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Treponema denticola , a bacterium that forms a “red complex” with Porphyromonas gingivalis and Tannerella forsythia , is associated with periodontitis, pulpitis, and other oral infections. The major surface protein (Msp) is a surface glycoprotein with a relatively well-established overall domain structure (N-terminal, central and C-terminal regions) and a controversial tertiary structure. As one of the key virulence factors of T. denticola , Msp is associated with adherence, immune response, and pore formation by the microorganism. It also mediates several pathological changes in histocytes, such as cytoskeleton disruption, neutrophil phagocytosis, and phosphoinositide balance interruption. In addition, the Msp of T. denticola is also an ortholog of the Treponema pallidum repeat (Tpr) proteins and Msp or Msp-like proteins that have been detected in other oral treponeme species. This review will discuss the structure, pathogenicity and homologs of Msp produced by T. denticola , illuminate the controversy regarding the structure and membrane topology of native Msp, explore the potential roles of Msp in the mechanism of T. denticola immune escape and provide an overview of the cytotoxicity and adherence ability of Msp. Further understanding of the structure and functions of Msp will offer new insights that will help promote further investigations of the pathogenic mechanisms of T. denticola and other treponemes, leading to more effective prophylactic or therapeutic treatments for relevant diseases.

Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola.

Polymerase chain reaction (PCR) is widely regarded as the gold standard for nucleic acid analysis; however, conventional thermal cycling limits its applicability in rapid and compact analytical systems. Here, we report an oscillating-flow microfluidic PCR method that enables rapid and flexible amplification by repeatedly shuttling the reaction mixture between two fixed-temperature zones. Unlike continuous-flow PCR, the proposed approach decouples PCR cycle number from microchannel geometry, allowing programmable cycling while reducing chip footprint. To enhance analytical reliability, polymer-assisted surface passivation using polyvinylpyrrolidone was employed to suppress nonspecific adsorption in polydimethylsiloxane (PDMS) microchannels, significantly improving amplification efficiency. Using Porphyromonas gingivalis and Treponema denticola as representative periodontal pathogens, 35-cycle amplification was completed within 20 min with reliable product yield. The proposed method advances oscillating-flow PCR toward a robust analytical strategy for rapid pathogen detection and related microfluidic nucleic acid analysis.

The purpose of this study was to compare microbial profiles of saliva, pooled and site-specific subgingival samples in patients with periodontitis. We tested the hypotheses that saliva can be an alternative to pooled subgingival samples, when screening for presence of periopathogens. Site specific subgingival plaque samples (n = 54), pooled subgingival plaque samples (n = 18) and stimulated saliva samples (n = 18) were collected from 18 patients with generalized chronic periodontitis. Subgingival and salivary microbiotas were characterized by means of HOMINGS (Human Oral Microbe Identification using Next Generation Sequencing) and microbial community profiles were compared using Spearman rank correlation coefficient. Pronounced intraindividual differences were recorded in site-specific microbial profiles, and site-specific information was in general not reflected by pooled subgingival samples. Presence of Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, Filifactor alocis, Tannerella forsythia and Parvimona micra in site-specific subgingival samples were detected in saliva with an AUC of 0.79 (sensitivity: 0.61, specificity: 0.94), compared to an AUC of 0.76 (sensitivity: 0.56, specificity: 0.94) in pooled subgingival samples. Site-specific presence of periodontal pathogens was detected with comparable accuracy in stimulated saliva samples and pooled subgingival plaque samples. Consequently, saliva may be a reasonable surrogate for pooled subgingival samples when screening for presence of periopathogens. Future large-scale studies are needed to confirm findings from this study.

Porphyromonas gingivalis and Treponema denticola are strongly associated with chronic periodontitis. These bacteria have been co-localized in subgingival plaque and demonstrated to exhibit symbiosis in growth in vitro and synergistic virulence upon co-infection in animal models of disease. Here we show that during continuous co-culture a P. gingivalis:T. denticola cell ratio of 6∶1 was maintained with a respective increase of 54% and 30% in cell numbers when compared with mono-culture. Co-culture caused significant changes in global gene expression in both species with altered expression of 184 T. denticola and 134 P. gingivalis genes. P. gingivalis genes encoding a predicted thiamine biosynthesis pathway were up-regulated whilst genes involved in fatty acid biosynthesis were down-regulated. T. denticola genes encoding virulence factors including dentilisin and glycine catabolic pathways were significantly up-regulated during co-culture. Metabolic labeling using 13C-glycine showed that T. denticola rapidly metabolized this amino acid resulting in the production of acetate and lactate. P. gingivalis may be an important source of free glycine for T. denticola as mono-cultures of P. gingivalis and T. denticola were found to produce and consume free glycine, respectively; free glycine production by P. gingivalis was stimulated by T. denticola conditioned medium and glycine supplementation of T. denticola medium increased final cell density 1.7-fold. Collectively these data show P. gingivalis and T. denticola respond metabolically to the presence of each other with T. denticola displaying responses that help explain enhanced virulence of co-infections.

Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis.

The increased incidence of periodontitis, the resistance of periodontal pathogens to antibiotics, and the adverse effects of certain drugs used in general dentistry present a strong rationale for seeking safe and effective plant-based treatments for periodontitis. HPLC-DAD analysis of a commercial herbal tincture confirmed the presence of rosmarinic acid (1102.79 ± 21.56 µg/mL), luteolin-7-O-glucoside (358.06 ± 5.64 µg/mL), and isorhamnetin (24.17 ± 0.49 µg/mL), bioactive phytochemicals known for their antimicrobial and anti-inflammatoryproperties. The randomized prospective study analyzed Tinctura paradentoica® as an adjunct to anti-infectious non-surgical periodontal therapy (NSPT) on clinical and microbiological parameters in patients with moderate periodontitis (Stage II, Grade A). All 60 recruited participants were randomly allocated to either the intervention group (NSPT + Tinctura paradentoica®) or the control group (NSPT alone). The rate of prevalence of the following periodontopathogenic microorganisms (Treponema denticola, Tannerella forsythensis), assessed by polymerase chain reaction (PCR) analysis, was significantly lower in the intervention group (p < 0.001), but no statistically significant difference was found for Porphyromonas gingivalis. The herbal tincture, combined with NSPT, produces a short-term reduction in periodontal clinical parameters (Green–Vermilion plaque index, bleeding on probing index (BOP), and clinical attachment level (CAL), without clinical relevance, and the prevalence of the following bacteria species (Tannerella forsythensis, Treponema denticola).

Polymicrobial diseases are caused by combinations of multiple bacteria, which can lead to not only mild but also life-threatening illnesses. Periodontitis represents a polymicrobial disease; Porphyromonas gingivalis , Treponema denticola and Tannerella forsythia , called ‘the red complex’, have been recognized as the causative agents of periodontitis. Although molecular interactions among the three species could be responsible for progression of periodontitis, the relevant genetic mechanisms are unknown. In this study, we uncovered novel interactions in comparative genome analysis among the red complex species. Clustered regularly interspaced short palindromic repeats (CRISPRs) of T. forsythia might attack the restriction modification system of P. gingivalis , and possibly work as a defense system against DNA invasion from P. gingivalis. On the other hand, gene deficiencies were mutually compensated in metabolic pathways when the genes of all the three species were taken into account, suggesting that there are cooperative relationships among the three species. This notion was supported by the observation that each of the three species had its own virulence factors, which might facilitate persistence and manifestations of virulence of the three species. Here, we propose new mechanisms of bacterial symbiosis in periodontitis; these mechanisms consist of competitive and cooperative interactions. Our results might shed light on the pathogenesis of periodontitis and of other polymicrobial diseases.

Periodontitis is a polymicrobial infection of tooth-supporting tissues. This cross-sectional study aimed to examine the associations between five target species and severe periodontitis in a Thai population. Using the CDC/AAP case definition, individuals diagnosed with no/mild and severe periodontitis were included. Quantitative analyses of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), and Prevotella intermedia (Pi) in subgingival plaque were performed using real-time polymerase chain reaction. The association between target species and severe periodontitis was examined using logistic regression analysis. The study subjects comprised 479 individuals with no/mild periodontitis and 883 with severe periodontitis. Bacterial prevalence and quantity were higher in subjects with severe periodontitis than in those with no/mild disease. In the fully adjusted model, all species except Tf showed a dose-dependent relationship with periodontitis. The mere presence of Pg, even in low amount, was significantly associated with severe periodontitis, while the amount of Aa, Td, and Pi had to reach the critical thresholds to be significantly associated with disease. Compared to individuals with low levels of both Td and Pi, high colonization by either Td or Pi alone significantly increased the odds of having severe periodontitis by 2.5 (95%CI 1.7-3.5) folds. The odds ratio was further increased to 14.8 (95%CI 9.2-23.8) in individuals who were highly colonized by both species. Moreover, the presence of Pg and high colonization by Aa were independently associated with severe periodontitis with odds ratios of 5.6 (95%CI 3.4-9.1) and 2.2 (95%CI 1.5-3.3), respectively. Our findings suggest that the presence of Pg and high colonization by Aa, Td, and Pi play an important role in severe periodontitis in this study population. We also demonstrate for the first time that individuals co-infected with Td and Pi were more likely to have periodontitis than were those infected with a single pathogen.

Background Periodontal diseases are polymicrobial diseases that cause the inflammatory destruction of the tooth-supporting (periodontal) tissues. Their initiation is attributed to the formation of subgingival biofilms that stimulate a cascade of chronic inflammatory reactions by the affected tissue. The Gram-negative anaerobes Porphyromonas gingivalis , Tannerella forsythia and Treponema denticola are commonly found as part of the microbiota of subgingival biofilms, and they are associated with the occurrence and severity of the disease. P. gingivalis expresses several virulence factors that may support its survival, regulate its communication with other species in the biofilm, or modulate the inflammatory response of the colonized host tissue. The most prominent of these virulence factors are the gingipains, which are a set of cysteine proteinases (either Arg-specific or Lys-specific). The role of gingipains in the biofilm-forming capacity of P. gingivalis is barely investigated. Hence, this in vitro study employed a biofilm model consisting of 10 subgingival bacterial species, incorporating either a wild-type P. gingivalis strain or its derivative Lys-gingipain and Arg-gingipan isogenic mutants, in order to evaluate quantitative and qualitative changes in biofilm composition. Results Following 64h of biofilm growth, the levels of all 10 species were quantified by fluorescence in situ hybridization or immunofluorescence. The wild-type and the two gingipain-deficient P. gingivalis strains exhibited similar growth in their corresponding biofilms. Among the remaining nine species, only the numbers of T. forsythia were significantly reduced, and only when the Lys-gingipain mutant was present in the biofilm. When evaluating the structure of the biofilm by confocal laser scanning microscopy, the most prominent observation was a shift in the spatial arrangement of T. denticola , in the presence of P. gingivalis Arg-gingipain mutant. Conclusions The gingipains of P. gingivalis may qualitatively and quantitatively affect composition of polymicrobial biofilms. The present experimental model reveals interdependency between the gingipains of P. gingivalis and T. forsythia or T. denticola .