Genetic relatedness and phenotypic characteristics of Treponema associated with human periodontal tissues and ruminant foot disease

Andrew M. Edwards1,{dagger}, David Dymock1, Martin J. Woodward2 and Howard F. Jenkinson1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Treponema have been implicated recently in the pathogenesis of digital dermatitis (DD) and contagious ovine digital dermatitis (CODD) that are infectious diseases of bovine and ovine foot tissues, respectively. Previous analyses of treponemal 16S rDNA sequences, PCR-amplified directly from DD or CODD lesions, have suggested relatedness of animal Treponema to some human oral Treponema species isolated from periodontal tissues. In this study a range of adhesion and virulence-related properties of three animal Treponema isolates have been compared with representative human oral strains of Treponema denticola and Treponema vincentii. In adhesion assays using biotinylated treponemal cells, T. denticola cells bound in consistently higher numbers to fibronectin, laminin, collagen type I, gelatin, keratin and lactoferrin than did T. vincentii or animal Treponema isolates. However, animal DD strains adhered to fibrinogen at equivalent or greater levels than T. denticola. All Treponema strains bound to the amino-terminal heparin I/fibrin I domain of fibronectin. 16S rDNA sequence analyses placed ovine strain UB1090 and bovine strain UB1467 within a cluster that was phylogenetically related to T. vincentii, while ovine strain UB1466 appeared more closely related to T. denticola. These observations correlated with phenotypic properties. Thus, T. denticola ATCC 35405, GM-1, and Treponema UB1466 had similar outer-membrane protein profiles, produced chymotrypsin-like protease (CTLP), trypsin-like protease and high levels of proline iminopeptidase, and co-aggregated with human oral bacteria Porphyromonas gingivalis and Streptococcus crista. Conversely, T. vincentii ATCC 35580, D2A-2, and animal strains UB1090 and UB1467 did not express CTLP or trypsin-like protease and did not co-aggregate with P. gingivalis or S. crista. Taken collectively, these results suggest that human oral-related Treponema have broad host specificity and that similar control or preventive strategies might be developed for human and animal Treponema-associated infections.


Abbreviations: CODD, contagious ovine digital dermatitis; CTLP, chymotrypsin-like protease; DD, digital dermatitis

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).

{dagger}Present address: Department of Pathology and Microbiology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Spirochaetes of the genus Treponema are anaerobic, motile Gram-negative rods with spiral morphology. They colonize a range of animal hosts and vary considerably in their pathogenic properties. For example, while Treponema pallidum subsp. pallidum infections may give rise to syphilis, Treponema phagedenis is a normal commensal of the human urogenital tract. Many Treponema species have been difficult to cultivate and this has seriously hampered taxonomic and phenotypic analyses. Increasing numbers of not yet cultivated bacterial species, particularly from the human oral cavity, have been assigned to the genus Treponema based on PCR-derived 16S rDNA sequences (Dewhirst et al., 2000; Paster et al., 2001).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
T. denticola strains ATCC 35405 and GM-1 were kindly provided by R. Lamont (University of Florida, Gainesville, FL, USA) and T. vincentii strains ATCC 35580 and D2A-2 were kindly provided by P. E. Greenberg (University of Iowa, USA). Spirochaete isolate UB1090 was obtained from a foot biopsy of a sheep with CODD (Naylor et al., 1998). Spirochaete isolates UB1466 (see Demirkan et al., 2001) and UB1467 were isolated from CODD and bovine DD lesions, respectively, using immunomagnetic enrichment (Demirkan et al., 1999a). Treponemes were grown and maintained in new oral spirochaete (NOS) medium at 37 °C (Haapasalo et al., 1991) in an anaerobic atmosphere of N2/CO2/H2 (8 : 1 : 1). Exponential-growth-phase cultures of Treponema spp. were preserved in NOS medium containing 15 % glycerol at -70 °C. Late-exponential-phase cultures of Treponema, corresponding to an OD600 of between 0·4 and 0·6, were obtained following a 3- to 4-day incubation at 37 °C. Porphyromonas gingivalis ATCC 33277 was grown in Trypticase Soy Broth (TSB, Difco) supplemented with 1 g yeast extract l-1, 5 mg haemin l-1 and 1 mg menadione l-1. Streptococcus crista (formerly cristatus) CC5A and fusobacteria were grown anaerobically as described above in TSB containing 5 g yeast extract l-1 and 0·5 % (w/v) glucose. Escherichia coli XL-1 Blue cells were grown in Luria–Bertani (LB) medium (Sambrook et al., 1989) or on LB agar, containing ampicillin (100 µg ml-1) when appropriate.

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 Jukes–Cantor 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 (0–0·5 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 {varepsilon}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 0–4 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Genetic relatedness of human and animal-derived Treponema isolates
To establish the relatedness of two new animal-derived strains of Treponema, 16S rRNA gene sequences were determined following PCR amplifications with universal 16S rDNA primers. Sequences were already available for human oral-derived T. denticola ATCC 35405 and T. vincentii ATCC 35580, and for Treponema ovine strain UB1090. PCR amplification using eubacterial primers 27F and 1392R generated 1259 bp 16S rDNA products from T. denticola GM-1, T. vincentii D2A-2 and ovine strain UB1466. 16S rDNA amplimers from bovine strain UB1467 were obtained following PCR amplification with eubacterial primers 63F and 1392R. The various 16S rDNA fragments were cloned into pGEM-T (Promega) and each was sequenced to obtain three times full sequence coverage for the entire fragment. To compare these sequences with 16S rDNA gene sequences in the GenBank database they were aligned and trimmed to the length of the smallest sequence used in the analysis (1223 bp) corresponding to the 155–1382 region within the E. coli 16S rRNA gene (accession no. NC 000913). The aligned sequences were then subjected to a cluster analysis to generate a phylogenetic tree (Fig. 1). As expected, the new sequences for T. denticola GM-1 and T. vincentii D2A-2 were most closely related to sequences from T. denticola ATCC 35405 and T. vincentii ATCC 35580, respectively (Fig. 1). The ovine isolate Treponema UB1090 was more closely related to T. vincentii, as previously observed (Collighan et al., 2000), and clustered with the new bovine isolate, Treponema UB1467, and with Treponema medium (Fig. 1). Conversely, the new ovine isolate Treponema spp. UB1466 (also designated G179) appeared more closely related to a bovine DD isolate 1-9185MED (Walker et al., 1995) and fell within a cluster that is phylogenetically more closely related to T. denticola (Fig. 1). These data support the evidence that bacteria closely related to T. denticola and T. vincentii are widely dispersed across animal species.



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Fig. 1. Phylogenetic tree of Treponema showing close relatedness of animal Treponema isolates to human oral species on the basis of 16S rDNA sequences. CLUSTAL W alignments of each sequence were analysed by the PHYLIP suite of programs (Felsenstein, 1993) (see Methods). The dendogram was drawn in TREEVIEW for Windows (v1.0a). Bootstrap values at each branch point are means of 100 replicates. Brachyspira hyodysenteriae is included as the outgroup. GenBank accession numbers are shown. Sequences derived from animal Treponema isolates or derived from direct PCR amplification of biopsy material from DD lesions are designated with an asterisk. The bar represents a 10 % difference in nucleotide sequence.

 
Outer-membrane protein profiles
Outer-membrane proteins from T. denticola, T. vincentii and Treponema animal isolates were extracted by partitioning into Triton X-114 and subjected to SDS-PAGE analysis. The outer-membrane protein profiles of T. denticola ATCC 35405 and GM-1 each contained, in unheated samples, a major band with an approximate molecular mass of 200 kDa (Fig. 2, lanes 1 and 2). This band was modified in samples that had been heated at 100 °C before loading and was replaced by a major band with an approximate molecular mass of 60 kDa and several other minor bands (Fig. 2, lanes 1 and 2). These results are consistent with the description by Rosen et al. (1999) of a T. denticola surface protein complex comprising the major surface protein (Msp, ~60 kDa) and a chymotrypsin-like protease (CTLP), the latter being composed of 72, 43 and 38 kDa polypeptides (Ishihara et al., 1996). In the heated sample profiles of T. denticola ATCC 35405 and GM-1 the putative Msps may be identified as 56 and 62 kDa bands, respectively (Fig. 2, lanes 1 and 2). The 72 kDa bands in these profiles are likely to represent the largest CTLP component. In T. vincentii extracts, which generally contained a much larger number of bands, an approximately 130 kDa heat-modifiable protein was identified (Fig. 2, lanes 3 and 4). The outer-membrane profiles from ovine strain UB1090 and bovine strain UB1467 were very similar to those of T. vincentii (Fig. 2, lanes 3, 4, 5 and 7). However, the ovine Treponema strain UB1466 had a distinct outer-membrane protein profile (Fig. 2, lane 6) that was more similar to T. denticola than to T. vincentii, or to the other animal isolates.



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Fig. 2. SDS-PAGE profiles of outer-membrane proteins of human or animal Treponema. Triton X-114-solubilized outer-membrane protein extracts were mixed with SDS sample buffer and incubated for 5 min at either 100 (+) or 20 °C (-) before subjecting them to SDS-PAGE. Lanes: 1, T. denticola ATCC 35405; 2, T. denticola GM-1; 3, T. vincentii ATCC 35580; 4, T. vincentii D2A-2; 5, Treponema UB1090 (ovine); 6, Treponema UB1466 (ovine); 7, Treponema UB1467 (bovine). Each lane contained 20 µg protein. The positions of molecular mass markers are indicated.

 
Adhesion of Treponema strains to fibronectin
Binding of T. denticola to extracellular matrix components is believed to play an essential role in treponemal invasion of oral tissues (Ellen et al., 1994). In particular, fibronectin binding provides a common means by which treponemes adhere to epithelial or endothelial cells (Dawson & Ellen, 1994). Accordingly, we determined the abilities of human- or animal-derived Treponema strains to adhere to immobilized human and bovine fibronectin. All strains tested showed saturable binding to immobilized fibronectins. For T. denticola ATCC 35405, which showed the highest affinity for binding fibronectin, a maximum of approximately 2·9x106 cells adhered to 0·1 µg immobilized human fibronectin (Fig. 3a). The adhesion levels of T. denticola GM-1 were lower than for strain ATCC 35405, while T. vincentii strains and bovine or ovine Treponema strains all bound fibronectin at levels <20 % of strain ATCC 35405 (Fig. 3a). Rather similar patterns of Treponema binding to immobilized bovine fibronectin were observed, but notably the adhesion levels of T. denticola were significantly lower (about 30 %) than to human fibronectin (Fig. 3b). In addition, Treponema UB1466 bound significantly better to human than to bovine fibronectin, while the converse was true for Treponema UB1090 (Fig. 3). These data reveal subtle differences in fibronectin recognition with the T. denticola-related strains showing a preference for human fibronectin over bovine fibronectin.



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Fig. 3. Binding curves for adhesion of biotinylated Treponema cells to 0·1 µg immobilized human plasma fibronectin (a) or bovine plasma fibronectin (b). Open circles, T. denticola ATCC 35405; filled circles, T. denticola GM-1; open triangles, T. vincentii ATCC 35580; filled triangles, T. vincentii D2A-2; open squares, ovine Treponema UB1090; filled squares, ovine Treponema UB1466; inverted triangles, bovine Treponema UB1467. Error bars indicate SD of triplicates from two independent experiments.

 
Region of fibronectin bound by Treponema
To determine the region(s) of fibronectin recognized by these Treponema strains, tryptic fragments of human fibronectin were Western-blotted onto nitrocellulose to generate a defined banding pattern corresponding to various fibronectin domains (Fig. 4). Binding of biotinylated Treponema cells to these bands was then visualized. All strains of Treponema tested were found to bind exclusively to a single 30 kDa band (Fig. 4) that contains the N-terminal heparin I/fibrin I binding region of fibronectin (Hayashi & Yamada, 1983). To corroborate this finding, specificities of binding to fibronectin by T. denticola ATCC 35405 (highest affinity, see Fig. 3) or Treponema UB1090 (lowest affinity) were assessed. Both strains adhered to immobilized 30 kDa fragment as well as, if not better than, to the intact fibronectin molecule (Table 1). Adhesion of cells to fibronectin was significantly reduced following pre-incubation of fibronectin with heparin (Table 1). Pre-treatment of fibronectin with collagen type I did not affect adhesion levels of strain UB1090 cells, but enhanced adhesion of T. denticola ATCC 35405 cells presumably by providing additional sites for attachment (Table 1). In further experiments it was observed that adhesion of strain ATCC 35405 cells to immobilized fibronectin was unaffected by pre-incubation of cells with fluid-phase fibronectin or the 30 kDa fragment, demonstrating higher affinity or specificity for immobilized fibronectin. Interestingly, adhesion levels of UB1090 cells to fibronectin were reduced in the presence of the fluid-phase 30 kDa fragment (Table 1), possibly reflecting the lower binding affinity of these cells for immobilized fibronectin. In addition, pre-incubation of Treponema cells with Arg-Gly-Asp-Ser (RGDS) peptide did not affect adhesion levels (Table 1). Therefore, these results are entirely consistent with the evidence that Treponema recognize the N-terminal heparin I/fibrin I domain of fibronectin.



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Fig. 4. Treponema cell adhesion to fragments of human fibronectin. Tryptic digest fragments encompassing various binding domains of fibronectin as indicated were separated by SDS-PAGE and blotted onto nitrocellulose. Blots were overlayered with biotinylated treponemal cells and binding was detected with streptavidin-linked HRP and colour reagent. Lanes: 1, T. denticola ATCC 35405; 2, T. denticola GM-1; 3, T. vincentii ATCC 35580; 4, T. vincentii D2A-2; 5, Treponema UB1090; 6, Treponema UB1466; 7, Treponema UB1467; 8, control (no biotinylated Treponema cells); 9, corresponding SDS-PAGE pattern of tryptic digest fragments and associated binding domains.

 

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Table 1. Specificity of Treponema binding to immobilized fibronectin

Values are the means±SD of quadruplicate determinations from a representative experiment repeated three times.

 
Adhesion of treponemes to tissue-associated proteins
To compare the adhesion properties of human- or animal-derived Treponema isolates in more detail their binding levels to a range of host-associated proteins were determined. The T. denticola strains adhered significantly better to all substrates tested, except to fibrinogen (Fig. 5). T. denticola cells bound with higher affinity to laminin than to fibronectin (Fig. 5) and showed significant levels of adhesion to fibrinogen, collagen type I, gelatin, keratin and lactoferrin. By contrast, T. vincentii and the animal-derived strains bound only weakly, or not significantly, to collagen, gelatin and keratin (Fig. 5). Low levels of binding of all Treponema strains tested were observed for heparin, hyaluronic acid and fetuin (Fig. 5). All strains, except UB1467, showed binding to lactoferrin. An important observation was that the animal-derived Treponema isolates all bound fibrinogen equally well, or better than, T. denticola (Fig. 5), and that T. vincentii was least adherent. These data suggest that different adhesion mechanisms are employed by Treponema in recognizing fibrinogen and fibronectin.



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Fig. 5. Treponema adhesion levels to proteins and host tissue components immobilized onto plastic wells. Biotinylated treponemal cells (6x107) were added to wells coated with 0·1 µg substrate and the number of bound cells was determined as described in Methods. Error bars indicate SD of triplicates from three independent experiments. Td, T. denticola; Tv, T. vincentii; T ov, Treponema ovine; T bov, Treponema bovine.

 
Enzymic characterization
The virulence properties of T. denticola have been linked to adhesion ability, motility and production of proteinases (Sela, 2001). Therefore, the activities of CTLP, trypsin-like peptidase and proline iminopeptidase were measured, together with neutral phosphatase as control. Both strains of T. denticola produced significant levels of CTLP and trypsin-like peptidase (OpdB) (Fenno et al., 2001) activities, whereas T. vincentii strains did not (Table 2). T. denticola expressed about 10-fold higher proline iminopeptidase activity than T. vincentii. Ovine strain UB1090 and bovine strain UB1467 showed enzymic levels similar to T. vincentii (Table 2). However, ovine strain UB1466 produced high levels of trypsin-like peptidase and proline iminopeptidase activities, and lower but easily detectable levels of CTLP (Table 2). All Treponema strains tested produced similar amounts of neutral phosphatase. These findings demonstrate further the close relatedness of Treponema ovine strain UB1466 to T. denticola.


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Table 2. Proteolytic enzymic activities associated with cells of human or animal Treponema strains

Values are the means±SD of triplicate determinations from a representative experiment repeated three times.

 
Coaggregation properties
In their natural habitats, Treponema exist in close associations with other bacteria and are co-isolated with specific organisms from diseased sites. Co-aggregation between bacteria may be important in these associations (Kolenbrander, 2000) and so the abilities of Treponema strains to co-aggregate with other relevant bacteria were determined. In periodontal disease, T. denticola is frequently isolated with P. gingivalis, while in CODD Fusobacterium necrophorum is present along with Treponema (Naylor et al., 1998). It is known that T. denticola specifically co-aggregates with P. gingivalis, S. crista and Fusobacterium nucleatum (Grenier, 1992; Yao et al., 1996) and this was confirmed for both T. denticola strains in this study (Table 3). T. vincentii did not co-aggregate with P. gingivalis or S. crista, and nor did animal strains UB1090 and UB1467. However, ovine Treponema strain UB1466 co-aggregated strongly with P. gingivalis and S. crista (Table 3), which is consistent with results demonstrating the closer relatedness of this strain to T. denticola. All Treponema strains co-aggregated strongly with F. nucleatum, and also with F. necrophorum but to more varied extents.


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Table 3. Coaggregation of human or animal Treponema strains with human oral bacteria

Coaggregation was scored visually across the range from 4 (bacterial clumps that freely sedimented) to 0 (no aggregates) as described in Methods.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Periodontal disease of humans, and DD or CODD in animals, are conditions that involve inflammatory processes and destruction of collagenous and connective tissues. These diseases are associated with mixed bacterial infections and Treponema species are strongly implicated in disease progression. There is growing evidence from in situ PCR analyses that multiple types or species of Treponema may be present within these lesions (Choi et al., 1994; Dewhirst et al., 2000; Stamm et al., 2002), but the activities of the different species in contributing to pathogenesis are not yet understood. In vitro studies suggest that T. denticola has a high capability to penetrate tissue layers of human gingival keratinocytes, whereas T. phagedenis does not (Lux et al., 2001). T. denticola also produces high levels of CTLP and trypsin-like protease activities, compared with other oral Treponema species, that provide potential for tissue penetration and destruction (Fenno et al., 1998; Ellen et al., 2000). These and other virulence-related properties of T. denticola tend to support the notion that this species may be a major periodontal pathogen. To understand more about the potential pathogenic and virulence properties of related Treponema species we have characterized in detail three strains of Treponema, independently isolated from foot lesion biopsies, and compared them with human oral isolates of T. denticola and T. vincentii.

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 16S–23S 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.


   ACKNOWLEDGEMENTS
 
We are grateful to J. Mawdsley for technical assistance, and to L. Moore, L. Green and R. Blowey for helpful advice and discussions. We thank R. Lamont, P. Greenberg and I. Demirkan for providing bacterial strains. A Seedcorn grant from the Veterinary Laboratories Agency and Defra funding through Project OD0213 are gratefully acknowledged.


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Received 8 November 2002; revised 28 January 2003; accepted 4 February 2003.



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