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Infection and Immunity, September 2007, p. 4364-4372, Vol. 75, No. 9
0019-9567/07/$08.00+0     doi:10.1128/IAI.00258-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Chymotrypsin-Like Protease Complex of Treponema denticola ATCC 35405 Mediates Fibrinogen Adherence and Degradation

Caroline V. Bamford,¹ J. Christopher Fenno,² Howard F. Jenkinson,^1* and David Dymock¹

Oral Microbiology Unit, Department of Oral and Dental Science, University of Bristol, Lower Maudlin Street, Bristol BS1 2LY, United Kingdom,¹ Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, Michigan²

Received 16 February 2007/ Returned for modification 25 April 2007/ Accepted 15 June 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Treponema denticola is an anaerobic spirochete strongly associated with human periodontal disease. T. denticola bacteria interact with a range of host tissue proteins, including fibronectin, laminin, and fibrinogen. The latter localizes in the extracellular matrix where tissue damage has occurred, and interactions with fibrinogen may play a key role in T. denticola colonization of the damaged sites. T. denticola ATCC 35405 showed saturable binding of fluid-phase fibrinogen to the cell surface and saturable adherence to immobilized fibrinogen. Levels of fibrinogen binding were enhanced in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride. The A and Bß chains of fibrinogen, but not the chains, were specifically recognized by T. denticola. Following fibrinogen affinity chromatography analysis of cell surface extracts, a major fibrinogen-binding component (polypeptide molecular mass, 100 kDa), which also degraded fibrinogen, was purified. Upon heating at 100°C, the polypeptide was dissociated into three components (apparent molecular masses, 80, 48, and 45 kDa) that did not individually bind or degrade fibrinogen. The native 100-kDa polypeptide complex was identified as chymotrypsin-like protease (CTLP), or dentilisin. In an isogenic CTLP^– mutant strain, CKE, chymotrypsin-like activity was reduced >90% compared to that in the wild type and fibrinogen binding and hydrolysis were ablated. Isogenic mutant strain MHE, deficient in the production of Msp (major surface protein), showed levels of CTLP reduced 40% relative to those in the wild type and exhibited correspondingly reduced levels of fibrinogen binding and proteolysis. Thrombin clotting times in the presence of wild-type T. denticola cells, but not strain CKE (CTLP^–) cells, were extended. These results suggest that interactions of T. denticola with fibrinogen, which may promote colonization and modulate hemostasis, are mediated principally by CTLP.

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Periodontitis is usually a chronic infection incorporating both bacterial factors and host responses. The communities of bacteria involved are polymicrobial, and a number of specific microorganisms, including Porphyromonas gingivalis, Tannerella forsythensis, and Treponema denticola, are strongly implicated in disease progression (57). Oral spirochetes, such as T. denticola, may be major components of complex pathogenic communities in deep periodontal pockets (22, 62). Many of the species present at diseased sites have not yet been cultivated, and consequently, most information on the potential role of spirochetes in periodontitis comes from studies of cultivable strains of T. denticola, "Treponema vincentii," and Treponema maltophilum.

T. denticola exhibits a broad repertoire of adhesive and cytopathic properties. Bacteria adhere to extracellular matrix components such as fibronectin, laminin, and collagen (15, 20, 31), and erythrocytes exposed to T. denticola become hemagglutinated, hemoxidized, and hemolyzed (11, 12). The adherence of T. denticola to epithelial cells or gingival fibroblasts results in the occurrence of profound morphological changes, cell detachment from surfaces, cytoskeletal rearrangement, and the inhibition of propagation (1, 3, 7, 16, 40, 59). A number of components in the outer membrane of T. denticola are known to act as adhesins or to have cytopathic properties. These components include the major surface protein (Msp) (24), the oligopeptide-binding protein (OppA) ortholog (26), and a chymotrypsin-like protease (CTLP), or dentilisin (24, 44). Msp (molecular mass in T. denticola ATCC 35405, approximately 53 kDa) forms high-molecular-mass oligomeric complexes embedded within the outer layers of T. denticola cells (20, 41). Msp is an abundant membrane protein that has both adhesive and cytotoxic properties and binds a range of host proteins, e.g., fibronectin, as well as receptors on human cells (24, 25). In addition, Msp acts as a porin, generating large pores in model and cell membranes (21, 47) and disrupting cell integrity. It also causes actin rearrangements, disrupts calcium signaling in human gingival fibroblasts (58), and induces the release of proteinases from neutrophils (17). The CTLP surface complex of T. denticola consists of a 72-kDa subtilisin-like protease (PrtP) (38) and two auxiliary stabilizing peptides, PrcA1 (40 kDa) and PrcA2 (30 kDa) (37, 43). Invasion by T. denticola through basement membranes and epithelial cell layers is mediated by the degradation of tight junctions by the CTLP complex (10, 23, 29).

Fibrinogen is a 340-kDa plasma-based protein consisting of pairs of A, Bß, and peptides, is essential for wound healing, and has a role in hemostasis (50). At sites of tissue damage, it is also found embedded within the extracellular matrix (52, 56). The virulence properties of some pathogenic bacteria have been linked to their interactions with fibrinogen. For example, Staphylococcus aureus mutants deficient in ClfA, which binds fibrinogen, produce reduced endocarditis compared to the wild type in a mouse model (49). Also, group A streptococcus strains capable of binding fibrinogen show reduced clearance through opsonization and phagocytosis (48). In periodontal tissues, fibrinogen will be abundant at sites of periodontal disease, where tissues are damaged and spontaneous bleeding is frequent. Interactions with fibrinogen may therefore be an important virulence mechanism for periodontal pathogens such as T. denticola.

In this study, we investigated the components of T. denticola that were involved in the interactions of bacterial cells with fibrinogen and determined the effects of inactivating Msp or CTLP functions on fibrinogen interactions. We demonstrate that the CTLP complex is primarily responsible for the binding and degradation of fibrinogen by T. denticola and for bacterial interference with the blood coagulation cascade.

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Bacterial strains. T. denticola ATCC 35405 and two isogenic mutants, MHE and CKE, generated by allelic replacement (27) were grown and maintained in new oral spirochete medium at 37°C (31) in an anaerobic atmosphere of N₂-CO₂-H₂ (8:1:1). Strain MHE carried an erm cassette inserted within the msp gene (27), while strain CKE carried the erm cassette replacing 908 bp within the prcA-prtP locus (27), thus disrupting CTLP production. Erythromycin (40 µg/ml) was included in the growth media of strains MHE and CKE to ensure the retention of the erm gene cassette. Exponential-phase cultures were regularly examined for purity by phase-contrast microscopy and harvested after 3 days of anaerobic incubation at 37°C by centrifugation at 10,000 x g (10 min at 4°C). Escherichia coli strains were grown in Luria-Bertani broth with 100 µg of ampicillin/ml at 37°C with shaking at 250 rpm.

Fluid-phase fibrinogen binding by T. denticola. To measure the binding of fibrinogen to T. denticola, cells from 3-day stationary-phase cultures were harvested, washed in phosphate-buffered saline (PBS; pH 7.0), and adjusted to an optical density at 600 nm (OD₆₀₀) of 0.25 and portions were immobilized onto the wells of a 96-well plastic plate (Greiner) at approximately 3 x 10⁷ cells per well with 0.25% glutaraldehyde. Wells were blocked with 2% bovine serum albumin (BSA), fraction V (Sigma), in PBS (0.2 ml) for 16 h at 4°C and then washed with 0.1% Tween 20 in PBS (PBST; 0.2 ml). Fixed cells were incubated in triplicate for 2 h at 20°C with human fibrinogen (Calbiochem, San Diego, CA) diluted in PBS. Unbound fibrinogen was removed by two washes with PBST containing 0.1% BSA (PBSTB) and one wash with PBS alone. To detect bound fibrinogen, horseradish peroxidase (HRP)-anti-human fibrinogen antibody (DakoCytomation, Glostrup, Denmark) diluted 1:1,000 in PBSTB was added and the plates were incubated for 2 h at 20°C. Wells were washed with PBSTB and PBS, color reagent o-phenylenediamine was added to each well, and the plates were incubated in the dark at 20°C for 10 min. Color development was stopped by adding 0.65 M H₂SO₄, the absorbance at 490 nm (A₄₉₀) was recorded, and the amount of bound fibrinogen was determined by reference to a standard curve relating micrograms of fibrinogen to A₄₉₀. Successful glutaraldehyde fixing of spirochetes onto plastic wells was confirmed using T. denticola whole-cell antibodies (20) in parallel control experiments.

Biotin labeling. The method of biotinylation of T. denticola cells was adapted from that of Edwards et al. (19). Briefly, T. denticola cells were washed three times in PBS and suspended in PBS at an OD₆₀₀ of 0.1 (2.4 x 10⁸ cells/ml). EZ-Link sulfo-NHS-LC-biotin (5 µg/ml; Pierce Rockford, IL) was added to the cell suspensions, and the suspensions were incubated for 30 min at 4°C. Excess biotin was removed by six rounds of sequential centrifugation and washing of the cells in PBS, and suspensions were readjusted to an OD₆₀₀ of 0.1 for adhesion assays and blot overlays.

Adherence of T. denticola to immobilized fibrinogen. Human fibrinogen, free of fibronectin as determined by an immunoassay, was dissolved in carbonate coating buffer (0.02 M NaHCO₃, 0.02 M Na₂CO₃, pH 9.3), and portions (0.05 ml) were applied to wells of Immulon 2 HB 96-well plastic plates (Thermo Labsystems, Franklin, MA) for 16 h at 4°C. Coated wells were incubated with 2% BSA in PBS for 16 h at 4°C to block nonspecific binding sites and then washed with PBST. Portions (0.05 ml) of suspensions of biotin-labeled T. denticola cells containing between 3 x 10⁷ and 2.4 x 10⁸ cells/ml were added in triplicate, and the plates were incubated for 2 h at 20°C. Unbound cells were removed by aspiration, wells were washed twice with PBSTB and once with PBS, and bound cells were detected by incubation with HRP-streptavidin (BD Biosciences Pharmingen, San Diego, CA) diluted 1:1,000 in PBSTB for 2 h. After washing with PBSTB and PBS (as described above), o-phenylenediamine reagent was added and color development (A₄₉₀) was measured as described above. Relative levels of biotinylation of the three strains employed in these studies were checked by reaction with HRP-streptavidin and found to be similar (within 10%).

Extraction of outer-sheath proteins. T. denticola cells were harvested as described above, washed twice in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) containing 0.05 mM phenylmethylsulfonyl fluoride (PMSF), suspended at an OD₆₀₀ of 1.5 (approximately 3.6 x 10⁹ cells/ml) in TE buffer containing 1% Triton X-114, with or without 0.05 mM PMSF, and incubated for 16 h at 4°C. Nonperiplasmic material (cytoplasmic cylinders) was sedimented by centrifugation at 21,000 x g for 1 h at 4°C, and the supernatant was retained. Portions were then incubated with sample buffer (10 mM Tris-HCl [pH 6.8] containing 2% sodium dodecyl sulfate [SDS] and 2 mM 2-mercaptoethanol) at either 100 or 20°C for 5 min before being subjected to SDS-polyacrylamide gel electrophoresis (PAGE).

Purification of recombinant fibrinogen chains. cDNA sequences encoding individual chains of human fibrinogen (4, 5, 46) were subcloned into pQE30 (QIAGEN) to generate expression vectors pQE30-, pQE30-ß, and pQE30-, kindly provided by E. J. Walsh (Trinity College Dublin, Dublin, Ireland). Individual His₆-tagged A, Bß, and fibrinogen peptide chains were purified from cultures of E. coli JM101. The expression of the fibrinogen chains was induced with isopropyl-ß-D-thiogalactopyranoside (1 mM) for 4 h at 37°C. Bacteria were harvested by centrifugation (5,000 x g for 15 min), suspended in 8 M urea buffer containing 0.1 mM PMSF and protease inhibitor cocktail (Sigma), and sonicated on ice at 100 W for six 30-s pulses at 30-s intervals with a Soniprobe sonicator. Cell debris was removed by centrifugation (10,000 x g for 30 min at 4°C), and the supernatant containing the His₆-tagged fibrinogen chains was incubated at 4°C with nickel-nitrilotriacetic acid resin (QIAGEN) with gentle agitation for 1 h. This mixture was then transferred into a plastic column, and fibrinogen chains were eluted with 0.2 M imidazole, pH 8, containing PMSF and protease inhibitor cocktail and dialyzed sequentially against 4 M urea, 2 M urea, and distilled H₂O. The purified fibrinogen chains were then freeze-dried in portions and reconstituted as required.

Gel electrophoresis and bacterial cell blot overlay. Fibrinogen or recombinant chains of fibrinogen were separated by SDS-PAGE and stained with Coomassie brilliant blue. For blot overlay, proteins were transferred onto nitrocellulose membrane (Amersham Biosciences) by electroblotting for 1 h at 100 V and nonspecific binding sites on the nitrocellulose were blocked for 16 h at 4°C with 5% BSA in TBS (10 mM Tris-HCl, 0.15 M NaCl, pH 8). Blots were washed for 5 min at 20°C with TBS containing 0.1% Tween 20 (TBST) and then incubated with approximately 1.2 x 10⁹ biotin-labeled T. denticola cells for 2 h at 20°C with gentle shaking. Unbound cells were removed by washing the blots twice with TBST containing 0.1% BSA (TBSTB) and once with TBS, and bound cells were detected with HRP-streptavidin (diluted 1:1,000 in TBSTB) for 2 h at 20°C. Blots were washed with TBSTB and then with TBS, and bound T. denticola cells were detected after color development with 4-chloro-1-naphthol as previously described (20). For controls, duplicate blots were incubated with no cells or with HRP-conjugated anti-human fibrinogen antibody (diluted 1:1,000) to visualize fibrinogen bands.

Fibrinogen blot overlay. Outer membrane proteins of T. denticola were extracted into Triton-X114 as described above, incubated at 20 or 100°C for 5 min in sample buffer, and separated by SDS-PAGE. Proteins were transferred onto nitrocellulose, and nonspecific binding sites were blocked for 16 h at 4°C with 10% skimmed milk powder in TBS. Blots were washed in TBST for 5 min and incubated with 5 µg of fibrinogen/ml in TBS for 3 h at 37°C. Blots were washed twice with TBST containing 1% skimmed milk powder (TBSTM) and once with TBS. Blots were then incubated with HRP-conjugated anti-human fibrinogen antibody (diluted 1:1,000 in TBSTM) for 2 h at 20°C and washed with TBSTM and TBS, and antibody binding was visualized by using an ECL detection system (Amersham Biosciences).

Affinity chromatography. CNBr-activated Sepharose 4B (Amersham Biosciences) was covalently linked to human fibrinogen or BSA (10 mg of protein/ml with 1 g of Sepharose) as recommended by the manufacturer. The linkage of proteins to Sepharose was confirmed by an enzyme-linked immunosorbent assay. Outer membrane proteins were extracted from T. denticola ATCC 35405 with 0.1% Triton X-114 for 16 h and mixed for 2 h by end-over-end rotation with protein-conjugated Sepharose beads. The Sepharose was sedimented by centrifugation at 500 x g for 2 min, and loosely bound proteins were removed from the beads by alternate washing and centrifugation with binding buffer (PBS containing 0.02% NaN₃ and 0.05% Nonidet P-40). To release bound proteins, the beads were mixed in a vortex with sample buffer (see above), the suspension was centrifuged as before to sediment the beads, and the supernatant was retained for SDS-PAGE or enzymatic analyses.

MS. For protein identification, bands were excised from SDS-PAGE gels and subjected to in-gel trypsin digestion and peptides were analyzed by liquid chromatography-tandem mass spectrometry (MS-MS) (61). Peptide sequences were aligned with the sequences translated from open reading frames within the T. denticola ATCC 35405 genome sequence (http://www.tigr.org) by using Bioworks v3.1 with TurboSEQUEST software (Thermo Electron).

Fibrinogen zymography. Proteins were separated by SDS-PAGE through gels containing 0.25 mg of fibrinogen/ml. After electrophoresis, gels were washed twice with 2.5% Triton X-100 in H₂O, twice with 2.5% Triton X-100 in 50 mM Tris-HCl, pH 7.4, to remove SDS, and twice with 50 mM Tris-HCl, pH 7.4, for 10 min at 20°C. The gels were then incubated for 18 h at 37°C in 50 mM Tris-HCl, pH 7.4, containing 10 mM CaCl₂ to allow proteolysis. Gels were stained with 0.04% Coomassie brilliant blue, and clear (unstained) regions of proteolytic activity appeared on the stained blue background after incubation for 30 min in destaining solution (methanol-glacial acetic acid-water, 40:10:50).

CTLP assay. Washed T. denticola cells were suspended in PBS (0.1 ml) at an OD₆₀₀ of 0.25 (6 x 10⁸ cells/ml) and incubated with 1 mM synthetic chromogenic chymotrypsin substrate N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanalide (SAAPFNA; Sigma) in Tris-HCl, pH 7.2, containing 2 mM dithiothreitol for 1 h at 37°C. Suspensions were centrifuged (10,000 x g for 10 min at 4°C), and the absorbance of the supernatants at 405 nm was measured to determine the p-nitroaniline concentration (proportional to enzymatic activity).

TCT assay. Fresh frozen human plasma diluted 1:1 in 0.9% NaCl (0.1 ml) was mixed with Owren's (sodium barbital) buffered saline, pH 7.35 (0.1 ml; Dade Behring), and warmed at 37°C for 5 min. Thrombin (5 U/ml; Diagnostic Reagents) in Owren's buffered saline containing 10 mg of BSA/ml (0.1 ml) was added, and the time (in seconds) to produce a clot was recorded. To determine the effects of T. denticola on the thrombin clotting time (TCT), washed exponential-phase cells in PBS (1.2 x 10⁹ cells/ml) were mixed with Owren's buffered saline (0.05 ml) and incubated with diluted fresh frozen plasma (0.1 ml) for 5 min at 37°C. Thrombin was added, and mean TCTs from assays run in triplicate were recorded.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Binding of fibrinogen by T. denticola. It is well established that within the human body, tissue proteins such as fibrinogen may be present in the fluid phase, e.g., mixed within plasma, or in an immobilized phase, e.g., associated with the extracellular matrix or bound to a surface. Accordingly, interactions of T. denticola with fibrinogen in fluid and immobilized phases were investigated. The binding of fluid-phase fibrinogen by T. denticola ATCC 35405 cells was dose dependent and saturable up to a maximum of 1 µg of added fibrinogen (Fig. 1A). Under these conditions, 3 x 10⁷ immobilized cells bound approximately 0.20 µg of fibrinogen. From a nonlinear regression analysis of saturation binding data (displayed as a Scatchard plot of the bound fibrinogen/free fibrinogen ratio [y axis] against the level of bound fibrinogen [x axis]), the maximum level of binding was calculated as 1.013 x 10⁵ molecules of bound fibrinogen per cell. In reverse-phase experiments, 0.15 µg of fibrinogen was immobilized onto plastic wells (on 96-well plates) and the numbers of bound biotinylated T. denticola cells were determined. The level of adherence to immobilized fibrinogen was proportional to the numbers of cells added, with binding sites essentially saturated when more than 5 x 10⁶ cells were added (Fig. 1C). Thus, T. denticola ATCC 35405 was able to bind fibrinogen in either the fluid or the solid phase, and the relative binding levels were not markedly different within the limitations of the assays.

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FIG. 1. Binding of fluid-phase or immobilized fibrinogen by T. denticola ATCC 35405 (), MHE (Msp–; ), or CKE (CTLP–; ). (A and B) Treponema cells immobilized onto plastic wells in the absence (A) or presence (B) of 0.05 mM PMSF serine protease inhibitor were incubated with increasing concentrations of fluid-phase fibrinogen, and binding was detected with HRP-conjugated anti-fibrinogen antibody and color reagent o-phenylenediamine (A490). In the absence of PMSF, MHE bound 15 to 20% less fluid-phase fibrinogen (P < 0.05) than wild-type T. denticola ATCC 35405. PMSF enhanced fibrinogen binding by both ATCC 35405 and MHE by approximately 35% (P < 0.05). (C and D) Increasing concentrations of biotin-labeled T. denticola cells were added to wells of microtiter plates coated with 0.15 µg of fibrinogen in the absence (C) or presence (D) of 0.05 mM PMSF. Numbers of bound cells were determined by utilizing HRP-streptavidin and o-phenylenediamine (A490). T. denticola ATCC 35405 and MHE adherence to immobilized fibrinogen in the presence of PMSF was increased by about 20% (P < 0.05). Error bars indicate standard deviations of the means from triplicates of a representative experiment.

Previous work (20) has suggested a potential role for the major surface protein (Msp) in fibrinogen binding by T. denticola. To determine if Msp was the primary mediator of fibrinogen interactions with T. denticola, we investigated the binding properties of an isogenic mutant strain, MHE, deficient in the production of Msp (27). Strain MHE cells showed dose-dependent saturable binding of fluid-phase fibrinogen (Fig. 1A) and bound 15 to 20% less overall than wild-type cells. In the reverse assay, strain MHE cells adhered dose dependently to fibrinogen and there was a 15 to 20% decrease in the level of adherence compared with that of wild-type cells (Fig. 1C). These results suggested that Msp played a minor role in mediating T. denticola interactions with fibrinogen.

Identification of fibrinogen-binding proteins. To isolate components of the T. denticola cell surface that bound fibrinogen, outer membrane protein extracts were incubated with fibrinogen-linked Sepharose or with BSA-linked Sepharose (control). Proteins that bound fibrinogen- or BSA-linked Sepharose were then desorbed with SDS sample buffer (see Materials and Methods). The eluate from the fibrinogen-linked Sepharose contained one major band migrating at 100 kDa (Fig. 2A). This band was resolved into 80-, 48-, and 45-kDa polypeptide bands when the sample was heated at 100°C for 5 min before SDS-PAGE (Fig. 2A). Immunoblot analysis with HRP-conjugated anti-fibrinogen antibody demonstrated the more diffuse areas of staining in Fig. 2A to represent fibrinogen degradation products (data not shown). No protein bands in the eluate from BSA-linked Sepharose were visualized. The 100-kDa band was excised from the polyacrylamide gel and subjected to trypsin digestion, and peptides were analyzed by liquid chromatography-MS-MS. Eight peptides (TELIIVGYDVANNR, TSAAAPLSNAIFGRVFVIMER, EKDIGEFKPVSR, SPVLTGKVMEAFTYSLK, TMGNNSSSGSNAVNGR, and three others) were identified as components of the CTLP complex (PrtP, PrcA1, and PrcA2) (2, 43).

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FIG. 2. Purification and enzymatic activity of major fibrinogen-binding protein from T. denticola ATCC 35405. Outer membrane proteins were extracted with 0.1% Triton X-114 solution and incubated with fibrinogen-linked Sepharose, and then tightly bound proteins were eluted with SDS sample buffer (see Materials and Methods). (A) Proteins stained with Coomassie blue; (B) fibrinogen-containing gel zymogram stained with Coomassie blue. The outer membrane extracts (applied) and the affinity-purified protein extracts (eluted) were incubated for 5 min at 20°C (–) or 100°C (+) before being subjected to SDS-PAGE. In panel B, the major eluted band with enzymatic activity in the unheated lane corresponds to an apparent molecular mass of 100 kDa. Positions of molecular mass markers are indicated.

The 100-kDa band eluted from fibrinogen-linked Sepharose had strong proteolytic activity as shown by fibrinogen zymography (Fig. 2B). When outer membrane extracts or eluate containing the 100-kDa polypeptide was heated at 100°C before SDS-PAGE, the proteolytic activity was destroyed (Fig. 2B). This result confirms that CTLP activity is heat sensitive and suggests that a major fibrinogen-binding component on the T. denticola cell surface is the CTLP complex.

A CTLP-deficient mutant does not bind fibrinogen. We then investigated the ability of an isogenic mutant deficient in the expression of the CTLP (dentilisin) complex, T. denticola CKE (27), to bind fibrinogen. In both fluid-phase and immobilized-phase assays, T. denticola CKE showed considerably reduced levels of fibrinogen binding compared to the wild type (Fig. 1). Strain CKE cells bound only 20% of wild-type levels of fluid-phase fibrinogen (Fig. 1A) and showed levels of adherence to immobilized fibrinogen reduced by >80% (Fig. 1C).

To determine if other components present among T. denticola outer membrane proteins bound fibrinogen, Western blots were incubated with fibrinogen in overlays and fibrinogen binding was detected with HRP-conjugated anti-fibrinogen antibody. The major fibrinogen-binding activity was localized to a 100-kDa band present in the wild-type strain and in msp mutant strain MHE (Fig. 3). No binding of fibrinogen to membrane proteins extracted from strain CKE in blot overlays was detected (Fig. 3). The heating of extracts at 100°C abolished the binding of fibrinogen. No binding of fibrinogen to the 80-, 48-, and 45-kDa components generated by heating at 100°C was observed, suggesting that the intact CTLP complex is necessary for fibrinogen binding. Taken collectively, these results show that the major fibrinogen-binding component present on the T. denticola cell surface is the CTLP complex.

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FIG. 3. Fibrinogen blot overlay of outer membrane protein extracts from T. denticola ATCC 35405, MHE, and CKE. Proteins were extracted with 1% Triton X-114 and incubated for 5 min at 20°C (–) or 100°C (+) before being subjected to SDS-PAGE. (A) Proteins stained with Coomassie blue. Arrowheads (lanes 1 and 2) identify the Msp complex and denatured Msp, respectively. (B) Corresponding Western blot incubated with fibrinogen (5 µg/ml). Fibrinogen binding was detected utilizing HRP-conjugated anti-human fibrinogen antibody and chemiluminescence (ECL detection system). A single band of bound fibrinogen (100 kDa) in unheated (–) extracts from ATCC 35405 and MHE was detected. A duplicate blot incubated with HRP-conjugated anti-human fibrinogen antibodies alone showed no reactive bands. Positions of molecular mass markers are indicated.

T. denticola recognizes A and Bß fibrinogen chains. When native fibrinogen is heated (100°C for 5 min) and subjected to SDS-PAGE, the molecule is dissociated into composite polypeptide chains designated A, Bß, and (Fig. 4A), with the A chain running as a doublet (13, 32). The A (80 kDa), Bß (60 kDa), and (55 kDa) chains were also expressed as recombinant polypeptides in E. coli (Fig. 4B). To determine the specificity of T. denticola interaction with fibrinogen, denatured human fibrinogen or purified recombinant polypeptides were blotted onto nitrocellulose and overlaid with biotinylated treponemal cells and bacterial binding was detected with HRP-streptavidin. T. denticola ATCC 35405 cells adhered principally to the A and Bß chains of denatured fibrinogen, and not to the chain (Fig. 4C). This specificity was confirmed by the observation that T. denticola cells adhered principally to the A and Bß recombinant polypeptides (Fig. 4C), although there was also some adherence to the recombinant chain polypeptide (Fig. 4C). Strain CKE cells did not adhere to fibrinogen or to fibrinogen chain polypeptides in similar blot overlay experiments (data not shown).

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FIG. 4. Adherence of T. denticola ATCC 35405 cells to denatured fibrinogen or recombinant fibrinogen polypeptides. (A) Human fibrinogen (N) or purified recombinant A, Bß, and chain polypeptides were heated for 5 min at 100°C and subjected to SDS-PAGE, and gels were stained with Coomassie blue. (B and C) Corresponding nitrocellulose blots reacted with HRP-conjugated anti-fibrinogen antibody (B) or biotin-labeled T. denticola ATCC 35405 cells followed by HRP-conjugated streptavidin (C). The apparent molecular masses of the A, Bß, and chain recombinant polypeptides were approximately 80, 60, and 55 kDa, respectively. Denatured human fibrinogen preparations show two A polypeptide bands (13, 32). Positions of molecular mass markers are indicated.

Proteolytic phenotypes of CTLP^– and Msp^– mutants. To visualize the proteolytic activities present within outer membrane extracts of wild-type, MHE, and CKE strains, proteins were subjected to fibrinogen zymography. As previously indicated, an unheated outer membrane extract from wild-type ATCC 35405 showed a strong proteolytic band at 100 kDa which was heat sensitive (Fig. 5). Strain MHE (Msp^–) also expressed the 100-kDa proteolytic band. However, this major band was absent in extracts from strain CKE (CTLP^–) (Fig. 5). A comparison of chymotrypsin-like enzyme activities expressed by cells utilizing the chromogenic substrate SAAPFNA showed that in fact the MHE mutant expressed about 75% (P < 0.05) of the wild-type level of enzymatic activity (data not shown). The SAAPFNA activity expressed by strain CKE cells was not above background levels, suggesting that CTLP accounts for the total chymotrypsin-like activity of T. denticola ATCC 35405. Interestingly, outer membrane extracts from strain CKE showed the presence of an additional band with fibrinogenolytic activity at an apparent molecular mass of 250 kDa. Following heating of the extract, the 250-kDa band with activity disappeared to be replaced by a 50-kDa band with activity (Fig. 5). Although these data are preliminary, they suggest that T. denticola may have the ability to express another protease that degrades fibrinogen, albeit with a much lower level of activity than CTLP, but that this protease is repressed, masked, or inactivated in the presence of CTLP production.

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FIG. 5. Fibrinogen zymograms of outer membrane proteins from T. denticola ATCC 35405, MHE (Msp–), and CKE (CTLP–). Triton X-114 extracts were incubated for 5 min at 20°C (–) or 100°C (+) before being subjected to SDS-PAGE through gels containing fibrinogen. Gels were developed for activity as described in Materials and Methods and stained with Coomassie blue. Areas of enzymatic activity are seen as clear bands. Positions of molecular mass markers are indicated.

Relationship between proteolytic activity and adherence. Previous work has suggested that the inactivation of CTLP with PMSF inhibits interactions of T. denticola with porcine periodontal ligament epithelial cells (24, 44). To determine if CTLP enzymatic activity was necessary for fibrinogen binding, assays in the presence or absence of 0.05 mM PMSF were conducted. This PMSF concentration was sufficient to inhibit >95% of the chymotrypsin-like activity of T. denticola ATCC 35405, as determined by a SAAPFNA assay. The inclusion of PMSF resulted in the enhanced binding of T. denticola ATCC 35405 to both fluid-phase (Fig. 1B) and immobilized (Fig. 1D) fibrinogen. Approximately 35% more fluid-phase fibrinogen became bound to cells, and the levels of adherence to immobilized fibrinogen increased by 20% (Fig. 1). Similar effects of PMSF on fibrinogen binding by strain MHE cells were observed, while PMSF did not enhance fibrinogen binding or adherence by strain CKE cells (Fig. 1). These results indicate that the overall levels of fibrinogen binding by T. denticola cells reflect a net result of adherence to and proteolysis of this substrate. However, the binding of, or adherence to, fibrinogen by T. denticola does not depend upon active proteolysis.

CTLP inhibits blood clot formation. Fibrinogen plays a vital role in platelet-platelet bridging and in fibrin clot formation at damaged tissue sites. The incubation of human plasma with thrombin in vitro induces clot formation, and the time taken for this to occur is known as the TCT. To test the hypothesis that CTLP may modulate clot formation, equal numbers of cells of strains ATCC 35405, MKE, and CHE were incubated with human plasma, thrombin was added, and the clotting times were compared with those for controls (no bacteria). The presence of wild-type or MHE cells significantly increased (P < 0.05) the TCTs compared to those for controls without bacterial cells, with longer clotting times recorded for the ATCC 35405 wild-type sample than the strain MHE sample (Fig. 6). Conversely, no significant differences in TCTs for strain CKE (CTLP^–) and control samples were observed (Fig. 6). These results suggest that the expression of CTLP by T. denticola may potentially inhibit hemostasis.

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FIG. 6. TCT in the absence or presence of T. denticola ATCC 35405, MHE (Msp–), or CKE (CTLP–). Human plasma was incubated with T. denticola (1.2 x 109 cells/ml) suspended in PBS or with PBS alone (control) for 5 min before the addition of 5 U of thrombin/ml, and the time required to induce clot formation was measured. Error bars indicate standard deviations of the means from triplicates of a representative experiment, while asterisks denote significant differences (P < 0.05) between TCTs for the indicated sample and the control.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fibrinogen is abundant at sites of periodontal disease, where tissues are destroyed and bleeding can occur spontaneously. Plasma fibrinogen levels are also elevated in the elderly (28) and in smokers (35), two population cohorts with increased risk for periodontal disease. Fibrinogen levels may therefore be of significance in modulating virulence strategies of potential periodontal pathogens, such as T. denticola. The characterization of the interactions of T. denticola with fibrinogen may assist in developing a better understanding of the relationship between these bacteria and the host. Although previous work has demonstrated the adherence of T. denticola to immobilized fibrinogen (31), the present study provides evidence that T. denticola ATCC 35405 binds both fluid-phase and immobilized fibrinogen and provides a molecular basis for these interactions. We anticipated that fluid-phase fibrinogen would be equivalent to the form of fibrinogen in plasma while immobilized fibrinogen may more closely resemble fibrinogen found embedded within the extracellular milieu or deposited onto oral surfaces. Adherence to both forms of fibrinogen was readily demonstrated, and this adherence may serve several potential functions in vivo. Firstly, soluble fibrinogen may provide a nutrient source by becoming degraded into smaller peptides and taken up by the cells. It has been suggested previously that OppA (a peptide-binding protein) of T. denticola (26), which binds soluble forms of fibronectin and plasminogen, may be involved in nutrient capture. Secondly, the detection of bacterial cells and their subsequent inactivation by host immune defense mechanisms may be diverted through the binding of fibrinogen to the bacterial cell surface. Thus, group A streptococci and Streptococcus equi subsp. equi are more slowly cleared by the host if they become coated with fibrinogen (6, 54, 60). Thirdly, T. denticola colonization and the subsequent invasion of host tissues may be promoted by adherence to immobilized fibrinogen, as has been shown previously for S. aureus (55) and Streptococcus agalactiae (30). Lastly, fibrinogen adherence and degradation may modulate fibrinogen interactions with platelets and therefore antagonize platelet functions.

By separating the component polypeptide chains of native fibrinogen by SDS-PAGE, it was shown that T. denticola bound to the A and Bß chains. Notably, the platelet IIa/IIIb glycoprotein binds the A and Bß chains of fibrinogen, and this effectively links platelets together. Interactions of microorganisms with fibrinogen may therefore potentially interfere with blood clotting. Staphylococcus epidermidis binds the Bß chain (51) at the position where thrombin cleaves fibrinopeptide B from the fibrinogen molecule to initiate clot formation (14). This finding indicates a major potential for interference with essential wound healing processes. S. aureus clumping factor A (ClfA) binds the tip of the C terminus of the chain (34), causing fibrinogen to clump and making it unavailable for thrombolytic conversion into fibrin (45). Fibrinogen adherence by various bacteria thus appears to occur at different sites on the fibrinogen molecule, effectively reducing competition for binding among the different organisms. Further work is under way to determine if specific regions or sequences within fibrinogen are recognized by T. denticola.

The major surface protein (Msp) of T. denticola has multiple adherence properties, and previous work with recombinant monomeric forms of this protein (20) showed that they bind fibrinogen. In keeping with this observation, strain MHE (deficient in Msp production) showed reduced binding of fluid-phase and immobilized fibrinogen. However, in strain CKE (CTLP deficient), fibrinogen adherence was abolished. This strain is also deficient in Msp production (27, 43), and so the results of these experiments do not rule out the possibility that fibrinogen interactions in situ involve a synergistic function of CTLP and Msp. It is possible that CTLP is required for the processing of Msp or the formation of an Msp complex within the outer membrane and that Msp is unstable or lost from the cell surface in the absence of CTLP. Alternatively, or in addition, there is evidence that CTLP may be involved in msp gene expression, since mutations in prcA or prtP have been reported to reduce msp transcription levels by about 50% (2). Cells of strain CKE show an approximately 50% reduction in levels of adherence to fibronectin and to human immunoglobulin G (data not shown) compared to wild-type cells, suggesting that CTLP, either directly or indirectly through the processing of Msp, also plays a role in binding these proteins.

The CTLP complex has been shown to have adherence and coaggregation potential (24, 33, 44). By using affinity chromatography, we detected a 100-kDa fibrinogen-binding protein in outer membrane extracts of T. denticola ATCC 35405. This protein had fibrinogenolytic activity and was highly sensitive to heating. Peptide MS analyses of this fibrinogen-binding and -degrading protein confirmed it to be the CTLP complex. A role for CTLP activity in the adherence of T. denticola to human cell lines was suggested following the observation that the serine protease inhibitor PMSF inhibits adherence (24, 44). However, levels of adherence of T. denticola ATCC 35405 and MHE cells to fibrinogen were significantly enhanced following the pretreatment of cells with PMSF. This result was not anticipated and suggests that adherence to fibrinogen may occur in the absence of functional enzymatic activity. In this respect, there is some similarity between the functions of CTLP and the gingipain proteases of P. gingivalis. The RgpA (arginine) and Kgp (lysine) proteases undergo posttranslational modifications, form surface-associated complexes, and mediate both adherence to and the degradation of host tissue proteins (9). Within the gingipains, separate regions may determine adherence and proteolytic functions (53). Evidence presented in this study suggests that an intact CTLP complex is required for both adherence and proteolysis. Neither the protease (PrtP) component nor the individual ancillary components PrcA1 and PrcA2 were capable individually of adherence to fibrinogen or fibrinogen degradation, but this capability was tested only with denatured polypeptides. Future studies involving active-site mutagenesis of PrtP or targeted mutagenesis of prcA-prtP may help identify the fibrinogen-binding regions. The ability to both adhere to and degrade fibrinogen may promote a cycle of attachment, proteolysis, and detachment, thus helping the bacteria to advance deeper into periodontal tissues.

The interactions of T. denticola with fibrinogen are also likely to influence pathogenicity through interference with hemostasis. The A and Bß chains of fibrinogen are normally cleaved by thrombin, while the C-terminal portions of the A chain bind activated IIa/IIIb integrin on the platelet surface to establish the formation of a platelet plug. The presence of T. denticola wild-type or MHE cells extended the time taken for thrombin-induced clot formation to occur, while CTLP mutant cells had no effect on the TCT. By degrading fibrinogen, the gingipains of P. gingivalis are also capable of inhibiting clotting (36). However, P. gingivalis apparently preferentially binds the chain of fibrinogen (42). Thus, it is possible that these two periodontal organisms, which are frequently found together at diseased sites (57), mount a combined operation on fibrinogen that effectively hampers healing processes and promotes bleeding and inflammation. T. denticola has also been detected at other body sites, e.g., atheromatous plaques (8), and may be disseminated to other organs via the vasculature (18, 39). In these environments, the ability to interact with fibrinogen may be vital in protection against host-mediated killing. Future studies on CTLP structure and function should help identify critical sequences or regions involved in host tissue interactions that may enable the development of effective inhibitors to better control Treponema-associated infections.

 
We are very grateful to K. Homer for MS analysis and to E. Walsh for providing plasmids. We thank J. L. Brittan and L. C. Dutton for expert technical assistance and A. M. Edwards, T. J. Foster, S. W. Kerrigan, and M. J. Woodward for helpful discussions.

 
* Corresponding author. Mailing address: Department of Oral and Dental Science, University of Bristol, Lower Maudlin St., Bristol BS1 2LY, United Kingdom. Phone: 44-117-928-4424. Fax: 44-117-928-4313. E-mail: howard.jenkinson{at}bristol.ac.uk

Published ahead of print on 25 June 2007.

Editor: J. N. Weiser

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Infection and Immunity, September 2007, p. 4364-4372, Vol. 75, No. 9
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