Background

The human periodontal ligament serves as a connector between the tooth and the surrounding alveolar bone proper. Its main role is to convert mechanical forces into chemical signals, which largely mediate tissue turnover through the expression of various proteases, primarily matrix metalloproteinases (MMPs) (Ho et al., 2007; Sokos et al., 2015; Takimoto et al., 2015; Jiang et al., 2016; Lin et al., 2017; Kim et al., 2020). Periodontal diseases are caused by bacterially derived factors, leading to a chronic infectious inflammatory disease that affects the periodontium and gradually destroys the tooth-supporting alveolar bone (Sela et al., 1997; Asai et al., 2003; Hayashi et al., 2010; Trindade et al., 2014; Cecil et al., 2017; Deng et al., 2017; Gao et al., 2020) (Figure 1). As periodontal disease progresses, periodontopathogenic bacteria invade deeper into the subgingival space, compromising the periodontal ligament (PDL) function and contributing to tooth loss.

Figure 1. Pathophysiology of periodontal disease

Among over 500 species of bacterium, the oral spirochete, Treponema denticola, is consistently found at significantly elevated levels in advanced lesions (Ateia et al., 2018; Solbiati and Frias-Lopez, 2018). Additionally, elevated T. denticola biofilm levels, combined with elevated MMP levels in host tissue, display robust combinatorial characteristics in predicting advanced periodontal disease severity. Thus, clinical data regarding the increased presence of T. denticola in periodontal lesions, together with basic research results involving the role of T. denticola products, suggest that it plays a pivotal role in driving periodontal disease progression. Therefore, delineation of causative mechanisms that T. denticola uses to drive host modulation may help to identify novel targets for better or alternative treatments for this chronic disease.

Conserved lipid moieties of the protease complex recognized by host receptor complexes may contribute to the activation of innate immune responses (Schenk et al., 2009). Because predominant host responses to lipoproteins are believed to be to their lipid moieties, most studies have focused on diacylated lipopeptide, Pam2CSK4, and triacylated lipopeptide, Pam3CSK4, which mimic bacterial lipoproteins for their potent immunostimulatory and osteoclastogenic activities (Schenk et al., 2009; Kim et al., 2013; Wilson and Bernstein, 2016). Recent studies have demonstrated that synthetic di- and tri-acylated lipopeptides, which preferentially activate TLR2/6 and TLR2/1-dependent pathways respectively, are sufficient to induce alveolar bone loss in mice (Kim et al., 2013; Souza et al., 2020), broadening the avenues of investigation into the role of lipoproteins underpinning the pathogenesis of periodontal disease. However, studies that utilize endogenously expressed bacterial lipopeptides are lacking.

Several proteinases and peptidases secreted by T. denticola have been identified as causative factors that likely contribute to periodontal disease pathogenesis, due to their roles in processing host tissue proteins and peptides to fulfill the nutritional requirements of these highly motile and invasive organisms (Veith et al., 2009; Ellis and Kuehn, 2010; Visser et al., 2011; Asai et al., 2003; Cecil et al., 2016). Of all T. denticola surface-expressed proteins that have been described, a chymotrypsin-like protease complex, also called dentilisin, has demonstrated a multitude of cytopathic effects consistent with periodontal disease pathogenesis, including changes in cellular adhesion activity (Bamford et al., 2007; Sano et al., 2014), degradation of various endogenous extracellular matrix–substrates (Bamford et al., 2007; Miao et al., 2011; Inagaki et al., 2016), and degradation of host chemokines and cytokines (Miyamoto et al., 2006; McDowell et al., 2012). Despite the many studies demonstrating its role at the protein level, few direct links have been reported between the activity of T. denticola ’s protease and the cellular and tissue processes driving periodontal tissue destruction. The following protocol delineates the experimental setup to study the interactions between T. denticola’s dentilisin protease and human periodontal ligament cells.

Materials and Reagents

1. 6-well clear multi-well plate (Fisher Scientific, catalog number: 25373-187)

2. 10 cm Falcon tissue culture plate (The Lab Depot, catalog number: 25373-10)

3. Minimal essential medium-α (MEM-α) (ThermoFisher Scientific, catalog number: 12571063)

4. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 14190-094)

5. Penicillin/streptomycin (P/S) (Thermo Fisher Scientific, Gibco, catalog number: 15140122)

6. Amphotericin B (Thermo Fisher Scientific, Gibco, catalog number: 15290018)

7. 0.25% trypsin with phenol red (Thermo Fisher, catalog number: 25200-05

8. Heat-inactivated fetal bovine serum (FBS) (Gibco, catalog number: 10-438-026)

9. BCA protein assay kit (Millipore Sigma, catalog number: 71285-3)

Equipment

1. 96-well SpectraMax Plus microplate reader (VWR, catalog number: 89212-396)

2. Labomed Lx400 phase contrast HD digital microscope (Microscope Central, catalog number: 9126017T-HDS)

3. SteriCycle 370 CO₂ incubator (Thermo Fisher Scientific, catalog number: TH-370N)

4. NanoDrop One Microvolume UV-Vis spectrophotometer (Fisher Scientific, catalog number: 13-400-519)

5. Bright line hemacytometer chamber (Carolina, catalog number: 700722)

6. Thermo Sorvall ST16R refrigerated centrifuge (Thermo Fisher Scientific, catalog number: 75-004-240)

Procedure