Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 5 rue JB Clément, 92296 Châtenay-Malabry cedex, France1
Institute of Infection and Immunity, Queens Medical Centre, University of Nottingham, University Park, Nottingham NG7 2RD, UK2
PHLS Central Public Health Laboratory, 61 Colindale Ave, London NW9 5HT, UK3
Author for correspondence: Marie-Claude Barc. Tel: +33 1 46 83 55 49. Fax: +33 1 46 83 58 83. e-mail: marie-claude.barc{at}cep.u-psud.fr
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ABSTRACT |
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Keywords: Clostridium difficile, flagella, flagellin, PCR, cloning
Abbreviations: GST, glutathione S-transferase
The GenBank accession numbers for the sequences reported in this paper are AF065259 (strain 79-685) and AF077341 (strain VPI 10463).
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INTRODUCTION |
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One aspect of C. difficile virulence that has been studied by us is its interaction with target cells (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.
We are interested in finding out whether flagella play a role in C. difficile colonization. In this study we undertook the isolation of C. difficile flagella from clinical strains and characterization and expression of the flagellin subunit gene. In addition, Southern analysis and PCR amplification of flagellin genes coupled with RFLP analysis were used in a preliminary attempt to differentiate between clinical isolates.
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METHODS |
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Flagellin protein of C. difficile: isolation, characterization and N-terminal sequencing.
Flagellin proteins were isolated by the procedure described by Delmée et al. (1990) . The strains were grown on blood agar plates under anaerobic conditions for 48 h. Bacteria were harvested in 5 ml distilled water; the suspensions were strongly shaken for 1 min and centrifuged at 5000 g for 30 min at 4 °C. The supernatants were centrifuged at 25000 g for 1 h at 4 °C and the pellets were suspended in 100 µl PBS (pH 7·4).
SDS-PAGE was carried out as described by Laemmli (1970) using an SDS-PAGE gel (7·5%, w/v, acrylamide). Gels were stained with Coomassie blue or used for electric transfer onto nitrocellulose membrane for immunoblotting. The nitrocellulose membrane was incubated for 30 min at room temperature in blocking buffer (0·2% Tween, 3% skim milk in PBS) and then overnight in a rabbit polyclonal antiflagellin serum to serogroup A (strain W1194), kindly provided by M. Delmée (UCL, Brussels, Belgium) (1:2000 dilution). The membranes were washed in 10 mM Tris/HCl, 150 mM NaCl, 0·05% Tween 20 buffer (TNT) and bound antibodies were detected with goat anti-rabbit IgG alkaline phosphatase conjugate (1:2500 dilution; Sigma) with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates.
For the N-terminal sequencing of the flagellin, proteins were electroblotted onto a Hybond-N membrane (Amersham) with CAPS buffer (Sigma) and stained with amido black. The bands of interest were excised and N-terminal analysis was performed (Polymer synthesis and Analysis unit, Department of Biochemistry, University of Nottingham). The method of Edman (1950) was used with an Applied Biosystems 473A amino acid sequencer on a PTH-RP-PHLC C18 column. Identification was performed using 610A analysis software (Applied Biosystems).
Electron and immunoelectron microscopy of flagella and strains.
Flagellum preparations or 24 h cultures of C. difficile strains as described above were resuspended in distilled water. They were negatively stained with a phosphotungstic acid solution for 5 min and adsorbed on carbon-stabilized nitrocellulose film copper specimen grids (2 min) by placing each grid on a drop of the bacterium-staining solution. After drying, the grids were observed with an EM 301 Philips transmission electron microscope.
For immunoelectron microscopy, after adsorption of bacteria or flagellum preparation to the grids for 5 min, the grids were floated on PBS with 1% BSA for 30 min, then incubated with antiflagellin antibodies diluted 1:100 for 1 h and washed three times in PBS. They were incubated with a 1:20 dilution of protein A labelled with 15-nm-diameter colloidal gold particles (British Biocen International) for 1 h, washed three times with PBS and subsequently fixed with 3% glutaraldehyde. After three washings the grids were stained with phosphotungstic acid before observation by transmission electron microscopy. A strain of B. subtilis was used as a negative control.
Motility assays.
Motility assays were conducted in BHI broth with 0·175% agar. The medium was placed in tubes which were inoculated from a colony by stabbing the agar with a toothpick. The tubes were incubated at 37 °C for 2 d under anaerobic conditions.
DNA techniques.
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 the vendors. The TSB (Transformation Storage Buffer) method was used for transformation of E. coli (Chung et al., 1989
).
PCR amplification and nucleotide sequencing of the C. difficile flagellin gene.
The N-terminal sequence of the C. difficile 79-685 flagellin and conserved motifs in the C-terminal sequences of flagellins from various bacteria (V. cholerae, Vibrio parahaemolyticus, B. subtilis, Clostridium tyrobutyricum) were used to design degenerate primers synthesized by Life Technologies. The N-terminal primer was ATGMGAGTWAATGTWTCWGCGCTY; the C-terminal primer was TTGWCGAAYTGTTGGTTWGCAGCWAGCAG. Genomic DNA was extracted from C. difficile strains as described by Karjalainen et al. (1994) or bacteria were harvested from blood agar plates, resuspended in sterile distilled water and boiled for 5 min and used directly in a standard amplification mixture. Amplifications were carried out in 100 µl volumes containing 1 µg genomic DNA, 2 µl MgCl2, 1 U Taq polymerase (Promega), 200 pmol of each deoxynucleotide triphosphate, 1 µM of each primer and 10 µl 10xpolymerase buffer for 34 cycles consisting of denaturation at 95 °C (1 min), annealing at 55 °C (1 min) and extension at 72 °C (2 min) (Perkin Elmer Thermocycler 480). At the end of the amplification, 20 µl samples were subjected to electrophoresis on a standard 1% agarose gel alongside a PCR size marker (100 bp ladder; Pharmacia) to confirm the presence of an amplified product. Amplified products were purified by a gel extraction kit (Wizard Gel Extraction kit; Promega).
The nucleotide sequences of both strands of amplified products of the C. difficile flagellin of strains 79-685 and VPI 10403 were obtained by using the Taq Dye Deoxy Terminator and Dye Primer cycle sequencing protocols developed by Applied Biosystems (Perkin Elmer) using fluorescence-labelled dideoxynucleotides and primers, respectively. The labelled extension products were analysed with an ABI PRISM 310 Genetic Analyzer (Perkin Elmer). More nucleotide sequence upstream and downstream of the first amplified region of the flagellin gene was obtained by amplification by PCR from the ZapII genomic library (Karjalainen et al., 1994
), converted into a plasmid library by excision en masse, using a gene-specific primer (ACGAACCTTCTGCTGTTTGTAC) and a primer (GGAAACAGCTATGACCATG) that hybridized with the pBluescript polylinker (M13rev). For the upstream region a PCR product of 500 bp was obtained. For the downstream region a PCR product of 1·1 kb was obtained with the gene-specific primer CTTTAGAGAATGTTACAGCAGC and a vector-specific primer GTAAAACGACGGCCAGT (M13 -20). Secondary structure of the flagellin protein was predicted by using the Chou and Fasman algorithm (Chou & Fasman, 1978
). Additional sequencing of the flagellin gene was performed with internal primers.
Southern blotting and RFLP analysis.
DNA of C. difficile strains was prepared as described by Karjalainen et al. (1994) . DNA was digested with HindIII and electrically transferred to a nylon membrane (Boehringer Mannheim). The 850 bp amplified PCR product was used as a flagellin gene specific probe. The DNA probe was labelled and detected by using the ECL direct nucleic acid labelling and detection system from Amersham. Washing of membranes was performed under low stringency (0·5xSSC at 42 °C). For RFLP amplified DNA was obtained by PCR from colonies grown on blood agar plates as described above. The DNA was digested with four restriction enzymes, HindIII, EcoRV, DraI and SacI, under the conditions recommended by the supplier (New England Biolabs). These digests were then subjected to electrophoresis on a 1% agarose gel alongside a PCR size marker (100 bp ladder; Pharmacia).
Cloning, expression, purification and identification of the fusion protein.
For the cloning of the C. difficile 79-685 flagellin gene into an expression vector, two oligonucleotide primers, CCCCTGGGATCCATGAGAGTTAATACAAATGTAAGTGC and CCGGGAATTCCTATCCTAATAATTGTAAAACTCC, incorporating the BamHI and EcoRI restriction site, respectively, were synthesized and used to amplify by PCR the full-length coding region of the fliC gene of strain 79-685 (Taq polymerase, Promega; 1 U per 100 µl reaction volume). The resulting 895 bp DNA product was digested with BamHI and EcoRI and cloned in-frame into the corresponding sites of pGEX-6P-1 (Pharmacia). The nucleotide sequence of the junction between vector and insert was confirmed by sequencing analysis to be correct. The plasmid was transformed into E. coli BL21.
For the expression and purification of the fusion protein, an overnight culture of E. coli BL21 containing pGEX-6P-1-fliC was diluted 1:100 into 4 l 2xYT medium (Life Technologies) containing ampicillin and the culture was grown to OD600 0·6 at 37 °C. The expression of the fusion protein was induced by adding IPTG at 1 mM for 2 h. Bacteria were collected by centrifugation and resuspended in 200 ml ice-cold PBS. The bacteria were lysed by sonication (intervals of 5 s for 1 h at 80% power; Bioblock Scientific 72442 Vibra Cell). Insoluble material was removed by centrifugation at 8000 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 FliC moiety bound to glutathioneSepharose with 80 units Prescission protease per 1 ml bed volume. Identification of the fusion protein was carried out by SDS-PAGE as described above and by immunoblot (serum dilution 1:2000).
A polyclonal antiflagellin serum was raised against the purified FliC recombinant protein. The gel band located at 39 kDa corresponding to the purified flagellin was cut out and injected into a rabbit. The polyclonal, monospecific antiserum was obtained according to a protocol described previously (Karjalainen et al., 1994 ) and used at a 1:2000 dilution in Western blots.
Computer analyses.
Nucleotide and protein sequence alignments were performed with the CLUSTALX program (Thompson et al., 1997 ). Homology searches were conducted with FASTA3 (EBI) or BLAST (National Institute for Biotechnology Information, Washington).
Adherence inhibition assays.
Cell culture and adherence inhibition assays were performed as previously described (Karjalainen et al., 1994 ). For adherence inhibition with antibodies, bacteria were incubated with preimmune serum (dilution 1:2) or immune serum (dilution 1:2) for 1 h prior to contact of 1 h with cultured Vero cells. 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 MayGrünwaldGiemsa (Sigma). The adhesion index is given as the mean number of adhering bacteria per cell (counted at a magnification of x1000) from at least three different assays.
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RESULTS |
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Detection of flagella by electron microscopy
Electron microscopy was used to observe the flagella of C. difficile strains, either on intact bacteria or in isolated flagellum preparations (Fig. 2). Some strains, such as Kohn and W1194, had numerous flagella, whereas other strains carried fewer flagella. It was evident by immunoelectron microscopy that the flagella were labelled by the Delmée flagellin antiserum A. B. subtilis and a C. sordellii strain, used as negative controls, showed no labelled flagella.
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DISCUSSION |
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Analysis of the promoter structure reveals the presence of a motif resembling the consensus sequence for 28 regulated promoters. However, the distance between the -10 and -35 motifs is 16 nt instead of the usual 15.
28, which is involved in transcription of the flagellar and chemotaxis genes, was originally found in B. subtilis (Gilman & Chamberlin, 1983
). Although transcription of some flagellar genes is initiated by
54, no consensus motif for this factor is present in the C. difficile fliC promoter.
The flagella of six strains of C. difficile were isolated. The molecular mass of C. difficile flagellin, 39 kDa, is in the middle of the range of other characterized flagellin molecules, which have been reported to have molecular masses ranging from 15 to 62 kDa (Arnold et al., 1998 ; Joys, 1988
; Sakamoto et al., 1992
; Wilson & Beveridge, 1993
). C. difficile flagellins did not display heterogeneity between the different strains studied nor did they have multiple molecular masses. Immunoblotting and immunogold labelling of strains with a polyclonal antiserum raised against purified flagellin demonstrated that all C. difficile strains reacted with the antiserum, in contrast to strains of C. sordellii and B. subtilis. This result suggests that the flagellin of each strain contains cross-reacting epitopes due to the presence of the flagellin monomers. Although flagellins generally are structurally conserved in the N- and C-termini, the internal region is divergent and accounts for serological distinctiveness. Prediction of antigenic determinants in FliC of C. difficile using the Hopp and Woods algorithm (Hopp & Woods, 1981
) revealed the highest probability for the presence of such motifs in the central, variable region between aa 100 and 130. The central region has been proposed to be the region that is exposed to the outer environment (Sakamoto et al., 1992
). Like other flagellins, the central portion has a surplus of acidic residues over basic residues and, by implication, a net negative charge. It appears that most bacterial cell surface proteins carry a net negative charge (Wilson & Beveridge, 1993
). We suspected that the ORF downstream of the fliC gene could encode a glycosyltransferase that could be involved in the in situ glycosylation of flagella. If FliC of C. difficile were glycosylated, this could explain the difference in molecular mass observed on SDS-PAGE (39 kDa) and that estimated from the nucleotide sequence (31 kDa). In fact, using the DIG Glycan Detection kit we demonstrated that C. difficile strain 79-685 flagella are non-glycosylated. The fact that the cloned gene expressed in E. coli produces a flagellin with almost the same molecular mass as the native protein from C. difficile suggests that the protein undergoes post-translational modification other than glycosylation, since E. coli is not able to glycosylate proteins. Flagellar filaments can contain phosphorylated tyrosines or serines,
-N-methyl-lysine, or they can be sulfated glycoproteins.
The isolation of the fliC gene and isolation of a monospecific antiserum will allow further investigations as to the role of flagella in the pathogenic process. It has been suggested that the virulence of C. difficile strains is not solely attributable to toxin production; other factors such as presence of flagella could contribute to virulence. The role of flagella in microbial pathogenesis factors such as virulence, adherence, invasiveness or colonization has been demonstrated for numerous bacteria (Grant et al., 1993 ; Scherer et al., 1993
; Zhang et al., 1993
; Grossman et al., 1995
; Pruckler et al., 1995
; Tamura et al., 1995
; Milton et al., 1996
; Mobley, 1996
; Bosshardt et al., 1997
; Kennedy et al., 1997
; Rosalski et al., 1997
; West et al., 1997
; Feldman et al., 1998
). In our experiments in vitro, no inhibition of adherence was shown with antibodies against the recombinant flagellum subunit. However, lack of adherence does not mean there is no role in colonization or virulence. We are planning to investigate further the role of the flagellin cap in adhesion (Arora et al., 1998
) and that of flagella in colonization and virulence using, for example, animal models.
The gene isolated here could be a potential biomarker to assess intraspecies genetic variation as there appears to be a divergent region in the amino acid sequence of the protein, a feature of surface-located proteins in bacteria (Whittam, 1995 ; Winstanley et al., 1996
; Winstanley & Morgan, 1997
). An epidemiological survey using the combined PCR-RFLP method and nucleotide sequencing is in progress on a larger number of C. difficile isolates.
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ACKNOWLEDGEMENTS |
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Received 22 September 1999;
revised 20 December 1999;
accepted 7 January 2000.