (pH 7·2)/5% skimmed milk (1 h, room temp.), followed by a 1 h incubation with protein A purified anti-GroEL antibodies (dilution 1:2000 in the same solution). The slide was washed with PBS (pH 7·2)/0·1% skimmed milk and incubated for 15 min with tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-mouse IgG antibody (Immunotech; 1:20 dilution in PBS/5% skimmed milk). After washing, the specimens were examined with a 100x oil-immersion objective using a Leitz Aristoplan microscope with epifluorescence coupled to an Image Analyzer Visiolab 1000 (Biocom).
Fractionation.
Bacterial proteins were separated into four compartments: supernatant, cell wall, membrane and cytoplasm, using a protocol described for Listeria monocytogenes (Jonquières et al., 1999 ). Equivalent amounts of each fraction, corresponding to 20 ml bacterial culture, were separated by SDS-PAGE (Laemmli, 1970
) using a 7·5% SDS-polyacrylamide gel, transferred to nitrocellulose and submitted to immunodetection as described below. The purity of the fractions was verified by studying two proteins with known cellular locations. Cwp66-C was used as a marker for the cell wall fraction (a peptidoglycan-attached protein, our unpublished data); PepC, a cytoplasmic peptidase reported to be adsorbed to the inner face of the cytoplasmic membrane in Lactococcus lactis (Jonquières et al., 1999
), was used as a marker for the cytoplasmic and membrane fractions.
The presence of GroEL in the supernatant of exponential-phase bacteria was investigated by precipitating proteins by 70% trichloroacetic acid as described by Schubert et al. (2000) and by detecting the presence of GroEL by immunoblotting as described below.
Immunoblotting.
Proteins separated by SDS-PAGE were transferred electrically onto nitrocellulose membrane for immunoblotting. The nitrocellulose membrane was incubated for 1 h at room temperature in blocking buffer (5% skimmed milk in TNT [10 mM Tris/HCl, pH 8·0, 150 mM NaCl, 0·05% Tween]) and then 1 h in the appropriate dilution of the specific antibody. The membranes were washed in TNT and bound antibodies were detected with goat anti-rabbit IgGalkaline phosphatase conjugate (1:2500 dilution; Sigma) with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Life Technologies) as substrates.
Cell-adherence and adherence-inhibition assays.
Cell-adherence assays were performed as previously described using the Vero cell line (Karjalainen et al., 1994 ). For adherence inhibition with antibodies, bacteria that had been washed twice in PBS and heat shocked at 48 °C or 60 °C for 20 min were preincubated with antibodies (1:10, 1:100 or 1:1000 dilution in TNT+5% skimmed milk) for 30 min before contact with cells (1 h at 37 °C under anaerobic conditions). When inhibitions were carried out with the purified GroEL protein or BSA (non-adhesive protein control), cells were preincubated for 15 min at 37 °C under a 5% CO2 atmosphere with 10, 50 and 75 µg protein ml-1 in Modified Eagles Medium (Life Technologies) before bacteria were put into contact with cells for 1 h. Non-adherent bacteria were eliminated by five washings in PBS (10 mM phosphate buffer, 150 mM NaCl, pH 7·0) and the cells were fixed and stained with May-Grünwald-Giemsa (Sigma). The adhesion index is given as the mean number of adhering bacteria per cell±SD (counted at a magnification of 1000x) from at least three different assays. The significance of differences between various treatments was assessed by a Students t-test.
Expression of GroEL induced by contact with target cells.
Exponential-phase C. difficile 79-685 (anaerobic culture in 100 ml TGY at 37 °C for 6 h) were collected by centrifugation and washed twice in PBS, pH 6·8, prior to application to the Vero cell monolayer. After 1 h adherence under anaerobic conditions, bacteria and cells were scraped off the tissue culture flasks with a rubber policeman and collected by centrifugation (10 min, 5000 g). After washing twice in PBS, the pellet was resuspended in 1·5 ml 0·1 M Tris/HCl, pH 8·6, and proteins were extracted by three successive freezethaw cycles. After centrifugation at 15000 g for 15 min, the supernatant was recovered and proteins present in it were separated by SDS-PAGE. GroEL was detected by immunoblotting as described above. The induction of expression of GroEL by contact with eukaryotic cells was evaluated by densitometric scanning of the bands. The C. difficile gelatinase (Poilane et al., 1998 ), a non-adhesive protein, served as a negative control.
DNA manipulations.
Plasmid isolations were performed by the alkaline lysis procedure using a kit from Qiagen. Ligations and restriction endonuclease digestions were done according to Sambrook et al. (1989) and protocols provided by vendors. The TSB method was used for transformation of E. coli (Chung et al., 1989
).
Computer analyses.
Nucleotide and amino acid sequence alignments were performed with the CLUSTALX program (Thompson et al., 1997 ). Homology searches were conducted with FASTA3 (European Bioinformatics Institute) or BLAST (National Institute for Biotechnology Information, Washington, DC). Hairpin loops in DNA were searched for using RNAdraw (Matzura & Wennborg, 1996
).
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RESULTS AND DISCUSSION |
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Analysis of the groESL operon from C. difficile
The 1940 bp groELS operon of C. difficile 79-685, isolated by PCR, is composed of the 285 bp groES gene which has the capacity of encoding a 10198 Da protein (pI=4·93) and the 1623 bp groEL gene with the capacity of encoding a 57677 Da protein (pI=4·72) (Fig. 1). The genes are separated by a 32 bp non-coding sequence. The G+C content of groES is 34 mol% and that of groEL is 33 mol%. Analysis of the deduced protein sequence of GroEL revealed the presence of the peptide sequence AAVEEGIVAGGG at position 403414 that is characteristic of the Hsp60 chaperone family [A-(AS)-X-(DEQ)-E-(X4)-G-G-(GA)]. Neither GroES nor GroEL possesses a signal peptide. Secondary structure prediction using the Chou & Fasman (1978)
algorithm revealed a predominantly
-helical conformation with one potential transmembrane domain in the GroEL protein (Fig. 1
). The 540 aa protein carries 28% charged residues. C. difficile GroEL displays best homology with that of C. acinetobutylicum (75% identity), C. thermocellum (75%), C. perfringens (74%) and Bacillus subtilis (73%).
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groEL copy number
In some bacteria groEL is found as multiple copies in the genome, so a genomic Southern blot was performed to determine if the C. difficile groEL gene is single or multi-copy. A 340 bp intragenic fragment was used to probe a Southern blot of genomic DNA isolated from 12 strains (Table 1) individually digested with two restriction enzymes. Employing low-stringency wash conditions, both enzymes produced only a single hybridizing band, suggesting that only a single genomic groEL locus is present (data not shown).
PCR amplification and RFLP analysis of amplified groEL gene products
As adhesins are attractive targets for the development of vaccines, we examined inter-strain variability of the groEL gene. Two internal oligonucleotide primers were used to amplify the groEL gene from 12 isolates (Table 1). All strains yielded an amplified product of 1·45 kb. The fragments were digested with AccI, AluI, DraI, EcoRV, HindIII, PvuII, ScaI and RsaI. All profiles obtained were identical except that of strain ATCC 53603 which with RsaI digestion exhibited a different pattern from the others (not shown). Sequencing of the gene from this strain showed an alteration of a nucleotide in an RsaI site but the gene was more than 99% homologous to the one isolated from the strain 79-685 at the nucleotide and amino acid level (data not shown). Thus GroEL is highly conserved between strains and could be used as a candidate in vaccine preparations.
Expression, purification, immunological detection and localization of GroEL
We decided to purify the GroEL protein in order to obtain a polyclonal, monospecific antiserum for GroEL permitting functional studies. groEL was expressed as a fusion with GST in E. coli and purified by affinity chromatography. As shown in Fig. 2(a), lane 7, a 58 kDa band corresponding to GroEL was observed in the eluate by SDS-PAGE. N-terminal sequencing of 21 aa of the purified protein revealed the sequence GPLGSPEFEGFKMAKEIKFSE, which is identical to the N-terminal amino acid sequence deduced from the groEL nucleotide sequence (residues in bold are the remaining GST residues encoded by pGEX-6P-1).
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Earlier studies had indicated that Hsp60 and Hsp70 (DnaK) are in general cytoplasmic proteins, but more recent studies suggest that they are membrane associated in some micro-organisms or secreted from the cells. If a protein is to serve as an adhesin, surface localization is required. GroEL of numerous bacteria, such as Legionella pneumophila (Garduno et al., 1998a , b
; Hoffman et al., 1990
), Helicobacter pylori (Cao et al., 1998
; Dunn et al., 1997
; Eschweiler et al., 1993
; Huesca et al., 1996
, 1998
; Kamiya et al., 1998
; Phadnis et al., 1996
; Vanet & Labigne, 1998
; Yamaguchi et al., 1996a
, b
, 1997
, 1998
), Haemophilus ducreyi (Frisk et al., 1998
; Parsons et al., 1997
), M. avium (Rao et al., 1994
), S. typhimurium (Ensgraber & Loos, 1992
), Actinobacillus actinomycetemcomitans (Goulhen et al., 1998
) and Borrelia burgdorferi (Kaneda et al., 1997
), has been shown to be involved in adhesion or invasion of various target cells or tissues and can be surface localized. C. difficile grown at 37 °C and heat shocked at 48 °C was fractionated to determine the localization of GroEL. The proteins in each fraction were separated by SDS-PAGE and analysed for GroEL by Western immunoblotting (Fig. 2b
). The GroEL protein was found mostly in the cytoplasmic and membrane fractions as well as extracellularly. A small amount of protein was found in the cell wall fraction.
Localization of GroEL on whole bacteria was examined by immunoelectron microscopy and immunofluorescence. In the non-heat-shocked condition (Fig. 3a), little immunolabelling of bacteria was observed [mean 1·2 grains (cm bacterial surface) -2]. More than twofold increase of grain density cm-2 was observed in bacteria shocked at 42 °C (mean 2·7 grains cm-2) (Fig. 3b
) and a more than sixfold increase at 48 °C (7·7 grains cm-2) (Fig. 3c
) compared with non-heat-shocked bacteria. The ratio of density of bacterial associated grains compared to the background labelling increased at each temperature. After heat shock at 48 °C the protein was associated with amorphous electron-dense, cell-surface associated material and was distributed in a relatively uniform fashion over the bacterial surface. After a heat shock, especially at 48 °C, the GroEL protein was also found to be localized within the extracellular space. Immunoblot analysis of proteins precipitated from the supernatant of heat shocked but intact and viable bacteria confirmed the presence of Hsp60 in this localization (data not shown).
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GroEL plays a role in cell adherence
The fact that C. difficile cell attachment is increased by stress and the presence of GroEL on the bacterial cell surface suggests a possible role for this protein in cell adherence. Involvement of the Hsp60 in adhesion of C. difficile to eukaryotic cells was investigated in competitive inhibition assays using anti-GroEL antibodies or the purified protein. As shown in Fig. 5, co-incubation of bacteria with antibodies at a dilution of 1:10 demonstrated a relative adherence of 55% compared with the control (incubation with preimmune serum at the same dilution), indicating that Hsp60 may indeed be involved in the adherence process. A significant reduction of adherence was also observed at 1:100 dilution of antibodies, whereas at 1:1000 dilution the decrease was not significant. Furthermore, the purified protein inhibited cell adherence by 50% at a concentration of 10 µg ml-1. In contrast, no inhibition was observed when competitive inhibition was carried out with the non-adhesive protein BSA. As in H. ducreyi (Frisk et al., 1998
), the partial inhibition can be explained by the likelihood of having multiple adhesins and mechanisms involved in adherence of C. difficile. We are in the process of characterizing some of the other adhesins.
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ACKNOWLEDGEMENTS |
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Received 1 June 2000;
revised 15 September 2000;
accepted 26 September 2000.