Periodontitis and peri-implantitis tissue levels of Treponema denticola-CTLP and its MMP-8 activating ability.

BACKGROUND AND AIMS: Chymotrypsin-like-proteinase of Treponema denticola (Td-CTLP) can stimulate the protein expression and activation of matrix metalloproteinase (MMP)-8 (or collagenase-2), a potent tissue destructive enzyme from gingival cells in vitro. The aims of this study were 1) to demonstrate the proMMP-8 (or latent MMP-8) activation by Td-CTLP in vitro and 2) to detect Td-CTLP and MMP-8 protein levels in the tissue samples of peri-implantitis and periodontitis patients. MATERIALS AND METHODS: proMMP-8 activation by Td-CTLP was analyzed by immunoblots. Tissue specimens were collected from 38 systemically healthy and non-smoking patients; 14 of whom had moderate to severe periodontitis, 10 of whom were suffering from peri-implantitis, and finally 14 of whom showed no sign of periodontal inflammation nor radiological bone decay (control group). The immune-expression levels of MMP-8 and Td-CTLP in the epithelium and the connective tissue were analyzed immunohistochemically. A pixel color-intensity analyze was performed with ImageJ software (version 1.46c; Rasband WS, National Institutes of Health, Bethesda, MD, USA) to obtain a comparable numeral score for each patient's epithelium and connective tissue MMP-8 and Td-CTLP enzyme level. RESULTS: Td-CTLP activated proMMP-8 in vitro by converting the 70-75 kDa proMMP-8 to 65 kDa active MMP-8. Also, lower molecular size 25-50 kDa parts of MMP-8 were formed. There was no statistically significant difference between the study groups in terms of their MMP-8 and Td-CTLP levels in the epithelium or in the connective tissue. CONCLUSION: Regarding the limits of this study, it can thus be said that the Td-CTLP enzyme can activate the host proMMP-8 enzyme. Tissue protein levels of MMP-8 and Td-CTLP do not seem to be changed in peri-implantitis and in periodontitis.

The Treponema denticola DgcA protein (TDE0125) is a functional diguanylate cyclase.

Periodontal disease (PD) is a progressive inflammatory condition characterized by degradation of the gingival epithelium, periodontal ligament, and alveolar bone ultimately resulting in tooth loss. Treponema denticola is a keystone periopathogen that contributes to immune dysregulation and direct tissue destruction. As periodontal disease develops, T. denticola must adapt to environmental, immunological and physiochemical changes in the subgingival crevice. Treponema denticola produces bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), an important regulatory nucleotide. While T. denticola encodes several putative diguanylate cyclases (DGCs), none have been studied and hence the biological role of c-di-GMP in oral treponemes remains largely unexplored. Here, we demonstrate that the T. denticola open reading frame, TDE0125, encodes a functional DGC designated as DgcA (Diguanylate cyclase A). The dgcA gene is universal among T. denticola isolates, highly conserved and is a stand-alone GGEEF protein with a GAF domain. Recombinant DgcA converts GTP to c-di-GMP using either manganese or magnesium under aerobic and anaerobic reaction conditions. Size exclusion chromatography revealed that DgcA exists as a homodimer and in larger oligomers. Site-directed mutagenesis of residues that define the putative inhibitory site of DgcA suggest that c-di-GMP production is allosterically regulated. This report is the first to characterize a DGC of an oral treponeme.

Multiple enzymes can make hydrogen sulfide from cysteine in Treponema denticola.

Treponema denticola is a spirochete that is involved in causing periodontal diseases. This bacterium can produce H2S from thiol compounds found in the gingival crevicular fluid. Determining how H2S is made by oral bacteria is important since this molecule is present at high levels in periodontally-diseased pockets and the biological effects of H2S can explain some of the pathologies seen in periodontitis. Thus, it is of interest to identify the enzyme, or enzymes, involved in the synthesis of H2S by T. denticola. We, and others, have previously identified and characterized a T. denticola cystalysin, called HlyA, which hydrolyzes cysteine into H2S (and pyruvate and ammonia). However, there have been no studies to show that HlyA is, or is not, the only pathway that T. denticola can use to make H2S. To address this question, allelic replacement mutagenesis was used to make a deletion mutant (ΔhlyA) in the gene encoding HlyA. The mutant produces the same amount of H2S from cysteine as do wild type spirochetes, indicating that T. denticola has at least one other enzyme that can generate H2S from cysteine. To identify candidates for this other enzyme, a BLASTp search of T. denticola strain 33520 was done. There was one gene that encoded an HlyA homolog so we named it HlyB. Recombinant His-tagged HlyB was expressed in E. coli and partially purified. This enzyme was able to make H2S from cysteine in vitro. To test the role of HlyB in vivo, an HlyB deletion mutant (ΔhlyB) was constructed in T. denticola. This mutant still made normal levels of H2S from cysteine, but a strain mutated in both hly genes (ΔhlyA ΔhlyB) synthesizes significantly less H2S from cysteine. We conclude that the HlyA and HlyB enzymes perform redundant functions in vivo and are the major contributors to H2S production in T. denticola. However, at least one other enzyme can still convert cysteine to H2S in the ΔhlyA ΔhlyB mutant. An in silico analysis that identifies candidate genes for this other enzyme is presented.

A di-iron protein recruited as an Fe[II] and oxygen sensor for bacterial chemotaxis functions by stabilizing an iron-peroxy species.

Many bacteria contain cytoplasmic chemoreceptors that lack sensor domains. Here, we demonstrate that such cytoplasmic receptors found in 8 different bacterial and archaeal phyla genetically couple to metalloproteins related to ß-lactamases and nitric oxide reductases. We show that this oxygen-binding di-iron protein (ODP) acts as a sensor for chemotactic responses to both iron and oxygen in the human pathogen Treponema denticola (Td). The ODP di-iron site binds oxygen at high affinity to reversibly form an unusually stable µ-peroxo adduct. Crystal structures of ODP from Td and the thermophile Thermotoga maritima (Tm) in the Fe[III]2-O22-, Zn[II], and apo states display differences in subunit association, conformation, and metal coordination that indicate potential mechanisms for sensing. In reconstituted systems, iron-peroxo ODP destabilizes the phosphorylated form of the receptor-coupled histidine kinase CheA, thereby providing a biochemical link between oxygen sensing and chemotaxis in diverse prokaryotes, including anaerobes of ancient origin.

Structure and Mechanism of Ergothionase from Treponema denticola.

Ergothioneine is a sulfur-containing histidine derivative that emerges from microbial biosynthesis and enters the human body through intestinal uptake and regulated distribution into specific tissues. Although the proteins involved in biosynthesis and uptake are well characterized, less is known about the degradative pathways of ergothioneine. This report describes the crystal structure of the active form of ergothionase from the oral pathogen Treponema denticola complexed with the substrate analogue desmethyl-ergothioneine sulfonic acid. This enzyme catalyzes the 1,2-elimination of trimethylamine from ergothioneine and ergothioneine sulfonic acid by using a unique mode of substrate activation combined with acid/base catalysis. This structural and mechanistic investigation revealed four essential catalytic residues, which are strictly conserved in homologous proteins from common gastrointestinal bacteria and numerous pathogenic bacteria, suggesting that bacterial activity may play an important role in determining the availability of ergothioneine in healthy and diseased human tissue.

Immunotopological Analysis of the Treponema denticola Major Surface Protein (Msp).

Treponema denticola, one of several recognized periodontal pathogens, is a model organism for studying Treponema physiology and host-microbe interactions. Its major surface protein Msp (or MOSP) comprises an oligomeric outer membrane-associated complex that binds fibronectin, has cytotoxic pore-forming activity, and disrupts several intracellular responses. There are two hypotheses regarding native Msp structure and membrane topology. One hypothesis predicts that the entire Msp protein forms a ß-barrel structure similar to that of well-studied outer membrane porins of Gram-negative bacteria. The second hypothesis predicts a bipartite Msp with distinct and separate periplasmic N-terminal and porin-like ß-barrel C-terminal domains. The bipartite model, based on bioinformatic analysis of the orthologous Treponema pallidum Tpr proteins, is supported largely by studies of recombinant TprC and Msp polypeptides. The present study reports immunological studies in both T. denticola and Escherichia coli backgrounds to identify a prominent Msp surface epitope (residues 229 to 251 in ATCC 35405) in a domain that differs between strains with otherwise highly conserved Msps. These results were then used to evaluate a series of in silico structural models of representative T. denticola Msps. The data presented here are consistent with a model of Msp as a large-diameter ß-barrel porin. This work adds to the knowledge regarding the diverse Msp-like proteins in oral treponemes and may contribute to an understanding of the evolutionary and potential functional relationships between Msps of oral Treponema and the orthologous group of Tpr proteins of T. pallidum.IMPORTANCETreponema denticola is among a small subset of the oral microbiota contributing to severe periodontal disease. Due to its relative genetic tractability, T. denticola is a model organism for studying Treponema physiology and host-microbe interactions. T. denticola Msp is a highly expressed outer membrane-associated oligomeric protein that binds fibronectin, has cytotoxic pore-forming activity, and disrupts intracellular regulatory pathways. It shares homology with the orthologous group of T. pallidum Tpr proteins, one of which is implicated in T. pallidum in vivo antigenic variation. The outer membrane topologies of both Msp and the Tpr family proteins are unresolved, with conflicting reports on protein domain localization and function. In this study, we combined empirical immunological data derived both from diverse T. denticola strains and from recombinant Msp expression in E. coli with in silico predictive structural modeling of T. denticola Msp membrane topology, to move toward resolution of this important issue in Treponema biology.

Design, Validation, and Application of an Enzyme-Coupled Hydrogen Sulfide Detection Assay.

Hydrogen sulfide (H2S) is a key metabolite in biosynthesis and is increasingly being recognized as an essential gasotransmitter. Owing to its diffusible and reactive nature, H2S can be difficult to quantify, particularly in situ. Although several detection schemes are available, they have drawbacks. In efforts to quantify sulfide release in the cross-linking reaction of the flagellar protein FlgE, we developed an enzyme-coupled sulfide detection assay using the Escherichia coli O-acetylserine sulfhydrylase enzyme CysM. Conversion of HS- to l-cysteine via CysM followed by derivatization with the thiol-specific fluorescent dye 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin enables for facile detection and quantification of H2S by fluorescent HPLC. The assay was validated by comparison to the well-established methylene blue sulfide detection assay and the robustness demonstrated by interference assays in the presence of common thiols such as glutathione, 2-mercaptoethanol, dithiothreitol, and l-methionine, as well as a range of anions. We then applied the assay to the aforementioned lysinoalanine cross-linking by the Treponema denticola flagellar hook protein FlgE. Overall, unlike previously reported H2S detection methods, the assay provides a biologically compatible platform to accurately and specifically measure hydrogen sulfide in situ, even when it is produced on long time scales.

The Treponema denticola PAS Domain-Containing Histidine Kinase Hpk2 Is a Heme Binding Sensor of Oxygen Levels.

Periodontal disease (PD) results from a shift in the composition of the microbial community of the subgingival crevice. As the bacterial population transitions from Gram-positive bacteria to predominantly Gram-negative anaerobes and spirochetes, dramatic changes occur in the physiological and immunological environment at diseased sites. Treponema denticola thrives in periodontal pockets, indicating that it has a unique ability to adapt to changing environmental conditions. Hpk2 (tde1970), a Per-Arnt-Sim motif (PAS) domain-containing histidine kinase (HK), is part of the T. denticola Hpk2-Rrp2 (tde1969) two-component regulatory (TCR) system. This TCR system is growth phase regulated and has been postulated to play a key role in adaptive responses. In this study, we employ predictive structural analyses and site-directed mutagenesis to investigate the functional role of specific amino acid residues located within the Hpk2 PAS domain. Specific substitutions impacted autophosphorylation (AP), phosphotransfer (PT), oligomerization, and hemin binding. The AP, PT, hemin binding, and oligomerization potential of some mutated Hpk2 proteins differed under aerobic versus anaerobic reaction conditions. The data presented here suggest that the regulatory activity of Hpk2 is linked to diatomic gas levels. In a broader sense, this study highlights the importance of studying proteins produced by anaerobes under conditions that approximate the environment in which they thrive.IMPORTANCE Periodontal disease affects nearly 60% of the global adult population. Its costs to individuals, and to society as a whole, are enormous. As periodontal disease develops, there is a shift in the composition of the oral microbial community. The bacteria that become dominant are able to cause significant damage to the tissues that support the teeth, leading to tooth loss. Treponema denticola is one of the keystone pathogens associated with periodontal disease. An earlier study demonstrated that the Hpk2 and Rrp2 proteins play an important role in adaptive responses. Here, we explore the role of specific Hpk2 amino acids in environmental sensing and function, using structural analyses and site-directed mutagenesis.

Treponema denticola enolase contributes to the production of antibodies against ENO1 but not to the progression of periodontitis.

Autoantibodies against alpha-enolase (ENO1) are often detected in various infectious and autoimmune diseases. Anti-ENO1 antibody titers were reported to be associated with the severity of periodontitis in patients with rheumatoid arthritis. Because the enolase of the periodontal pathogen Treponema denticola (TdEno) has the highest homology with ENO1 among the enolases of human-associated bacteria, we hypothesized that anti-ENO1 autoantibodies produced during the immune response to TdEno may contribute to the progression of periodontitis and tested it in human and mouse systems. In human subjects with healthy periodontium or chronic periodontitis, a strong positive correlation between the levels of anti-TdEno and anti-ENO1 antibodies was observed. In addition, the purified anti-TdEno antibodies recognized ENO1 as well as TdEno in a dot blot, confirming the cross-reactivity between TdEno and ENO1. However, anti-ENO1 antibody titers were not associated with the severity of periodontitis. To further investigate the role of TdEno in the production of anti-ENO1 antibodies and the progression of periodontitis, mice received an oral gavage of P. gingivalis alone, subcutaneous immunization with TdEno alone, or both P. gingivalis oral gavage and TdEno immunization. Immunization with TdEno induced not only anti-TdEno but also anti-mouse Eno1 (mEno1) antibodies and increased the expression of TNFα in the gingival tissues. However, alveolar bone loss was not increased by TdEno immunization. In conclusion, autoreactive anti-ENO1/mEno1 antibodies that are produced as byproducts during the antibody response to TdEno play a minimal role in the progression of periodontitis in the absence of rheumatoid arthritis.

Treponema denticola chymotrypsin-like protease as associated with HPV-negative oropharyngeal squamous cell carcinoma.

BACKGROUND: An opportunistic oral pathogen, Treponema denticola (Td), has been linked to orodigestive carcinogenesis, but its role in oropharyngeal squamous cell carcinoma (OPSCC) has remained open. We evaluated the presence of Td chymotrypsin-like protease (Td-CTLP) in a series of 201 unselected consecutive OPSCC patients, and the relation of the Td-CTLP to human papillomavirus (HPV) status, to expression of toll-like receptors (TLR) 5, 7, and 9, and to clinical parameters and patient outcome. METHODS: Clinicopathological data came from hospital registries. The expression of cell surface-bound Td-CTLP was evaluated by immunohistochemistry. Immunoexpression of TLRs 5, 7, and 9, and HPV status we studied earlier in this patient series. RESULTS: We detected Td-CTLP in 81% of the OPSCC, and especially in HPV-negative tumours (48% of all OPSCCs). Among the HPV-positive tumours (52% of all OPSCCs), low Td-CTLP expression associated with low TLR 5 and high TLR 7 expression. Among those HPV-negative, higher TLR 5 and lower TLR 7 expression associated with high Td-CTLP expression. Strong Td-CTLP expression associated with poor disease-specific survival, but no similar association among HPV-positive and HPV-negative subgroups emerged. CONCLUSIONS: Td-CTLP was highly expressed in OPSCC and was associated with the HPV status of tumour tissue.

Treponema denticola chymotrypsin-like proteinase may contribute to orodigestive carcinogenesis through immunomodulation.

BACKGROUND: Periodontal pathogens have been linked to oral and gastrointestinal (orodigestive) carcinogenesis. However, the exact mechanisms remain unknown. Treponema denticola (Td) is associated with severe periodontitis, a chronic inflammatory disease leading to tooth loss. The anaerobic spirochete Td is an invasive bacteria due to its major virulence factor chymotrypsin-like proteinase. Here we aimed to investigate the presence of Td chymotrypsin-like proteinase (Td-CTLP) in major orodigestive tumours and to elucidate potential mechanisms for Td to contribute to carcinogenesis. METHODS: The presence of Td-CTLP within orodigestive tumour tissues was examined using immunohistochemistry. Oral, tonsillar, and oesophageal squamous cell carcinomas, alongside gastric, pancreatic, and colon adenocarcinomas were stained with a Td-CTLP-specific antibody. Gingival tissue from periodontitis patients served as positive controls. SDS-PAGE and immunoblot were used to analyse the immumodulatory activity of Td-CTLP in vitro. RESULTS: Td-CTLP was present in majority of orodigestive tumour samples. Td-CTLP was found to convert pro MMP-8 and -9 into their active forms. In addition, Td-CTLP was able to degrade the proteinase inhibitors TIMP-1, TIMP-2, and α-1-antichymotrypsin, as well as complement C1q. CONCLUSIONS: Because of its presence within tumours and regulatory activity on proteins critical for the regulation of tumour microenvironment and inflammation, the Td-CTLP may contribute to orodigestive carcinogenesis.

Butyrate production under aerobic growth conditions by engineered Escherichia coli.

Butyrate is an important industrial platform chemical. Although several groups have reported butyrate production under oxygen-limited conditions by a native producer, Clostridium tyrobutylicum, and by a metabolically engineered Escherichia coli, efforts to produce butyrate under aerobic growth conditions have met limited success. Here, we constructed a novel butyrate synthetic pathway that functions under aerobic growth conditions in E. coli, by modifying the 1-butanol synthetic pathway reported previously. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, and endogenous thioesterase(s) of E. coli. To evaluate the potential of this pathway for butyrate production, culture conditions, including pH, oxygen supply, and concentration of inorganic nitrogen sources, were optimized in a mini-jar fermentor. Under the optimal conditions, butyrate was produced at a concentration of up to 140 mM (12.3 g/L in terms of butyric acid) after 54 h of fed-batch culture.

Treponema denticola, Porphyromonas gingivalis, and Tannerella forsythia induce cell death and release of endogenous danger signals.

OBJECTIVE: The aim of this study was to analyze whether periodontopathogens induced inflammatory cell death and the release of diverse endogenous danger molecules in THP-1-derived macrophages. METHODS: The macrophages were treated with Treponema denticola, Porphyromonas gingivalis, and Tannerella forsythia. Activation of caspase-1 and caspase-4 was detected by Western blotting. Cell death of bacteria-stimulated macrophages was examined using a lactate dehydrogenase (LDH) assay and propidium iodide (PI)/annexin V (AV) staining. Levels of endogenous danger signals, including adenosine triphosphate (ATP), uric acid, heat shock protein 60 (HSP60), high-mobility group box protein 1 (HMGB1), and fibronectin in the culture supernatants were determined using an ATP bioluminescence assay kit, a uric acid assay kit, and Western blotting, respectively. RESULTS: T. denticola, P. gingivalis, and T. forsythia induced activation of caspase-1 and caspase-4. The LDH assay and PI/AV staining showed that all three pathogens induced pyroptotic cell death. All three bacteria induced release of ATP, which is an important ligand for inflammasome activation; the increase in ATP ultimately leads to caspase-1 activation. T. denticola induced release of HSP60 and fibronectin, while T. forsythia induced release of HMGB1 in addition to HSP60 and fibronectin. None of the endogenous molecules except for fibronectin were detected in P. gingivalis-infected cells, possibly due to degradation of these factors by the proteolytic activity of the bacteria. Interestingly, P. gingivalis induced uric acid release. CONCLUSION: Inflammatory cell death and endogenous danger molecules released from cells infected with periodontopathogens may play critical roles in the pathogenesis and progression of periodontitis by augmenting immune and inflammatory responses.

Treponema denticola invasion into human gingival epithelial cells.

Host cell invasion is important for periodontal pathogens in evading host defenses and spreading into deeper areas of the periodontal tissue. Treponema denticola has been implicated in a number of potentially pathogenic processes, including periodontal tissue penetration. Here we tested the ability of T. denticola strains to invade human gingival epithelial cells (HGEC). After 2 h infection, intracellular location of T. denticola cells was confirmed by confocal laser scanning microscopy (CLSM). Results from an antibiotic protection assay following [(3)H]uridine labeling indicated that invasion efficiency reached a maximum at 2 h after infection. Internalized T. denticola cells were still observed in HGEC at 24 h by CLSM. A dentilisin deficient mutant exhibited significantly decreased invasion (p < 0.05) compared with the wild-type strain. In inhibition assays, phenylmethylsulfonyl fluoride and metabolic inhibitors such as methyl-ß-cyclodextrin and staurosporine significantly reduced T. denticola invasion. Under CLSM, T. denticola colocalized with GM-1 ganglioside-containing membrane microdomains in a cholesterol-dependent manner. These results indicated that T. denticola has the ability to invade into and survive within HGECs. Dentilisin activity of T. denticola and lipid rafts on HGEC appear to play important roles in this process.

Selenium utilization in thioredoxin and catalytic advantage provided by selenocysteine.

Thioredoxin (Trx) is a major thiol-disulfide reductase that plays a role in many biological processes, including DNA replication and redox signaling. Although selenocysteine (Sec)-containing Trxs have been identified in certain bacteria, their enzymatic properties have not been characterized. In this study, we expressed a selenoprotein Trx from Treponema denticola, an oral spirochete, in Escherichia coli and characterized this selenoenzyme and its natural cysteine (Cys) homologue using E. coli Trx1 as a positive control. (75)Se metabolic labeling and mutation analyses showed that the SECIS (Sec insertion sequence) of T. denticola selenoprotein Trx is functional in the E. coli Sec insertion system with specific selenium incorporation into the Sec residue. The selenoprotein Trx exhibited approximately 10-fold higher catalytic activity than the Sec-to-Cys version and natural Cys homologue and E. coli Trx1, suggesting that Sec confers higher catalytic activity on this thiol-disulfide reductase. Kinetic analysis also showed that the selenoprotein Trx had a 30-fold higher Km than Cys-containing homologues, suggesting that this selenoenzyme is adapted to work efficiently with high concentrations of substrate. Collectively, the results of this study support the hypothesis that selenium utilization in oxidoreductase systems is primarily due to the catalytic advantage provided by the rare amino acid, Sec.

Targeting cystalysin, a virulence factor of treponema denticola-supported periodontitis.

Cystalysin from Treponema denticola is a pyridoxal 5'-phosphate dependent lyase that catalyzes the formation of pyruvate, ammonia, and sulfide from cysteine. It is a virulence factor in adult periodontitis because its reaction contributes to hemolysis, which sustains the pathogen. Therefore, it was proposed as a potential antimicrobial target. To identify specific inhibitors by structure-based in silico methods, we first validated the crystal structure of cystalysin as a reliable starting point for the design of ligands. By using single-crystal absorption microspectrophotometry, we found that the enzyme in the crystalline state, with respect to that in solution, exhibits: 1) the same absorption spectra for the catalytic intermediates, 2) a close pKa value for the residue controlling the keto enamine ionization, and 3) similar reactivity with glycine, L-serine, L-methionine, and the nonspecific irreversible inhibitor aminoethoxyvinylglycine. Next, we screened in silico a library of 9357 compounds with the Fingerprints for Ligands and Proteins (FLAP) software, by using the three-dimensional structure of cystalysin as a template. From the library, 17 compounds were selected and experimentally evaluated by enzyme assays and spectroscopic methods. Two compounds were found to competitively inhibit recombinant T. denticola cystalysin, with inhibition constant (Ki ) values of 25 and 37 µM. One of them exhibited a minimum inhibitory concentration (MIC) value of 64 µg mL(-1) on Moraxella catarrhalis ATCC 23246, which proves its ability to cross bacterial membranes.

Oxantel disrupts polymicrobial biofilm development of periodontal pathogens.

Bacterial pathogens commonly associated with chronic periodontitis are the spirochete Treponema denticola and the Gram-negative, proteolytic species Porphyromonas gingivalis and Tannerella forsythia. These species rely on complex anaerobic respiration of amino acids, and the anthelmintic drug oxantel has been shown to inhibit fumarate reductase (Frd) activity in some pathogenic bacteria and inhibit P. gingivalis homotypic biofilm formation. Here, we demonstrate that oxantel inhibited P. gingivalis Frd activity with a 50% inhibitory concentration (IC50) of 2.2 µM and planktonic growth of T. forsythia with a MIC of 295 µM, but it had no effect on the growth of T. denticola. Oxantel treatment caused the downregulation of six P. gingivalis gene products and the upregulation of 22 gene products. All of these genes are part of a regulon controlled by heme availability. There was no large-scale change in the expression of genes encoding metabolic enzymes, indicating that P. gingivalis may be unable to overcome Frd inhibition. Oxantel disrupted the development of polymicrobial biofilms composed of P. gingivalis, T. forsythia, and T. denticola in a concentration-dependent manner. In these biofilms, all three species were inhibited to a similar degree, demonstrating the synergistic nature of biofilm formation by these species and the dependence of T. denticola on the other two species. In a murine alveolar bone loss model of periodontitis oxantel addition to the drinking water of P. gingivalis-infected mice reduced bone loss to the same level as the uninfected control.

A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochaete Treponema denticola.

Neuraminidases (sialidases) catalyse the removal of terminal sialic acid from glycoconjugates. Bacterial pathogens often utilize neuraminidases to scavenge host sialic acid, which can be utilized either as a nutrient or as a decorating molecule to disguise themselves from host immune attacks. Herein, a putative neuraminidase (TDE0471) was identified in Treponema denticola, an oral spirochaete associated with human periodontitis. TDE0471 is a cell surface-exposed exo-neuraminidase that removes sialic acid from human serum proteins; it is required for T.denticola to grow in a medium that mimics gingival crevice fluid, suggesting that the spirochaete may use sialic acid as a nutrient in vivo. TDE0471 protects T.denticola from serum killing by preventing the deposition of membrane attack complexes on the bacterial cell surface. Animal studies revealed that a TDE0471-deficient mutant is less virulent than its parental wild-type strain in BALB/C mice. However, it causes a level of tissue damage similar to the wild type in complement-deficient B6.129S4-C3(tm1) (Crr) /J mice albeit the damage caused by both bacterial strains is more severe in these transgenic mice. Based on these results, we propose that T.denticola has evolved a strategy to scavenge host sialic acid using its neuraminidase, which allows the spirochaete to acquire nutrients and evade complement killing.

Inactivation of cyclic Di-GMP binding protein TDE0214 affects the motility, biofilm formation, and virulence of Treponema denticola.

As a ubiquitous second messenger, cyclic dimeric GMP (c-di-GMP) has been studied in numerous bacteria. The oral spirochete Treponema denticola, a periodontal pathogen associated with human periodontitis, has a complex c-di-GMP signaling network. However, its function remains unexplored. In this report, a PilZ-like c-di-GMP binding protein (TDE0214) was studied to investigate the role of c-di-GMP in the spirochete. TDE0214 harbors a PilZ domain with two signature motifs: RXXXR and DXSXXG. Biochemical studies showed that TDE0214 binds c-di-GMP in a specific manner, with a dissociation constant (Kd) value of 1.73 µM, which is in the low range compared to those of other reported c-di-GMP binding proteins. To reveal the role of c-di-GMP in T. denticola, a TDE0214 deletion mutant (TdΔ214) was constructed and analyzed in detail. First, swim plate and single-cell tracking analyses showed that TdΔ214 had abnormal swimming behaviors: the mutant was less motile and reversed more frequently than the wild type. Second, we found that biofilm formation of TdΔ214 was substantially repressed (∼6.0-fold reduction). Finally, in vivo studies using a mouse skin abscess model revealed that the invasiveness and ability to induce skin abscesses and host humoral immune responses were significantly attenuated in TdΔ214, indicative of the impact that TDE0214 has on the virulence of T. denticola. Collectively, the results reported here indicate that TDE0214 plays important roles in motility, biofilm formation, and virulence of the spirochete. This report also paves a way to further unveil the roles of the c-di-GMP signaling network in the biology and pathogenicity of T. denticola.

Sequence divergence in the Treponema denticola FhbB protein and its impact on factor H binding.

Treponema denticola is an anaerobic spirochete whose abundance in the subgingival crevice correlates with the development and severity of periodontal disease. The ability of T. denticola to survive and thrive in the hostile environment of the periodontal pocket is due, at least in part, to its ability to bind factor H (FH), a negative regulator of the alternative complement pathway. The FH binding protein of T. denticola has been identified as FhbB and its atomic structure has been determined. The interaction of FH with T. denticola is unique in that FH bound to the cell surface is cleaved by the T. denticola protease, dentilisin. It has been postulated that FH cleavage by T. denticola leads to immune dysregulation in periodontal pockets. In this study, we conduct a comparative assessment of the sequence, properties, structure and ligand binding kinetics of the FhbB proteins of strains 33521 and 35405. The biological outcome of the interaction of these strains with FH could differ significantly as 33521 lacks dentilisin activity. The data presented here offer insight into our understanding of the interactions of T. denticola with the host and its potential to influence disease progression.