Periodontal disease is characterized by the destruction of the hard and soft tissues comprising the periodontium. This destruction translates to a degradation of the extracellular matrices (ECM), mediated by bacterial proteases, host-derived matrix metalloproteinases (MMPs), and other proteases released by host tissues and immune cells. Bacterial pathogens interact with host tissue, triggering adverse cellular functions, including a heightened immune response, tissue destruction, and tissue migration. The oral spirochete Treponema denticola is highly associated with periodontal disease. Dentilisin, a T. denticola outer membrane protein complex, contributes to the chronic activation of pro-MMP-2 in periodontal ligament (PDL) cells and triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells. Despite these advances, no mechanism for dentilisin-induced MMP-2 activation or PDL cytopathic behaviors leading to disease is known. Here, we describe a method for purification of large amounts of the dentilisin protease complex from T. denticola and demonstrate its ability to activate MMP-2, a key regulator of periodontal tissue homeostasis. The T. denticola dentilisin and MMP-2 activation model presented here may provide new insights into the dentilisin protein and identify potential therapeutic targets for further research. Key features • This protocol builds upon a method described by Cunningham et al. [1] for selective release of Treponema outer membrane proteins. • We adapted the protocol for the purification of biologically active, detergent-stable outer membrane protein complexes from large batch cultures of T. denticola. • The protocol involves large-scale preparative electrophoresis using a Model 491 Prep Cell. • We then use gelatin zymography to demonstrate the activity of the purified dentilisin complex by its ability to activate matrix metalloproteinase 2 (MMP-2).

Dentilisin is a surface protease synthesized by the cell wall of Treponema denticola. This protein aids in the invasion of the periodontal tissue by causing infection. To identify drug molecules that have better results, homology modeling of the dentilisin protein was constructed, and molecular docking was performed with the oxazole compounds (1-6) taken from previous studies that are not yet clinically used. Data shows that compounds 1, 2, 3 show better inhibiting properties.

The oral spirochete Treponema denticola is a keystone periodontal pathogen that, in association with members of a complex polymicrobial oral biofilm, contributes to tissue damage and alveolar bone loss in periodontal disease. Virulence-associated behaviors attributed to T. denticola include disruption of the host cell extracellular matrix, tissue penetration and disruption of host cell membranes accompanied by dysregulation of host immunoregulatory factors. T. denticola dentilisin is associated with several of these behaviors. Dentilisin is an outer membrane-associated complex of acylated subtilisin-family PrtP protease and two other lipoproteins, PrcB and PrcA, that are unique to oral spirochetes. Dentilisin is encoded in a single operon consisting of prcB-prcA-prtP. We employ multiple approaches to study mechanisms of dentilisin assembly and PrtP protease activity. To determine the role of each protein in the protease complex, we have made targeted mutations throughout the protease locus, including polar and nonpolar mutations in each gene (prcB, prcA, prtP) and deletions of specific PrtP domains, including single base mutagenesis of key PrtP residues. These will facilitate distinguishing between host cell responses to dentilisin protease activity and its acyl groups. The boundaries of the divergent promoter region and the relationship between dentilisin and the adjacent iron transport operon are being resolved by incremental deletions in the sequence immediately 5\' to the protease locus. Comparison of the predicted three-dimensional structure of PrtP to that of other subtilisin-like proteases shows a unique PrtP C-terminal domain of approximately 250 residues. A survey of global gene expression in the presence or absence of protease gene expression reveals potential links between dentilisin and iron uptake and homeostasis in T. denticola. Understanding the mechanisms of dentilisin transport, assembly and activity of this unique protease complex may lead to more effective prophylactic or therapeutic treatments for periodontal disease.

Treponema denticola is a proteolytic anaerobic spirochete and key contributor to periodontal disease of microbial etiology. As periodontal disease develops and progresses, T. denticola thrives in the hostile environment of the subgingival crevice by exploiting the negative regulatory activity of the complement protein, factor H (FH). FH bound to the cell surface receptor, FhbB (FH binding protein B), is competent to serve as a cofactor for the Factor I mediated-cleavage of the opsonin C3b. However, bound FH is ultimately cleaved by the T. denticola protease, dentilisin. As the T. denticola population expands, the rate of FH cleavage may exceed its rate of replenishment leading to local FH depletion and immune dysregulation culminating in tissue and ligament destruction and tooth loss. The goal of this study was to develop a T. denticola FhbB based-vaccine antigen that can block FH binding and cleavage and kill cells via antibody-mediated bactericidal activity. Tetra (FhbB-ch4) and pentavalent fhbB (FhbB-ch5) chimerics were engineered to have attenuated FH binding ability. The chimerics were immunogenic and elicited high-titer bactericidal and agglutinating antibody. Anti-Fhb-ch4 antisera blocked FH binding and cleavage by the T. denticola protease, dentilisin, in a dose dependent manner. Precedent for the use of FH binding proteins comes from the successful development of two FDA approved vaccines for type B Neiserria meningitidis. This study is the first to extend this approach to the development of a preventive or therapeutic vaccine (or monoclonal Ab) for periodontal disease.

Objective Treponema denticola is involved in \'chronic\' periodontitis pathogenesis. The mechanism underlying the regulation of the expression of its virulence factors, such as major surface protein (Msp) and prolyl-phenylalanine specific protease (dentilisin) is yet to be clarified. We determined the gene expression profiles of Msp- and dentilisin-deficient mutants of T. denticola to identify the regulation network of gene expression concomitant with the inactivation of these virulence genes. Methods Gene expression profiles of T. denticola ATCC 35405 (wild type), dentilisin-deficient mutant K1, and msp-deficient mutant DMSP3 were determined using DNA microarray analysis and quantitative real-time reverse transcription PCR (qRT-PCR). Msp and dentilisin protein levels were determined by immunoblotting and proteolytic activity assays. Results In addition to several differentially expressed genes, dentilisin expression was reduced in DMSP3; msp expression was significantly reduced in K1 (p < 0.05), both at the gene and protein levels. To identify the regulatory system involved, the expression levels of the potential regulators whose expression showed changes in the mutants were evaluated using qRT-PCR. Transcriptional regulators TDE_0127 and TDE_0814 were upregulated in K1, and the potential repressor, TDE_0344, was elevated in DMSP3. Conclusions Dentilisin and Msp expression were interrelated, and gene expression regulators, such as TDE_0127, may be involved in their regulation.

Treponema denticola is a potent periodontal pathogen that forms a red complex with Porphyromonas gingivalis and Tannerella forsythia. It has many virulence factors, yet there are only a few reports detailing these factors. Among them, dentilisin is a well-documented surface protease. Dentilisin is reported to be involved in nutrient uptake, bacterial coaggregation, complement activation, evasion of the host immune system, inhibition of the hemostasis system, and cell invasion as a result of its action, in addition to its original proteolysis function. Therefore, characterization of dentilisin, and clarifying the relationship between T. denticola and the onset of periodontal disease will be important to better understanding this disease. In this chapter, we explain the methods for analysis of dentilisin activity and pathogenicity.

In oropharyngeal squamous cell carcinoma (OPSCC), toll-like receptors (TLR) 5 and 7 associate with the tumor\'s human papilloma virus (HPV) status (Jouhi et al., 2017). TLR 2, on the other hand, has been linked to head and neck squamous cell carcinoma (HNSCC), and to oral carcinogenesis (Farnebo et al., 2015; Binder Gallimidi et al., 2015). Here we investigated the presence of TLR 2 and 4 in HPV-positive and HPV-negative OPSCC, and their relationship to opportunistic oral pathogen Treponema denticola chymotrypsin-like protease (Td-CTLP) immunoexpression, clinical parameters, and patient outcome.
Clinicopathological data of 198 unselected consecutive OPSCC patients came from hospital registries. Immunoexpression of TLRs 2 and 4 we evaluated by immunohistochemistry, and earlier in this patient series we studied immunoexpression of Td-CTLP and HPV DNA, HPV mRNA, and p16 status.
Immunoexpression of both TLRs 2 and 4 showed a significant association with HPV-status. Strong expression was associated with HPV-positivity and mild expression with HPV-negativity. Patients with strong TLR 2 immunoexpression in the HPV negative subgroup had significantly poorer 5-year DSS (58%) than did patients with mild TLR 2 expression (77%), and strong TLR 2 immunoexpression remained as an independent factor linked to increased disease mortality in the multivariable setting (P = 0.019). No association existed between TLR 2 or 4 and Td-CTLP expression.
Our results support the role of TLR 2 receptor as a possible target for development of therapeutics as earlier proposed (Farnebo et al., 2015). The involvement of Td and other oral pathogens in carcinogenesis of OPSCC, remains open and calls for further study.

Treponema denticola is a proteolytic-anaerobic spirochete whose abundance in the subgingival crevice correlates with periodontal disease severity. Treponema denticola evades serum-mediated killing through the binding of factor H (FH), a negative regulator of the complement system. The T. denticolaFH receptor has been identified as FhbB, an 11.4kDa immunodominant lipoprotein. Three distinct subfamilies of FhbB proteins have been delineated and designated as FhbB1, FhbB2 and FhbB3. In this study we demonstrate that all FhbB variants bind human plasminogen (Plg). Competitive binding analyses revealed that FH and Plg do not compete for binding. Binding studies with FhbB135405 site-directed amino acid substitution mutants demonstrated that the interaction domains for FH and Plg on FhbB are separable. Inhibition of Plg-FhbB binding by ε-aminocaproic acid (a lysine analog) indicates that binding is mediated by electrostatic interactions that presumably occur with Lys binding sites contained within Plg \"Kringle\" domains 1, 2, 4 or 5. Similar to that demonstrated for FH, Plg can also serve as a substrate for the T. denticola protease, dentilisin. The in vivo consequences of dentilisin-mediated cleavage of Plg remained to be determined. The data presented demonstrate that FhbB is a multi-functional protein that may contribute to virulence through several mechanisms including immune evasion, manipulation of the host immune response, adherence or tissue invasion.

OBJECTIVE: In the gingival crevice, the interaction between epithelial cells and periodontopathic bacteria is important for the development of periodontitis. Treponema denticola is a major pathogen of chronic periodontitis and possesses several virulence factors, such as major surface protein (Msp) and prolyl-phenylalanine-specific protease (dentilisin). Here, we investigated the behaviours of epithelial cells infected with T. denticola by measuring the expression of interleukin (IL)-1β, IL-6, β defensin 2 (BD-2) and heat-shock protein 70 (HSP70).
METHODS: Epithelial cells were infected with T. denticola wild-type strain, Msp-deficient mutant or dentilisin-deficient mutant, and the expression levels of the above targets were analysed by polymerase chain reaction.
RESULTS: Infection with T. denticola wild-type strain and mutants induced the production of IL-6 and HSP70. The level of BD-2 induced by T. denticola wild-type strain at 24 hr was significantly higher than that of the dentilisin-deficient mutant. The level of IL-1β mRNA in the wild-type strain and dentilisin-deficient mutant was slightly lower than that in the uninfected control.
CONCLUSIONS: These results suggest that the levels of BD-2 were affected by Msp and dentilisin. This effect may contribute to the disruption of the response of epithelial cells to eradicate T. denticola.

Treponema denticola is an important periodontal pathogen capable of tissue invasion. Its chymotrypsin-like proteinase (CTLP) can degrade a number of basement membrane components in vitro, thus suggesting a contribution to tissue invasion by the spirochete. The aim of this study was to analyze the localization of CTLP in chronic periodontitis tissues ex vivo. A polyclonal antibody specific to T. denticola cell-bound CTLP was used to detect the spirochetes in the gingival tissues of patients with moderate to severe chronic periodontitis (n=25) by immunohistochemistry and periodic acid-Schiff staining (PAS). The presence of T. denticola in the periodontal tissue samples was analyzed by PCR. Periodontal tissue samples of 12 of the 25 patients were found to be positive for T. denticola by PCR. Moreover, CTLP could be detected in the periodontal tissues of all these patients by immunohistochemistry. In the epithelium, the CTLP was mostly intracellular. Typically, the positive staining could be seen throughout the whole depth of the epithelium. When detected extracellularly, CTLP was localized mainly as granular deposits. The connective tissue stained diffusely positive in four cases. The positive staining co-localized with the PAS stain in nine cases. T. denticola and its CTLP could be detected in diseased human periodontium both intra- and extracellularly. The granular staining pattern was suggestive of the presence of T. denticola bacteria, whereas the more diffused staining pattern was indicative of the recent presence of the bacterium and shedding of the cell-bound proteinase.