The chymotrypsin-like protease complex of Treponema denticola ATCC 35405 mediates fibrinogen adherence and degradation

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 Aalpha and Bbeta chains of fibrinogen, but not the gamma chains, were specifically recognized by T. denticola. Following fibrinogen affinity chromatography analysis of cell surface extracts, a major fibrinogen-binding component (polypeptide molecular mass, approximately 100 kDa), which also degraded fibrinogen, was purified. Upon heating at 100 degrees 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.     

The Treponema denticola chymotrypsin-like protease dentilisin induces matrix metalloproteinase-2-dependent fibronectin fragmentation in periodontal ligament cells

Periodontal disease is a bacterially mediated chronic inflammatory disease that results in destruction of the periodontal ligament (PDL) and alveolar bone that surround and support the dentition. While their precise roles are not well understood, periodontal pathogens, including Treponema denticola, are believed to initiate the destructive inflammatory responses and dysregulation of tissue homeostasis that characterize the disease. These responses are believed to result from both proinflammatory effects of acylated bacterial membrane components (lipopolysaccharides and lipoproteins) and degradative effects of secreted bacterial proteases. Host-derived matrix metalloproteinases (MMPs) are key enzymes both in tissue homeostasis and tissue destruction. MMP expression is modulated in part by specific proteolytic fragments of fibronectin (FN), which are associated with periodontal disease. FN is a predominant extracellular matrix component in the periodontium. We examined the ability of Treponema denticola and its acylated outer membrane PrtP protease complex to induce both activation of MMP-2 and generation of FN fragments in human PDL cell culture supernatants. T. denticola parent and isogenic mutant strains, as well as MMP-2 small interfering RNA and specific inhibitors of MMP-2 and PrtP activity, were used to examine protein expression, gelatinolytic activity, and FN fragmentation in culture supernatants. T. denticola and its purified protease induced both MMP-2 activation and FN fragmentation. Here, we demonstrate that PrtP proteolytic activity induces the activation of MMP-2 and that active MMP-2 is required for FN fragmentation. These results suggest a specific mechanism by which the T. denticola protease may disrupt homeostatic processes required for the maintenance of periodontal health.     

Characterization of fibrinolytic activities of Treponema denticola.

Several fibrinolytic activities of Treponema denticola, an oral spirochete associated with gingivitis and periodontal disease, were identified and characterized following phase partitioning with the nonionic detergent Triton X-114. The apparent molecular masses of the proteases ranged from 91 to 228 kDa when analyzed in sodium dodecyl sulfate-polyacrylamide gels containing fibrinogen as the protease substrate. A qualitative analysis of zymograms showed that the proteases were highly enriched in the detergent phase, although the 91-, 173-, and 228-kDa proteases were also found in the aqueous phase. Zymograms of crude outer sheaths prepared by repeated freezing-thawing revealed that the proteases may be associated with this subcellular compartment. The proteases displayed substrate specificity towards fibrinogen, were susceptible to sulfhydryl group reagents, and had a pH optimum between 7 and 8. The similarities in their sensitivity to inhibitors, temperature stability, pH optimum, and laddered protein profiles suggest that these hydrolytic enzymes may be part of a family of oligomeric proteases that may play an important role in the invasiveness of and tissue damage caused by the spirochete.     

Tip-oriented adherence of Treponema denticola to fibronectin.

The adherence of Treponema denticola to ligands on cell surfaces or in basement membranes of periodontal tissues might play an important role in its pathogenicity. A direct microscopic assay was used to examine the binding of T. denticola to fibronectin and other protein substrates adsorbed on plastic cover slips. All strains of T. denticola that were tested adhered to fibronectin but to different degrees. The strains which bound in high numbers frequently bound by their tips. Type strain ATCC 33520 bound to fibronectin in high numbers (149 +/- 11.3 bacteria per microscopic field), with 60% bound by the tips. Strain e' bound in high numbers (140 +/- 10.2) and had the highest percentage of tip binding (98%); strain e bound in lowest numbers (39 +/- 8.2) and had the lowest percentage of tip binding (15%). Laminin supported binding at a level similar to that of fibronectin, as did fibronectin fragments which contained the cell binding domain peptides, RGDS. Type IV collagen and non-RGDS peptides did not support binding. Binding to fibronectin and laminin was inhibited by the addition of antifibronectin and antilaminin antibodies. By lowering the incubation temperature from 37 to 4 degrees C, the number of cells that attached decreased by 60% and tip binding was reduced by 50%. Pretreatment of the cells with collagen did not affect binding, whereas fibronectin pretreatment enhanced binding by 50% and laminin pretreatment resulted in a decrease of 60%. T. denticola adheres by its tips to fibronectin-coated surfaces, which suggests that fibronectin-specific adhesins cluster at the tips.     

Antagonistic effect of oral bacteria towards Treponema denticola.

This study was designed to isolate oral bacteria exhibiting antagonism towards Treponema denticola and to characterize the inhibitory activity. Eleven bacterial isolates obtained from subgingival sites and identified as either Staphylococcus aureus or Streptococcus mutans were found to inhibit the growth of T. denticola. When the activity spectra of these isolates were analyzed, two additional periodontopathogens (Porphyromonas gingivalis and Prevotella intermedia) were found to be affected, whereas most gram-positive bacteria were not. Strains of S. aureus produce a bacteriocin-like inhibitory substance (heat stable and protease sensitive), whereas the inhibitory effect of S. mutans appears to be related to the production of lactic acid. The negative interactions reported in this paper may govern population shifts observed in subgingival sites.     

Proteases of Treponema denticola outer sheath and extracellular vesicles.

Electron microscopical observations of the oral periodontopathogen Treponema denticola show the presence of extracellular vesicles bound to the bacterial surface or free in the surrounding medium. Extracellular vesicles from T. denticola ATCC 35404, 50 to 100 nm in diameter, were isolated and further characterized. Protein and proteolytic patterns of the vesicles were found to be very similar to those of isolated T. denticola outer sheaths. They were enriched with the major outer sheath polypeptides (molecular sizes, 113 to 234 kDa) and with outer sheath proteases of 91, 153, 173, and 228 kDa. These findings indicate that treponemal outer sheath vesicles contain the necessary adhesins and proteolytic arsenal for adherence to and damage of eucaryotic cells and mammalian matrix proteins. The major outer sheath- and vesicle-associated protease of T. denticola ATCC 35404 was purified and characterized. The purified enzyme had a molecular size of 91 kDa, and it dissociated into three polypeptides of 72, 38, and 35 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The activity of the enzyme could be inhibited by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and phenylboronic acid. The value of the second-order rate constant of the protease inactivation by phenylmethylsulfonyl fluoride was 0.48 x 10(4) M(-1) min-1. Inhibition of the enzyme by phenylboronic acid was rapid (< 1 min) and pH dependent. These data strongly suggest that this major surface proteolytic activity belongs to a family of serine proteases.     

Sulfhydryl-dependent attachment of Treponema denticola to laminin and other proteins.

Attachment of Treponema denticola ATCC 35405 to laminin, a major basement membrane protein, and to other proteins was studied. Microdilution plates were coated with the proteins, and the attachment of T. denticola was measured by the enzyme-linked immunosorbent assay technique. Compared with bovine serum albumin (BSA), T. denticola had a high affinity to laminin, fibronectin, fibrinogen, and gelatin, as well as to type I and type IV collagens. Attachment to RGD peptide (Gly-Arg-Gly-Asp-Ser, the integrin recognition sequence) was only about 30% of that to laminin and was comparable to attachment to BSA. Tests with laminin fragments obtained through elastase digestion showed that the spirochetes attached well to an A-chain 140-kDa fragment involved in eukaryote cell attachment but did not attach to a 50-kDa fragment that includes the heparin binding site. Pretreatment of T. denticola with soluble laminin, fibronectin, gelatin, BSA, or fibrinogen had no effect on the attachment of the bacteria to laminin or fibronectin. A wide variety of compounds were tested for their possible inhibitory actions on the attachment. While most treatments of T. denticola ATCC 35405 had little or no effect on the attachment to proteins, sulfhydryl reagents p-chloromercuribenzoic acid (pCMBA) and oxidized glutathione inhibited the attachment by 70 to 99%, depending on the protein. When T. denticola was first allowed to attach to proteins, addition of pCMBA or oxidized glutathione could no longer reverse the attachment. Heat treatment of the spirochetes also markedly reduced the attachment to laminin, gelatin, and fibrinogen but not to BSA. Mixed glycosidase treatment of the spirochetes inhibited the attachment by 20 to 80%. None of the above treatments of the substrate proteins had any marked effect on the spirochete attachment. The results indicate that T. denticola has the capacity to bind to many different kinds of proteins by utilizing specific attachment mechanisms. The binding appears to involve protein SH groups and/or carbohydrate residues on the surface of T. denticola.     

Quantitative relationship of Treponema denticola to severity of periodontal disease.

The Treponema denticola content of plaque was quantitatively estimated for samples taken from periodontitis patients as well as periodontally healthy subjects among two separate human populations. The populations studied included military volunteers and civilians at a university dental clinic. The plaque samples from each population were grouped according to pocket depth measurements at the collection site. A biotin-avidin enzyme-linked immunosorbent assay procedure was developed with a monoclonal antibody specific for a serovariety of T. denticola. T. denticola was present at significantly elevated levels in plaque samples collected from deep-pocket sites of patients with severe periodontitis relative to the healthy controls and a group with moderate disease. The ratio of T. denticola content per milligram of plaque in the deep pocket groups to that of the other two groups was about 2:1 for both populations. This is the first quantitative evidence of a positive relationship between a specific spirochete species and severe periodontitis.     

Cloning and expression of a neutral phosphatase gene from Treponema denticola.

We have isolated and characterized a neutral phosphatase gene, phoN, from Treponema denticola ATCC 35405. The gene was isolated from a T. denticola clone bank constructed in the medium-copy-number plasmid vector pMCL19. Subcloning and nucleotide sequencing of the DNA insert from one phosphatase clone, pTph14, revealed that the activity corresponded to an open reading frame consisting of 1,027 bp coding for a 37.9-kDa protein. Hydrophobicity analysis indicated that the protein exhibits some hydrophobic regions. Indeed, partial purification of the phosphatase suggested that the enzyme was membrane associated both in T. denticola and in the Escherichia coli clone. The pH optimum of the enzyme, approximately pH 6.4, indicated that it corresponded to a neutral phosphatase activity from T. denticola. An examination of possible natural substrates for the enzyme suggested that this enzyme hydrolyzes nucleoside di- and triphosphates. Northern (RNA) blot analysis revealed that this phosphatase gene is not likely to be present in an operon structure.     

Treponema denticola induces actin rearrangement and detachment of human gingival fibroblasts.

Spirochetes are associated with destructive periodontal diseases, and one cultivatable oral species, Treponema denticola, binds to mammalian cells and perturbs metabolism. To evaluate the cytoskeletal responses and attachment functions of human gingival fibroblasts (HGF) exposed to T. denticola, monolayers of HGF were incubated with T. denticola strains ATCC 35405, e, and e' in serum-free medium. HGF retracted pseudopods, rounded up, and ultimately detached from the substratum. Scanning electron microscopy showed spirochetes in close contact with HGF surfaces; occasionally, bacteria were partially submerged between folds in the HGF membrane. Blebbing and numerous microvilli formed on the cell surface as the HGF retracted. By confocal microscopy, spirochetes were detected in contact with the HGF surface but were never found on the ventral surface of fibroblasts between the substratum and cell. Morphological alterations were associated with and preceded by actin assembly, as measured by microscopic fluorimetry: there was a 263% increase in actin fluorescence over controls within 30 min. Detachment of fibroblasts from the substratum was related to incubation time and was dependent on the concentration of T. denticola. Detachment was observed for all strains tested and was also dependent on the viability of T. denticola: UV light, heat, and metronidazole treatment markedly reduced the HGF detachment response. Detachment was also significantly reduced by the protease inhibitor phenylmethylsulfonyl fluoride. HGF viability was not significantly affected by coincubation with spirochetes, as measured by lactate dehydrogenase release. Thus, T. denticola induces rapid cytoskeletal remodelling followed by cell detachment, which might be stimulated by a bacterially associated protease but is not likely directly mediated by proteolytic degradation at the cell-substratum adhesive contact points.     

Characterization of hemolysis and hemoxidation activities by Treponema denticola

This study determined the presence of two hemolytic activities in the oral treponeme, Treponema denticola, strains ATCC 35404 (TD-4), ATCC 33520, GM-1, and MS25. These activities, referred to as hemolytic and hemoxidative (HeA, HeO, respectively), were found to be both secreted into the extracellular environment, and cell associated. The extracellular activity was associated with small molecules with relative molecular weights of <1000 Da, and its activity was cysteine independent; the cell associated HeA and HeO activities were associated with a molecular weight fraction >10 kDa, and were cysteine dependent. The HeO activity of the fractionated material observed was due to the oxidation of hemoglobin to methemoglobin, and preceded the HeA lysis of the RBCs by approximately 2 h. Heating at 80°C and treatment with proteinase K resulted in the complete destruction of these activities in the fraction >10 kDa, while lipase at high concentration (800 μg/ml) reduced the HeA and HeO activities in the extracellular fraction by approximately 50%. Proteinase inhibitors had a variable effect on HeA and HeO activities in both extracellular and cell-associated fractions. Scanning and transmission electron microscopy revealed a progressive destruction of the RBC membrane, with membrane protrusions formed early in the interaction, which progressed to irregular holes in the membrane, and the complete loss of membrane integrity.     

Composition and localization of Treponema denticola outer membrane complexes

The Treponema denticola outer membrane lipoprotein-protease complex (dentilisin) contributes to periodontal disease by degrading extracellular matrix components and disrupting intercellular host signaling pathways. We recently demonstrated that prcB, located upstream of and cotranscribed with prcA and prtP, encodes a 22-kDa lipoprotein that interacts with PrtP and is required for its activity. Here we further characterize products of the protease locus and their roles in expression, formation, and localization of outer membrane complexes. PrcB migrates in native gels as part of a >400-kDa complex that includes PrtP and PrcA, as well as the major outer sheath protein Msp. PrcB is detectable as a minor constituent of the purified active protease complex, which was previously reported to consist of only PrtP and auxiliary polypeptides PrcA1 and PrcA2. Though it lacks the canonical ribosome binding site present upstream of both prcA and prtP, PrcB is present at levels similar to those of PrtP in whole-cell extracts. Immunofluorescence microscopy demonstrated cell surface exposure of the mature forms of PrtP, PrcA1, PrcB, and Msp. The 16-kDa N-terminal acylated fragment of PrtP (predicted to be released during activation of PrtP) was present in cell extracts but was detected neither in the purified active protease complex nor on the cell surface. PrcA2, detectable on the surface of Msp-deficient cells but not that of wild-type cells, coimmunoprecipitated with Msp. Our results indicate that PrcB is a component of the outer membrane lipoprotein protease complex and that Msp and PrcA2 interaction may mediate formation of a very-high-molecular-weight outer membrane complex.     

Quantitative immunoassay of Treponema denticola serovar C in adult periodontitis.

Murine monoclonal antibodies specific for Treponema denticola serovar C were produced and characterized in this study. An immunoassay was then developed by using these monoclonal antibodies, and the T. denticola serovar C antigen content of subgingival plaque was quantitated for samples taken from patients with periodontitis and healthy volunteers. The human subgingival plaque samples were grouped by severity of disease and pocket depth measurements at the collection site. The T. denticola serovar C content per milligram of subgingival plaque from deep pockets (greater than 6 mm) of patients with severe periodontitis was found to be twice that of samples collected from deep pockets (4 to 6 mm) of patients with moderate periodontitis or samples collected from healthy subjects (pocket depth, less than 4 mm).     

Molecular cloning and sequence analysis of antigen gene tdpA of Treponema denticola.

We isolated and characterized an immunogenic protein of an oral spirochete, Treponema denticola Johnson. A genomic DNA library constructed with bacteriophage lambda EMBL3 as a vector was immunologically screened with a rabbit antiserum against the whole cells. Using Western immunoblot analysis, we found a particular clone encoding an antigen with a molecular weight of 53,000; we designated the antigen as T. denticola protein A (TdpA). Complete sequence determination revealed an open reading frame of 1,419 bp and a signal peptide sequence that was homologous to that of bacterial lipoprotein. Southern hybridization analysis revealed that the tdpA gene is highly conserved in six tested strains of T. denticola species. Furthermore, we found that sera from some periodontitis patients contained antibody against the TdpA protein, although the reactivities of the antibodies varied among individuals.     

Monoclonal antibodies that recognize a specific surface antigen of Treponema denticola.

Spirochetes have been implicated as potential etiologic agents of periodontitis in humans. Murine monoclonal antibodies (MAbs) specific for a serogroup of Treponema denticola, an oral spirochete, were developed and characterized in this study. Antibodies secreted by clone IAA11 were judged to be the most useful, since they were able to detect 8 of 15 T. denticola strains. This MAb consisted of an immunoglobulin G3 heavy chain and a kappa light chain. MAb IAA11 was found to react with an epitope target located on the outer sheath of the cell wall. This MAb should be of diagnostic and scientific value in the study of T. denticola populations in human periodontitis.     

The opdB locus encodes the trypsin-like peptidase activity of Treponema denticola.

High levels of Treponema denticola in subgingival dental plaque are associated with severe periodontal disease. T. denticola, along with Porphyromonas gingivalis and Bacteroides forsythus, are the only cultivatable oral microorganisms that produce significant amounts of "trypsin-like" peptidase activity. The ability of subgingival plaque to hydrolyze N-alpha-benzoyl-DL-arginine-2-naphthylamide (BANA) is associated with high levels of one or more of these organisms. The purpose of this study was to identify the gene encoding trypsin-like activity in T. denticola and thus facilitate molecular-level studies of its potential role in disease. Using published peptide sequences of a T. denticola surface-associated oligopeptidase with BANA-hydrolyzing activity, we identified the gene, designated opdB, in an apparently noncoding region of the T. denticola genome unannotated contigs (11/2000; http://www.tigr.org). The opdB gene begins with a TTG start codon and encodes a 685-residue peptide with high homology to the oligopeptidase B family in prokaryotes and eukaryotes. An isogenic T. denticola opdB mutant was constructed by allelic replacement mutagenesis using an ermF/AM gene cassette. The mutant lacked BANA-hydrolyzing activity and had a slightly slower growth rate than the parent strain. This mutant will be used in future studies of interactions of T. denticola with host cells and tissue.     

Nutritional interactions between two suspected periodontopathogens, Treponema denticola and Porphyromonas gingivalis.

A mutual symbiotic enhancement of growth of Porphyromonas gingivalis and Treponema denticola is described in this report. Brain heart infusion broth supplemented with vitamin K did not support the individual growth of P. gingivalis or T. denticola. However, when inoculated as a mixture, both bacterial species did grow significantly. The growth-stimulating factors produced by P. gingivalis and T. denticola were dialyzable and heat stable and were further identified as isobutyric acid and succinic acid, respectively. Since some forms of periodontal disease are associated with the presence, in affected sites, of high numbers of P. gingivalis and spirochetes, it is suggested that the bacterial interaction described in this report might be of utmost importance in the initiation and progression of the disease.     

Activation of murine macrophages by lipoprotein and lipooligosaccharide of Treponema denticola

We have recently demonstrated that the periodontopathogenic oral spirochete Treponema denticola possesses membrane-associated lipoproteins in addition to lipooligosaccharide (LOS). The aim of the present study was to test the potential of these oral spirochetal components to induce the production of inflammatory mediators by human macrophages, which in turn may stimulate tissue breakdown as observed in periodontal diseases. An enriched lipoprotein fraction (dLPP) from T. denticola ATCC 35404 obtained upon extraction of the treponemes with Triton X-114 was found to stimulate the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and interleukin-1 (IL-1) by mouse macrophages in a dose-dependent manner. Induction of NO by dLPP was at 25% of the levels obtained by Salmonella typhosa lipopolysaccharide (LPS) at similar concentrations, while IL-1 was produced at similar levels by both inducers. dLPP-mediated macrophage activation was unaffected by amounts of polymyxin B that neutralized the induction produced by S. typhosa LPS. dLPP also induced NO and TNF-alpha secretion from macrophages isolated from endotoxin-unresponsive C3H/HeJ mice to an extent similar to the stimulation produced in endotoxin-responsive mice. Purified T. denticola LOS also produced a concentration-dependent activation of NO and TNF-alpha in LPS-responsive and -nonresponsive mouse macrophages. However, macrophage activation by LOS was inhibited by polymyxin B. These results suggest that T. denticola lipoproteins and LOS may play a role in the inflammatory processes that characterize periodontal diseases.