Proteolytic activity among various oral Treponema species and cloning of a prtP-like gene of Treponema socranskii subsp. socranskii

The proteolytic activity of 11 treponemal strains representing different phylogenetic groups was investigated by SDS-polyacrylamide gel electrophoresis with copolymerised casein, gelatin and fibrinogen as substrates. The activity was specified to be trypsin- and chymotrypsin-like by the cleavage of synthetic substrates BAPNA and SAAPFNA, respectively. Nine strains degrade casein and the synthetic substrate BAPNA. Chymotrypsin-like activity specifically inhibited by phenylmethylsulfonyl fluoride was found in four treponemes. Southern blot analysis using a Treponema socranskii subsp. socranskii-specific prtP probe confirmed the presence of prtP homologous genes in these four strains. The internal fragments of the chymotrypsin-like protease genes were cloned and sequenced after PCR amplification. Here we report the cloning of the complete prtP-like gene of T. socranskii subsp. socranskii, an organism shown to possess epidemiologic relevance in periodontitis.     

Cleavage of Treponema denticola PrcA polypeptide to yield protease complex-associated proteins Prca1 and Prca2 is dependent on PrtP

Analysis of potential virulence factors of oral spirochetes focuses on surface and secreted proteins. The Treponema denticola chymotrypsin-like protease (CTLP) is implicated in degradation of host cell molecules and contributes to tissue invasion. The CTLP complex, composed of the 72-kDa PrtP protein and two auxiliary proteins with molecular masses of approximately 40 and 30 kDa, is also involved in localization and oligomerization of the T. denticola major surface protein (Msp). The larger auxiliary protein was reported to be encoded by an open reading frame (ORF2) directly upstream of prtP. The deduced 39-kDa translation product of ORF2 contains a sequence matching the N-terminal sequence determined from one of the CTLP complex proteins. No proteins with significant homology are known, nor was information available on the third protein of the complex. DNA sequence analysis showed that ORF2 extended an additional 852 bp upstream of the reported sequence. The complete gene, designated prcA, encodes a predicted N-terminally-acylated polypeptide of approximately 70 kDa. Isogenic mutants with mutations in prtP, prcA, and prcA-prtP all lacked CTLP protease activity. The prcA mutant lacked all three CTLP proteins. The prcA-prtP mutant produced only a C-terminally-truncated 62-kDa PrcA protein. The prtP mutant produced a full-length 70-kDa PrcA. Immunoblot analysis of recombinant PrcA constructs confirmed that PrcA is cleaved to yield the two smaller proteins of the CTLP complex, designated PrcA1 and PrcA2. These data indicate that PrtP is required for cleavage of PrcA and suggest that this cleavage may be required for formation or stability of outer membrane complexes.     

Surface protease of Treponema denticola hydrolyzes C3 and influences function of polymorphonuclear leukocytes

Treponema denticola is a dominant microorganism in human periodontal lesions. One of the major virulence factors of this microorganism is its chymotrypsin-like surface protease, dentilisin. The purpose of this study was to evaluate the effect of dentilisin on human polymorphonuclear leukocytes (PMNs). We used chemiluminescence to assess production of O(-)(2) by PMNs against T. denticola ATCC 35405 and dentilisin-deficient mutant K1. T. denticola ATCC 35405 induced production of O(-)(2), whereas dentilisin-deficient K1 did not. We found that chymostatin, a protease inhibitor, strongly reduced the ability of T. denticola ATCC 35405 to induce production of, O(-)(2), whereas K1 was relatively unaffected. We also used Immunoblot and ELISA to evaluate the activation of complement by this microorganism in relation to PMNs. T. denticola ATCC 35405 hydrolyzed the alpha-chain of C3, producing iC3b. Furthermore, strain ATCC 35405 induced a larger release of MMP-9 from PMNs than strain K1. Dentilisin activated PMNs via complement pathways and may play a role in establishing periodontal lesions.     

A 43-kDa protein of Treponema denticola is essential for dentilisin activity

A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43- and 38-kDa proteins. A sequence encoding a 43-kDa protein was located in the 3' region of the prcA gene upstream of the dentilisin gene (prtP). The 43-kDa protein was apparently generated from digestion of PrcA. To clarify the function of the protein, we constructed a mutant of the 43-kDa protein following homologous recombination. The mutant lacked detectable dentilisin activity. Immunoblot analysis demonstrated that the dentilisin protein was degraded in the mutant. The results of real-time polymerase chain reaction suggested that prtP mRNA expression in the mutant was somewhat decreased compared with the wild-type strain. These data suggest that the 43-kDa protein is involved in the stabilization of the dentilisin protein.     

Binding of Porphyromonas gingivalis fimbriae to Treponema denticola dentilisin

Treponema denticola has been reported to coaggregate with Porphyromonas gingivalis and localize closely together in matured subgingival plaque. In this study of the interaction of T. denticola with P. gingivalis, the P. gingivalis fimbria-binding protein of T. denticola was identified by two-dimensional electrophoresis followed by a ligand overlay assay with P. gingivalis fimbriae, and was determined to be dentilisin, a chymotrypsin-like proteinase of T. denticola. The binding was further demonstrated with a ligand overlay assay using an isolated GST fusion dentilisin construct. Our results suggest that P. gingivalis fimbriae and T. denticola dentilisin are implicated in the coaggregation of these bacteria.     

Phylogenetic analysis of the spirochetes.

The 16S rRNA sequences were determined for species of Spirochaeta, Treponema, Borrelia, Leptospira, Leptonema, and Serpula, using a modified Sanger method of direct RNA sequencing. Analysis of aligned 16S rRNA sequences indicated that the spirochetes form a coherent taxon composed of six major clusters or groups. The first group, termed the treponemes, was divided into two subgroups. The first treponeme subgroup consisted of Treponema pallidum, Treponema phagedenis, Treponema denticola, a thermophilic spirochete strain, and two species of Spirochaeta, Spirochaeta zuelzerae and Spirochaeta stenostrepta, with an average interspecies similarity of 89.9%. The second treponeme subgroup contained Treponema bryantii, Treponema pectinovorum, Treponema saccharophilum, Treponema succinifaciens, and rumen strain CA, with an average interspecies similarity of 86.2%. The average interspecies similarity between the two treponeme subgroups was 84.2%. The division of the treponemes into two subgroups was verified by single-base signature analysis. The second spirochete group contained Spirochaeta aurantia, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirochaeta litoralis, and Spirochaeta isovalerica, with an average similarity of 87.4%. The Spirochaeta group was related to the treponeme group, with an average similarity of 81.9%. The third spirochete group contained borrelias, including Borrelia burgdorferi, Borrelia anserina, Borrelia hermsii, and a rabbit tick strain. The borrelias formed a tight phylogenetic cluster, with average similarity of 97%. THe borrelia group shared a common branch with the Spirochaeta group and was closer to this group than to the treponemes. A single spirochete strain isolated fromt the shew constituted the fourth group. The fifth group was composed of strains of Serpula (Treponema) hyodysenteriae and Serpula (Treponema) innocens. The two species of this group were closely related, with a similarity of greater than 99%. Leptonema illini, Leptospira biflexa, and Leptospira interrogans formed the sixth and most deeply branching group. The average similarity within this group was 83.2%. This study represents the first demonstration that pathogenic and saprophytic Leptospira species are phylogenetically related. The division of the spirochetes into six major phylogenetic clusters was defined also by sequence signature elements. These signature analyses supported the conclusion that the spirochetes represent a monophylectic bacterial phylum.     

Insertional inactivation of the prtP gene of Treponema denticola confirms dentilisin's disruption of epithelial junctions

The purified chymotrypsin-like protease of Treponema denticola, designated dentilisin or PrtP (DDBJ accession no. D83264), can disrupt cell-cell junctions and impair the barrier function of epithelial monolayers in vitro. Serine protease inhibitors block these effects. Yet, the protease is apparently less significant in perturbing intracellular signaling pathways and cytoskeletal rearrangement in fibroblasts. The purpose of this study was to use a PrtP-deficient mutant of T. denticola to confirm that the cytopathic effects of whole bacteria and its outer membrane on epithelial cell junctions were primarily accounted for by the activity of this protease. The prtP gene of ATCC 35405 was inactivated by insertion of an erythromycin-resistance cassette, yielding mutant K1. In contrast to wildtype ATCC 35405, mutant K1 grew in tight cell aggregates; the cells had a disrupted outer sheath, as determined by electron microscopy. When compared by silver stained SDS-PAGE of sonicated extracts of whole cells, the extract of mutant K1 was missing a band at approximately 90 kDa that was present in the wildtype ATCC 35405 strain. Whole cells and Triton X-100 outer membrane (OM) extracts of K1 and the wildtype strains were compared 1) for SAAPNA degrading activity by a colorimetric assay, 2) for stress fiber disruption in human gingival fibroblasts (HGF) by fluorescence microscopy of TRITC-phalloidin stained cells, and 3) the OM extracts only for perturbation of HEp-2 epithelial monolayers by electrical cell-substrate impedance sensing (ECIS). Mutant K-1 cells and OM had no SAPPNA degrading activity that is characteristic of dentilisin. K1 cells had HGF stress fiber disrupting activity (86 +/- 4.5% of HGFs affected) equivalent to both 35405 wildtype strains (84 +/- 3.9% and 71 +/- 14.1% of HGF, respectively). Yet, mutant K1 OM had diminished stress fiber disrupting activity (12.9 +/- 4.6% of HGF) compared with its parent 35405's OM (94.6 +/- 2.9%). The major cytopathogenic difference between the K1 mutant and wildtype strains was in their OM's effect on epithelial cell junctions. ATCC 35405 OM completely disrupted epithelial resistance in a concentration - dependent manner; mutant K1 OM had negligible effects. These data confirm that inactivation of the prtP gene completely reverses T. denticola's disruption of epithelial junctions, but there are pleiotropic effects of the mutation that may account for its apparently diminished effects on the cytoskeleton of HGF when the cells were challenged with OM extracts.     

Isolation and characterization of cytoplasmic fibrils from treponemes

Electron microscopy of Triton X-100-treated whole cells of an oral treponeme, Treponema sp. strain E-21, revealed that six cytoplasmic fibrils (CFs) helically wound as a bundle in the cytoplasm. The CFs were isolated and purified by disruption and solubilization of the cells followed by CsCl density gradient centrifugation. The purified CF preparation contained mostly fibrils of about 9 nm in width and very small amounts of thinner strands of about 3 nm in diameter. The CFs were apparently seen to be a tubular structure, but the isolated CFs had narrowed sites of about 4-5 nm in width lacking lumen-like images, possibly representing twisted sites. Thus, the CF did not seem to be a tubular structure. The purified CFs were composed of one major 82 kDa protein and a few minor proteins. The CFs were destructed by treatment with proteases, 8 M urea or 4 M guanidine hydrochloride. Very low tyrosine content (0.76 mol %) and lack of methionine were characteristic features for the 82 kDa protein. The CF preparations from the other five treponemes including Treponema phagedenis and T. denticola also had 82 kDa proteins as a major component, and the 82 kDa proteins of all of the treponemes had a common antigen when examined by using antiserum against the 82 kDa protein from Treponema sp. strain E-21. Furthermore, the 82 kDa protein was demonstrated to be a principal component of the CFs of all the treponemes by immunoelectron microscopy.     

Pathological observation on experimental swine dysentery

Experimental swine dysentery caused by 4 cultured strains (S73/2, DJ183, DJ70 and DK762) of Treponema hyodysenteriae was studied pathologically. The distribution and quantity of treponemes were examined on tissue sections stained by the Warthin-Starry method. Of the organs the colon contained the largest number of treponemes and the cecum and rectum the second largest number. Histopathological lesions were restricted to the large intestine. They ranged from mild catarrhal colitis in the mild case to desquamative, hemorrhagic colitis in the severest case. The severity of lesion was closely associated with the quantity of treponemes present. There was no difference in quality of the lesion between any two of the strains used in this study. Electron microscope revealed a large number of free treponemes present in the intestinal lumen and crypts. Treponemes were seen more frequently in the cytoplasm of goblet cells than in that of intestinal epithelial cells. They were also observed in desquamated degenerative epithelia. A small number of them were found in intact epithelia. Morphologically, the treponeme had a granular protoplasmic cylinder at the center which was surrounded by a thin envelope. Between the cylinder and the envelope there were axial fibrils.     

Biochemical and molecular characterization of serine proteases from larvae of Chrysomya bezziana, the Old World Screwworm fly

The diversity of serine proteases secreted from Chrysomya bezziana larvae was investigated biochemically and by PCR and sequence analysis. Cation-exchange chromatography of purified larval serine proteases resolved four trypsin-like activities and three chymotrypsin-like activities as discerned by kinetic studies with benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Amino-terminal sequencing of the three most abundant fractions gave two sequences, which were homologous to other Dipteran trypsins and chymotrypsins. Analysis of products generated by PCR of cDNA from whole larvae using specific primers based on the amino-terminal sequences and generic serine protease primers identified 22 different sequences, while phylogenetic analysis of the deduced amino acid sequences differentiated two trypsin-like and four chymotrypsin-like families. Phylogenetic comparisons with Dipteran and mammalian serine protease sequences showed that all the Chrysomya bezziana sequences clustered with Dipteran sequences. The Chrysomya bezziana chymotrypsin-like sequences segregated within a Dipteran cluster of chymotrypsin sequences, but were well dispersed amongst these sequences. The largest Chrysomya bezziana serine protease family, the trypB family, clustered tightly as a group, and was closely related to a Lucilia cuprina trypsin but distinct from Drosophila melanogaster alpha and beta trypsins. The trypB family contains ten highly homologous sequences and probably represents an example of concerted evolution of a trypsin gene in Chrysomya bezziana.     

Physical map of the Treponema denticola circular chromosome.

A physical map of the Treponema denticola ATCC 33520 genome was constructed by pulsed-field gel electrophoresis and DNA hybridization. The organism possesses a single, circular chromosome of approximately 3.0 Mbp and a 2.6-kbp circular plasmid, pTD1. The physical map of the A+T-rich genome was constructed with the rare-cutting restriction enzymes AscI, NotI, and SrfI, which have 8-bp G+C-rich recognition sites. The genes flgE, tdpA, and prtB encoding the flagellar hook protein, a 53-kDa immunogenic protein, and chymotrypsinlike protease, respectively, were located on the map. This treponeme was found to have two copies of each of the rRNA genes, as has been found to be the case for both Treponema phagedenis and Treponema pallidum.     

Fatty acids synthesized by oral treponemes in chemically defined media

OMIZ-W68, a chemically defined medium that contains no long-chain fatty acids and yet supports in vitro proliferation of a wide range of fastidious oral anaerobes, is described. The type strains of Treponema denticola, Treponema lecithinolyticum, Treponema maltophilum, Treponema pectinovorum, Treponema socranskii, and an as yet unpublished canine Treponema species could be propagated indefinitely in this medium with sugar supplements for the saccharolytic species. Analysis of the cellular fatty acids (CFA) of these treponemes by gas chromatography demonstrated the synthesis of C14, C15, C16, and C17 fatty acids (linear-, iso-, and anteiso-forms) in various proportions, but neither hydroxy- nor unsaturated fatty acids. However, between 0% and 40% of the eluted material could not be identified. The proportions of CFAs differed not only between species but also between the eight strains of Treponema denticola investigated. Replacing OMIZ-W68 by a derivative minimal essential medium (OMIZ-M/TDCDK) developed for Treponema denticola had little effect on the CFA profiles. In contrast, the CFA profiles of treponemes grown in OMIZ-W68 showed at best minor similarity to the strains from the Moore library of the Virginia Polytechnic Institute, which had been grown in media containing serum, peptones, and yeast extract.     

Mutagenesis of outer membrane virulence determinants of the oral spirochete Treponema denticola

The pore-forming major surface protein (Msp) and the chymotrypsin-like protease (CTLP) of Treponema denticola ATCC 35405 have been implicated in periodontal pathogenicity. Allelic replacement mutations were constructed at two sites in the msp gene and at one site in the CTLP locus. All mutant strains lacked the Msp hexagonal array outer membrane ultrastructure. Strain CKE, in which the locus encoding CTLP was disrupted, produced no CTLP and had aberrant Msp expression. The msp mutant MHE lacked Msp, and produced increased levels of CTLP. In contrast, msp mutant MPE made small amounts of a truncated Msp, but produced no CTLP. Interactions between Msp and CTLP in transport or assembly of the outer membrane complex are proposed.     

Molecular pathogenesis of the cell surface proteins and lipids from Treponema denticola

Treponema denticola, frequently isolated from the human oral cavity, is thought to be a major pathogen of human periodontal disease. Recent developments in molecular analysis have clarified the surface structure of this microorganism and the characteristics of its pathogenic factors. Structural analysis of the outer sheath showed T. denticola to have a new type of outer membrane lipid. Limited exposure of the major outer sheath protein is suggested by electron-microscopic analysis. A protease-deficient mutant has revealed the roles of the protease in the organization of the outer sheath material and in T. denticola pathogenicity. The surface features that contribute to the pathogenicity of T. denticola in periodontal disease are gradually being elucidated, and are reviewed.     

Treponema denticola induces interleukin-8 and macrophage chemoattractant protein 1 production in human umbilical vein epithelial cells

Treponema denticola, a major pathogen of periodontitis, has also been detected in the lesions of atherosclerosis. The aim of this study was to investigate induction of chemokine production in human umbilical vein endothelial cells (HUVECs) by T. denticola and determine whether those chemokines were degraded by a protease, dentilisin. T. denticola ATCC35405 or dentilisin-deficient mutant K1 were added to HUVECs and levels of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatants were determined by enzyme-linked immunosorbent assay. T. denticola ATCC35405 induced production of IL-8 in a time-dependent manner, with both production of IL-8 and expression of IL-8 mRNA showing higher levels than with exposure to dentilisin-deficient mutant K1. Although exposure to ATCC35405 induced expression of MCP-1 mRNA in the HUVECs, MCP-1 levels were remained similar to that in unstimulated cells. IL-8 and MCP-1 showed partial hydrolysis with exposure to T. denticola ATCC35405, but not with T. denticola K1. These results suggest that T. denticola can evade host defense mechanisms by modulating production of IL-8 and MCP-1, and that this play a role in the development of chronic infections such as periodontitis. The association of T. denticola infection to atherosclerosis was also discussed based on the present study.     

Dissecting the molecular diversity and commonality of bovine and human treponemes identifies key survival and adhesion mechanisms

Here, we report the first complete genomes of three cultivable treponeme species from bovine digital dermatitis (DD) skin lesions, two comparative human treponemes, considered indistinguishable from bovine DD species, and a bovine gastrointestinal (GI) treponeme isolate. Key genomic differences between bovine and human treponemes implicate microbial mechanisms that enhance knowledge of how DD, a severe disease of ruminants, has emerged into a prolific, worldwide disease. Bovine DD treponemes have additional oxidative stress genes compared to nearest human-isolated relatives, suggesting better oxidative stress tolerance, and potentially explaining how bovine strains can colonize skin surfaces. Comparison of both bovine DD and GI treponemes as well as bovine pathogenic and human non-pathogenic saprophyte Treponema phagedenis strains indicates genes encoding a five-enzyme biosynthetic pathway for production of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, a rare di-N-acetylated mannuronic acid sugar, as important for pathogenesis. Bovine T. phagedenis strains further differed from human strains by having unique genetic clusters including components of a type IV secretion system and a phosphate utilisation system including phoU, a gene associated with osmotic stress survival. Proteomic analyses confirmed bovine derived T. phagedenis exhibits expression of PhoU but not the putative secretion system, whilst the novel mannuronic acid pathway was expressed in near entirety across the DD treponemes. Analysis of osmotic stress response in water identified a difference between bovine and human T. phagedenis with bovine strains exhibiting enhanced survival. This novel mechanism could enable a selective advantage, allowing environmental persistence and transmission of bovine T. phagedenis. Finally, we investigated putative outer membrane protein (OMP) ortholog families across the DD treponemes and identified several families as multi-specific adhesins capable of binding extra cellular matrix (ECM) components. One bovine pathogen specific adhesin ortholog family showed considerable serodiagnostic potential with the Treponema medium representative demonstrating considerable disease specificity (91.6%). This work has shed light on treponeme host adaptation and has identified candidate molecules for future diagnostics, vaccination and therapeutic intervention.     

The Gastrointestinal Tract as a Potential Infection Reservoir of Digital Dermatitis-Associated Treponemes in Beef Cattle and Sheep

Digital dermatitis (DD) is an important cause of lameness in dairy cattle worldwide. It has now been reported in beef cattle and also sheep (contagious ovine digital dermatitis [CODD]). Three Treponema phylogroups are consistently isolated from lesions, Treponema medium-like, Treponema phagedenis-like, and Treponema pedis. The gastrointestinal (GI) tract and feces are suggested sites of treponemal infection in dairy cattle; however, isolation of DD-associated treponemes from these areas has previously failed. This study surveyed gingival tissues, rectal tissues, and feces of beef cattle and sheep for the molecular presence (PCR) and isolation of the three cultivable DD-treponeme phylogroups. Of the sheep gingival (n = 40) and rectal (n = 40) tissues, 1/40 gingival tissues was positive for DD-associated treponemes (T. pedis), as were 3/40 rectal tissues (one containing T. medium-like and two containing T. pedis). No DD-associated treponeme DNA was amplified from beef cattle rectal tissues (n = 40); however, 4/40 beef gingival tissues were positive for DD-associated treponemes (all containing T. phagedenis-like). A T. phagedenis-like DD-associated treponeme was isolated from the rectal tissue of a CODD symptomatic sheep. Beef cattle (n = 41) and sheep (n = 79) feces failed to amplify DD-associated Treponema DNA. Twenty-two treponemes were isolated from sheep feces; however, upon phylogenetic analysis, these clustered with the considered nonpathogenic treponemes. This study detected DD-associated treponemes in the GI tract tissues of sheep and beef cattle and successfully isolated a DD-associated treponeme from ruminant rectal tissue. This gives evidence that the GI tract is an important infection reservoir of DD-associated treponemes in multiple DD-infected species.     

Characterization of novel bovine gastrointestinal tract Treponema isolates and comparison with bovine digital dermatitis treponemes

This study aimed to isolate and characterize treponemes present in the bovine gastrointestinal (GI) tract and compare them with bovine digital dermatitis (BDD) treponemes. Seven spirochete isolates were obtained from the bovine GI tract, which, on the basis of 16S rRNA gene comparisons, clustered within the genus Treponema as four novel phylotypes. One phylotype was isolated from several different GI tract regions, including the omasum, colon, rumen, and rectum. These four phylotypes could be divided into two phylotype pairs that clustered closest with each other and then with different, previously reported rumen treponemes. The treponemes displayed great genotypic and phenotypic diversity between phylotypes and differed considerably from named treponeme species and those recently reported by metagenomic studies of the bovine GI tract. Phylogenetic inference, based on comparisons of 16S rRNA sequences from only bovine treponemes, suggested a marked divergence between two important groups. The dendrogram formed two major clusters, with one cluster containing GI tract treponemes and the other containing BDD treponemes. This division among the bovine treponemes is likely the result of adaptation to different niches. To further differentiate the bovine GI and BDD strains, we designed a degenerate PCR for a gene encoding a putative virulence factor, tlyC, which gave a positive reaction only for treponemes from the BDD cluster.     

Dentilisin Activity Affects the Organization of the Outer Sheath of Treponema denticola

Prolyl-phenylalanine-specific serine protease (dentilisin) is a major extracellular protease produced by Treponema denticola. The gene, prtP, coding for the protease was recently cloned and sequenced (K. Ishihara, T. Miura, H. K. Kuramitsu, and K. Okuda, Infect. Immun. 64:5178–5186, 1996). In order to determine the role of this protease in the physiology and virulence of T. denticola, a dentilisin-deficient mutant, K1, was constructed following electroporation with a prtP-inactivated DNA fragment. No chymotrypsin-like protease activity was detected in the dentilisin-deficient mutant. In addition, the high-molecular-mass oligomeric protein characteristic of the outer sheath of the organism decreased in the mutant. Furthermore, the hydrophobicity of the mutant was decreased, and coaggregation of the mutant with Fusobacterium nucleatum was enhanced compared to that of the wild-type organism. The results obtained with a mouse abscess model system indicated that the virulence of the mutant was attenuated relative to that of the wild-type organism. These results suggest that dentilisin activity plays a major role in the structural organization of the outer sheath of T. denticola. The loss of dentilsin activity and the structural change in the outer sheath affect the pathogenicity of T. denticola.