1 Oral Microbiology Unit, Department of Oral and Dental Science, University of Bristol Dental School, Lower Maudlin Street, Bristol BS1 2LY, UK
2 Molecular Genetics Unit, Department of Bacterial Diseases, Veterinary Laboratories Agency (Weybridge), Woodham Lane, Addlestone, Surrey KT15 3NB, UK
Correspondence
Howard F. Jenkinson
howard.jenkinson{at}bris.ac.uk
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ABSTRACT |
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The GenBank accession numbers for the 16S rDNA sequences reported in this paper are AY119692 (Treponema denticola GM-1), AY119690 (Treponema vincentii D2A-2), AF363634 (Treponema sp. UB1466, previously designated G179) and AY119691 (Treponema sp. UB1467).
Present address: Department of Pathology and Microbiology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.
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INTRODUCTION |
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Treponema denticola is strongly associated with progression of adult human periodontitis (Sela, 2001), a chronic inflammatory disease of the gums and gingival tissues that can lead to bone resorption and tooth loss (Page et al., 1978
). Disease severity is correlated with the presence, within periodontal lesions, of T. denticola in association with obligately anaerobic Gram-negative bacteria such as Porphyromonas gingivalis and Bacteroides forsythus (Tannerella forsythensis) (Socransky et al., 1998
). The interactions of these organisms with periodontal tissues involves adhesion to epithelial cells and extracellular matrix components, proteolysis and modulation of host immune functions (Loesche, 1993
; Lamont & Jenkinson, 1998
). A number of studies have demonstrated that T. denticola adheres avidly to epithelial cells (Ellen et al., 1994
), causes cytopathic effects mediated in part via the combined activities of proteases (Grenier et al., 1990
; Fenno et al., 1998
) and an outer-membrane protein designated Msp (Fenno et al., 1996
), and actively penetrates tissue layers by chemotaxis (Lux et al., 2001
). However, on the basis of 16S rDNA analysis data from periodontal pocket samples, it appears that T. denticola may be only one of more than 20 different species of Treponema found within a single active disease site (Choi et al., 1994
). The contributions of these other species, including the more well-characterized species Treponema pectinovorum and Treponema vincentii, to periodontal disease pathogenesis is currently not understood.
Digital dermatitis (DD), which is a bovine foot disease, and contagious ovine digital dermatitis (CODD, formerly known as severe virulent ovine footrot) are both ulcerative conditions that lead to lameness in affected animals (Blowey & Sharp, 1988; Naylor et al., 1998
). Lesions originate in the interdigital cleft and if left unchecked, spread to encompass the entire foot (Blowey et al., 1994
). Outbreaks of these diseases within herds or flocks are common, particularly in winter, and result in profound welfare and economic problems (Read & Walker, 1998
). Spirochaetes have been identified within these lesions and based upon 16S rDNA sequence data, have been shown to be related to human oral isolates of T. denticola and T. vincentii (Choi et al., 1997
; Collighan et al., 2000
). Although aetiological roles for Treponema in bovine or ovine foot diseases have yet to be proven, treponemes tend to be found only in active lesions (Dopfer et al., 1997
). Furthermore, serum samples from diseased animals contain elevated antibody levels to Treponema antigens (Demirkan et al., 1999b
). There are thus many parallels with the pathology, immunology and inferred bacterial aetiology of these human and animal diseases of collagenous tissues (Edwards et al., 2003
).
In this article we describe the genotypic and phenotypic properties of three Treponema isolates that we have successfully cultivated from animal foot disease lesions. The properties of these isolates have been compared with T. denticola and T. vincentii strains originating from the human oral cavity. The results show that the animal isolates are taxonomically distinct from the major periodontal pathogen T. denticola. However, two of the animal strains are very closely related to T. vincentii, while the other is more similar to T. denticola. These organisms have a number of common adhesive and putative virulence properties that may be related to broad host specificity of colonization.
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METHODS |
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DNA purification.
Treponema chromosomal DNA was extracted essentially as described by Nelson & Selander (1994). Bacteria from late-exponential-phase cultures (10 ml) were harvested by centrifugation at 10 000 g for 5 min, suspended in 0·5 ml TE buffer (10 mM Tris/HCl, pH 7·5, 1 mM EDTA) containing 0·25 % (w/v) SDS, and the suspension was incubated at 65 °C for 1 h. RNase A (10 µl, 5 mg ml-1) was added and the mixture incubated at 37 °C for 30 min, followed by addition of Proteinase K (5 µl, 10 mg ml-1). To the lysate was then added 5 M NaCl (0·1 ml) and 0·08 ml 10 % (w/v) CTAB (cetyltrimethylammonium bromide) solution in 0·7 M NaCl and the mixture was incubated at 65 °C for 10 min to facilitate precipitation of bacterial lipopolysaccharides. The suspension was cooled at 4 °C, emulsified with an equal volume of TE-saturated chloroform and centrifuged at 12 000 g for 10 min. The aqueous phase containing DNA was recovered, subjected to phenol/chloroform (1 : 1) extraction and DNA within the aqueous phase was precipitated by adding an equal volume of propan-2-ol. The recovered DNA was suspended in TE buffer, precipitated with 95 % (v/v) ethanol in the presence of 3·25 M ammonium acetate, washed with 70 % (v/v) ethanol and suspended in TE buffer.
PCR and rDNA analysis.
Universal eubacterial 16S rDNA primers 27F (5'-GTGCTGCAGAGAGTTTGATCCTGGCTCAG-3'), 63F (5'-CAGGCCTAACACATGCAAGTC-3') and 1392R (5'-CACGGATCCACGGGCGGTGTGTRC-3') were utilized in PCR amplifications as described by Harper-Owen et al. (1999) to isolate Treponema 16S rDNA fragments. The amplimers generated were cloned into pGEM-T (Promega) in Escherichia coli XL-1 Blue. Plasmids containing 16S rDNA inserts were purified with the Concert rapid extraction kit (Life Technologies) and inserts were sequenced using an automated ABI sequencer. An edited CLUSTAL W alignment of 1196 nt of each sequence was phylogenetically analysed using the PHYLIP suite of programs (Felsenstein, 1993
) available at the UK Human Genome Mapping Project Resource Centre (http://menu.hgmp.mrc.ac.uk). More specifically, DNADIST was used to compare sequences by the JukesCantor algorithm, NEIGHBOR was used for neighbour-joining cluster analysis (Saitou & Nei, 1987
), and SEQBOOT and CONSENSE were used for bootstrap analysis.
Protein extraction and electrophoresis.
Treponemal surface proteins were extracted by the method of Cunningham et al. (1988) as modified by Fenno et al. (1998)
. Briefly, bacteria from late-exponential-phase cultures were harvested by centrifugation and washed twice with TE buffer. Bacteria were resuspended at a concentration of approximately 2·5x109 cells ml-1 in Triton X-114 solution (1 %, v/v, in TE buffer) and incubated with shaking on a rotary mixer at 4 °C for 16 h. The suspension was then centrifuged at 21 000 g at 4 °C for 1 h to sediment non-periplasmic material (cytoplasmic cylinders) and to obtain an outer-membrane-protein-enriched supernatant. A portion of supernatant was mixed with an equal volume of sample buffer (10 mM Tris/HCl, pH 6·8, containing 2 % SDS and 2 mM 2-mercaptoethanol) and incubated for 5 min at either 100 or 20 °C. Proteins were separated by SDS-PAGE in 10 % (w/v) acrylamide and stained with Coomassie blue. Molecular masses of proteins were estimated by reference to the mobilities of pre-stained molecular mass marker proteins (Bio-Rad).
Biotinylation of cells.
Late-exponential-phase cells were harvested by centrifugation, washed three times in ice-cold PBS and resuspended at an OD600 of 0·1 (approx. 2x109 cells ml-1) in ice-cold PBS. Sulfo-NHS-LC-biotin (0·5 mg; Pierce Biochemicals) was added and the suspension was incubated at 4 °C for 30 min. Non-conjugated biotin was removed by three rounds of alternate centrifugation and washing of cells with ice-cold PBS. Cells were stored on ice and assayed for adhesion properties (see below) as soon as possible and within 4 h of labelling. A standard reference plot was generated for each Treponema strain relating cell numbers of immobilized biotin-labelled cells to HRP-conjugated streptavidin reactivities (A490).
Adhesion assays.
Human plasma fibronectin and human laminin were obtained from Roche Molecular Biochemicals, and human collagen type I was purchased from BD Biosciences. Bovine fibronectin, BSA, bovine fetuin, human fibrinogen, porcine gelatin, human keratin, human lactoferrin, porcine heparin and human hyaluronan (hyaluronic acid) were obtained from Sigma. Substrates to be immobilized were dissolved in coating buffer (50 µl; 0·02 M NaHCO3, 0·02 M Na2CO3, pH 9·3), added to the wells of Immulon 2HB (Dynex Technologies) 96-well plastic plates (00·5 µg per well) and incubated at 4 °C for 16 h. Non-specific binding sites were blocked with 1 % (w/v) BSA in PBS at 4 °C for 16 h. Wells were washed once with PBS and then Treponema cell suspensions in PBS (50 µl) containing between 1x107 and 2x108 biotinylated cells were applied in triplicate wells and incubated at 20 °C for 2 h. Unbound cell suspensions were aspirated and wells were washed twice with PBS (0·2 ml). Horseradish-peroxidase-conjugated streptavidin (Dako) diluted 1 : 4000 in PBSTB (PBS containing 0·1 %, v/v, Tween 20 and 0·1 %, w/v, BSA) was added to each well and incubated at 37 °C for 45 min. Liquid was aspirated from the wells, which were then washed once in PBSTB and twice with PBS. Colour reagent (o-phenylenediamine) was then added to the wells (50 µl), incubated in the dark for 10 min, developed by the addition of 0·65 M H2SO4 and the A490 measured with a Bio-Rad Benchmark plate reader. Numbers of cells bound were calculated from standard curves relating A490 to cell numbers (microscopic count) for each Treponema isolate.
Blot overlay assay.
To generate tryptic fragments of fibronectin, human plasma fibronectin (30 µg) was suspended in 0·1 M Tris/HCl, pH 8·1, containing 10 mM L-cysteine hydrochloride, 10 mM CaCl2 and 11 U trypsin (Sigma), and incubated at 37 °C for 1 h. The reaction was stopped by addition of PMSF (final concentration 0·25 mM) and then stored at -20 °C. Portions of trypsin digest were mixed with sample buffer, heated at 100 °C for 5 min and subjected to SDS-PAGE as described above. Identification of fibronectin fragments was based on the banding patterns described by Hayashi & Yamada (1983). Proteins were electroblotted onto nitrocellulose membrane (Amersham) at 8 V cm-1 for 1·5 h. To assay bacterial cell binding to protein bands, the nitrocellulose was blocked with 1 % (w/v) BSA in PBS containing 0·1 % (v/v) Tween 20 at 4 °C for 16 h. The blot was washed twice (5 min each) with PBS, cut into 5 mm wide strips and strips were incubated with biotinylated Treponema cells (or no cells, controls) at 20 °C for 2 h with shaking. The blots were then washed three times (5 min each) with PBS, incubated with HRP-conjugated streptavidin (diluted 1 : 2000 in PBSTB) at 20 °C for 1·5 h, washed once with PBSTB (5 min) and twice with PBS (5 min each). Biotinylated Treponema cells that were bound to the blots were detected following development with 3·5 mM 4-chloro-1-naphthol solution containing 0·03 % (v/v) H2O2. Protein bands on control strips (no cells added) did not bind streptavidin-HRP.
Enzyme assays.
Washed cell suspensions (0·1 ml, 5x108 cells) were mixed with 0·4 ml of a solution of chromogenic substrate and incubated at 37 °C for 1 h. Activities were assayed as follows: chymotrypsin-like activity with 1 mM N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide in 0·5 M Tris/HCl, pH 7·2, containing 2 mM dithiothreitol (Grenier et al., 1990); trypsin-like activity with 1 mM N-benzoyl-DL-arginine p-nitroanilide hydrochloride in 0·1 M Tris/HCl, pH 8·1, containing 10 mM cysteine and 10 mM CaCl2 (Aduse-Opuku et al., 1998
); proline iminopeptidase activity with 1 mM proline p-nitroanilide in 0·05 M Tris/HCl, pH 7·8, containing 150 mM NaCl (Makinen et al., 1996
); neutral phosphatase activity with 1 mM p-nitrophenyl phosphate in 0·025 M cacodylate buffer, pH 6·4 (Ishihara & Kuramitsu, 1995
). Following incubation with substrate, Treponema cells were collected by centrifugation and the A405 of the supernatant was measured against a blank (no cells) taken through the assay. Enzyme activities were expressed in Units (U) where 1 U is defined as the amount of enzyme that produced 1 µmol p-NP min-1 at 37 °C, based on a value of
405 for p-NP of 3·8x103 M-1 cm-1. All assays were performed in triplicate on at least three individual bacterial cultures.
Coaggregation assay.
Late-exponential-phase suspensions of bacteria were prepared at densities of approximately 1010 cells ml-1 in coaggregation buffer (1 mM Tris/HCl, pH 8·0, containing 0·1 mM CaCl2, 0·1 mM MgCl2, 0·02 %, w/v, NaN3 and 0·15 M NaCl) (Cisar et al., 1979). Equal volumes of each cell type (0·2 ml) were mixed in clear plastic cuvettes and incubated at room temperature for 15 min. Coaggregation was scored visually as described by Kolenbrander et al. (1990)
on a scale of 04 defined as follows: 0, no coaggregation; 1, finely dispersed aggregates; 2, definite aggregates but not settled; 3, larger clumps settling but suspension remaining turbid; 4, flocculant coaggregates settling to give a clear supernatant.
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RESULTS |
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DISCUSSION |
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Phylogenetic data, based on 16S rDNA sequence analyses, suggest that Treponema present in DD or CODD lesions are closely related to T. denticola, T. vincentii and T. medium that are found at human periodontal sites. Interestingly, the isolates obtained from sheep and cattle did not themselves form a distinct phylogenetic group. However, more sensitive methods than 16S rDNA sequence analysis, such as 16S23S rDNA intergenic spacer region analysis (Stamm et al., 2002), could be used to determine if there were distinct lines of descent for strains isolated from the different hosts. Thus, ovine strain UB1466 was more closely related to T. denticola, while ovine strain UB1090 and bovine strain UB1467 appeared very closely related to T. medium, and clustered with T. vincentii (Fig. 1
). The various genetic relationships were corroborated by phenotypic data on outer-membrane protein profiles and proteolytic enzymic activities. Unfortunately, T. medium was not available to us at the time, and a more detailed comparison of strains UB1090 and UB1467 with T. medium would be most valuable. These results indicate that mixed Treponema species ecologies occur at diseased sites in both humans and animals, and that isolates from DD or CODD lesions share common ancestors with human oral strains. They also raise the possibility that Treponema strains may be able to move between sheep and cattle, which has implications for foot disease progression in mixed husbandry situations.
The precise mechanisms by which T. denticola and other oral Treponema adhere to epithelial cells in the process of colonizing oral tissue sites are largely unknown. Different species vary widely in their attachment properties (Carranza et al., 1997) and in their abilities to induce changes in host-cell morphology and cytopathic effects (Ellen et al., 1994
). T. denticola has been shown to bind avidly to fibronectin via the cell tips (Dawson & Ellen, 1994
) and this interaction may be important in facilitating adhesion to epithelial tissues via bridging of integrins on the epithelial cell membrane. In addition, the abilities of Treponema cells to bind laminin and collagen (Haapasalo et al., 1991
), as well as fibronectin, may promote treponemal invasion of basement membrane connective tissue. In this article we have confirmed the abilities of two strains of T. denticola to bind immobilized human fibronectin with high affinity and to bovine fibronectin with slightly lower affinity. Much lower (5- to 10-fold) binding levels to fibronectin were observed for T. vincentii and animal Treponema strains. All Treponema were shown to bind the fibrin I/heparin I binding domain of fibronectin and, consistent with these findings, adhesion of Treponema cells to fibronectin was inhibitable by heparin, but not by collagen. By contrast with another study (Dawson & Ellen, 1990
), we obtained no evidence for recognition by Treponema of the cell-binding domain of fibronectin, as evidenced by the inability to achieve inhibition of adhesion to fibronectin in the presence of RGDS peptide, and inability to demonstrate treponemal cell adhesion to this domain fragment in blot overlays. The high affinity of T. denticola binding to fibronectin may be related to the expression of the CTLP/Msp cell-surface complex by this species in contrast to the T. vincentii-like strains that did not express CTLP. Since Msp has been shown to also bind laminin (Fenno et al., 1996
) and collagen (Umemoto & Namikawa, 1994
), this could also account for the higher binding levels of T. denticola to these substrates.
For all substrates tested, with the exception of fibrinogen, highest levels of adhesion were obtained for T. denticola. However, binding of T. denticola to heparin, hyaluronic acid and fetuin was especially weak. Fetuin was included in these experiments because it has been reported that Treponema may recognize sialic acid residues on the host-cell surface (Keulers et al., 1993). In our analyses fetuin, which is highly sialylated, did not support significant levels of Treponema cell adhesion. In general, our data suggest that Treponema do not have a high affinity for these negatively charged substrates (Haapasalo et al., 1996
). On the other hand, six of the seven strains tested in this article demonstrated relatively high levels of adhesion to lactoferrin. This has been reported for T. denticola (Staggs et al., 1994
) and may be relevant to oral cavity colonization either in inactivating innate defence properties of lactoferrin or in acquisition of iron. We have also shown a unique ability of T. denticola cells to bind keratin. This was surprising since we anticipated that this property might be exhibited by Treponema isolated from DD and CODD lesions, where there is an abundance of keratin associated with the hoof. Clearly, these results add further support to the notion that the animal isolates have not acquired a specificity in adhesion properties that sets them apart from human periodontal isolates. Interestingly though, all strains tested showed relatively high binding abilities to fibrinogen, and the animal isolates all bound to fibrinogen at higher levels than did T. vincentii. The molecules that mediate Treponema adhesion to fibrinogen are unknown and are currently under investigation. However, the evidence suggests that the mechanism is broadly distinct from the mechanisms mediating adhesion of T. denticola to fibronectin, laminin, collagen and keratin. Adhesion to fibrinogen may be of major importance in Treponema pathogenesis, since modulation of fibrinogen functions at diseased sites could promote bleeding, avoidance of immune cell recognition and further tissue destruction.
In summary, we have described in detail a range of adhesion properties for Treponema strains isolated from human periodontal tissues and from animal foot tissues. Our data indicate that there are no fundamental differences between these animal isolates and human isolates, and that the animal isolates show no obvious predisposition for interaction with animal-derived proteins or animal-derived bacteria such as F. necrophorum. Indeed, one of the animal isolates designated strain UB1466, from a CODD lesion, more closely resembled T. denticola in a number of properties. This strain, unlike T. vincentii and the other animal isolates, produced CTLP, which is a major virulence factor for T. denticola (Fenno et al., 1998). It also produced high levels of trypsin-like protease which, in T. denticola, has been identified as a prolyl oligopeptidase (OpdB) that may function as a convertase to modulate host inflammatory response proteins (Fenno et al., 2001
). Furthermore, strain UB1466 co-aggregated specifically with P. gingivalis and S. crista. Interactions of T. denticola with these bacteria may be significant in the establishment of oral bacterial communities associated with the initiation and progression of periodontal disease (Yao et al., 1996
). Taken collectively, these results suggest that Treponema related to the designated species denticola, vincentii and medium have broad host specificity. Indeed, it seems reasonable to suggest that these Treponema represent a cluster, within this heterologous genus, the members of which may be transmissible from humans to animals (and vice versa). This raises the intriguing possibility that anti-treponemal strategies currently being developed to control or prevent human oral periodontal disease may be directly applicable, or adaptable, to the control or prevention of ruminant foot diseases.
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ACKNOWLEDGEMENTS |
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Received 8 November 2002;
revised 28 January 2003;
accepted 4 February 2003.
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