Characterization of a novel potential peptide import system in Treponema denticola.

Treponema denticola is a major etiologic agent of chronic periodontitis. On the outer sheath of T. denticola, several proteins, such as the major outer sheath protein and dentilisin were detected, and among them, a 95 kDa protein which has not yet been characterized. The aim of this study was to characterize the function of this 95 kDa protein containing gene cluster. A gene encoding this 95 kDa protein (TDE_1072) of T. denticola was inactivated by homologous recombination. We compared growth curves between the TDE_1072 mutant and wild-type strains as well as differences in gene expression by DNA microarray analysis. Differential expression of genes identified by microarray analysis was confirmed by quantitative reverse transcription-polymerase chain reaction. The proteins encoded by TDE_1072, TDE_1073, TDE_1074, TDE_1075, and TDE_1076 shared respective similarities to the substrate-binding domain (DppA) of an ABC-type dipeptide/oligopeptide/nickel transport system, and to the permease components (DppB and DppC) and ATPase components (DppD and DppF) of an ABC-type dipeptide/oligopeptide/nickel transport system. Inactivation of dppA attenuated the growth of T. denticola and dppA-dppF were co-transcribed. In contrast, expression of oppB-oppF was up-regulated in the mutant. Our findings indicate that TDE_1072 may be a potential periplasmic solute binding protein encoded by dppA that is involved in the organization of a peptide uptake system with dppB-dppF.

Litsea japonica Leaf Extract Suppresses Proinflammatory Cytokine Production in Periodontal Ligament Fibroblasts Stimulated with Oral Pathogenic Bacteria or Interleukin-1ß.

Periodontal disease, a chronic disease caused by bacterial infection, eventually progresses to severe inflammation and bone loss. Regulating excessive inflammation of inflamed periodontal tissues is critical in treating periodontal diseases. The periodontal ligament (PDL) is primarily a connective tissue attachment between the root and alveolar bone. PDL fibroblasts (PDLFs) produce pro-inflammatory cytokines in response to bacterial infection, which could further adversely affect the tissue and cause bone loss. In this study, we determined the ability of Litsea japonica leaf extract (LJLE) to inhibit pro-inflammatory cytokine production in PDLFs in response to various stimulants. First, we found that LJLE treatment reduced lipopolysaccharide (LPS)-induced pro-inflammatory cytokine (interleukin-6 and interleukin-8) mRNA and protein expression in PDLFs without cytotoxicity. Next, we observed the anti-inflammatory effect of LJLE in PDLFs after infection with various oral bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. These anti-inflammatory effects of LJLE were dose-dependent, and the extract was effective following both pretreatment and posttreatment. Moreover, we found that LJLE suppressed the effect of interleukin-1 beta-induced pro-inflammatory cytokine production in PDLFs. Taken together, these results indicate that LJLE has anti-inflammatory activity that could be exploited to prevent and treat human periodontitis by controlling inflammation.

Periodontal pathogens invade gingiva and aortic adventitia and elicit inflammasome activation in αvß6 integrin-deficient mice.

The American Heart Association supports an association between periodontal diseases and atherosclerosis but not a causal association. This study explores the use of the integrin ß6(-/-) mouse model to study the causality. We investigated the ability of a polymicrobial consortium of Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum to colonize the periodontium and induce local and systemic inflammatory responses. Polymicrobially infected Itgß6(-/-) mice demonstrate greater susceptibility to gingival colonization/infection, with severe gingival inflammation, apical migration of the junctional epithelium, periodontal pocket formation, alveolar bone resorption, osteoclast activation, bacterial invasion of the gingiva, a greater propensity for the bacteria to disseminate hematogenously, and a strong splenic T cell cytokine response. Levels of atherosclerosis risk factors, including serum nitric oxide, oxidized low-density lipoprotein, serum amyloid A, and lipid peroxidation, were significantly altered by polybacterial infection, demonstrating an enhanced potential for atherosclerotic plaque progression. Aortic gene expression revealed significant alterations in specific Toll-like receptor (TLR) and nucleotide-binding domain- and leucine-rich-repeat-containing receptor (NLR) pathway genes in response to periodontal bacterial infection. Histomorphometry of the aorta demonstrated larger atherosclerotic plaques in Itgß6(-/-) mice than in wild-type (WT) mice but no significant difference in atherosclerotic plaque size between mice with polybacterial infection and mice with sham infection. Fluorescence in situ hybridization demonstrated active invasion of the aortic adventitial layer by P. gingivalis. Our observations suggest that polybacterial infection elicits distinct aortic TLR and inflammasome signaling and significantly increases local aortic oxidative stress. These results are the first to demonstrate the mechanism of the host aortic inflammatory response induced by polymicrobial infection with well-characterized periodontal pathogens.

Deficiency of cathepsin K prevents inflammation and bone erosion in rheumatoid arthritis and periodontitis and reveals its shared osteoimmune role.

Using rheumatoid arthritis (RA) and periodontitis mouse models, we demonstrate that RA and periodontitis share many pathological features, such as deregulated cytokine production, increased immune-cell infiltration, increased expression of Toll-like receptors (TLRs), and enhanced osteoclast activity and bone erosion. We reveal that genetic deletion of cathepsin K (Ctsk) caused a radical reduction in inflammation and bone erosion within RA joint capsules and periodontal lesions, a drastic decrease in immune-cell infiltration, and a significant reduction in osteoclasts, macrophages, dendritic and T-cells. Deficiency of Ctsk greatly decreased the expression of TLR-4, 5, and 9 and their downstream cytokines in periodontal gingival epithelial lesions and synovial RA lesions. Hence, Ctsk may be targeted to treat RA and periodontitis simultaneously due to its shared osteoimmune role.

Characterization of Treponema denticola mutants defective in the major antigenic proteins, Msp and TmpC.

Treponema denticola, a gram-negative and anaerobic spirochete, is associated with advancing severity of chronic periodontitis. In this study, we confirmed that two major antigenic proteins were Msp and TmpC, and examined their physiological and pathological roles using gene-deletion mutants. Msp formed a large complex that localized to the outer membrane, while TmpC existed as a monomer and largely localized to the inner membrane. However, TmpC was also detected in the outer membrane fraction, but its cell-surface exposure was not detected. Msp defects increased cell-surface hydrophobicity and secretion of TNF-α from macrophage-like cells, whereas TmpC defects decreased autoagglutination and chymotrypsin-like protease activities. Both mutants adhered to gingival epithelial cells similarly to the wild-type and showed slightly decreased motility. In addition, in Msp-defective mutants, the TDE1072 protein, which is a major membrane protein, was abolished; therefore, phenotypic changes in the mutant can be, at least in part, attributed to the loss of the TDE1072 protein. Thus, the major antigenic proteins, Msp and TmpC, have significant and diverse impacts on the characteristics of T. denticola, especially cell surface properties.

Early bacterial colonization and soft tissue health around zirconia and titanium abutments: an in vivo study in man.

AIM: To compare the early bacterial colonization and soft tissue health of mucosa adjacent to zirconia (ZrO(2)) and titanium (Ti) abutment surfaces in vivo. MATERIALS AND METHODS: Twenty edentulous subjects received two endosseous mandibular implants. The implants were fitted with either a ZrO(2) or a Ti abutment (non-submerged implant placement, within-subject comparison, left-right randomization). Sulcular bacterial sampling and the assessment of probing pocket depth, recession and bleeding on probing were performed at 2 weeks and 3 months post-surgery. Wilcoxon matched-pairs, sign-rank tests were applied to test differences in the counts of seven marker bacteria and the clinical parameters that were associated with the ZrO(2) and Ti abutments, at the two observation time points. RESULTS: ZrO(2) and Ti abutments harboured similar counts of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Peptostreptococcus micros, Fusobacterium nucleatum and Treponema denticola at 2 weeks and 3 months. Healthy clinical conditions were seen around both ZrO(2) and Ti abutments at all times, without significant differences in most clinical parameters of peri-implant soft tissue health. Mean probing depths around Ti abutments were slightly deeper than around ZrO(2) abutments after 3 months (2.2 SD 0.8 mm vs. 1.7 SD 0.7 mm, P=0.03). CONCLUSIONS: No difference in health of the soft tissues adjacent to ZrO(2) and Ti abutment surfaces or in early bacterial colonization could be demonstrated, although somewhat shallower probing depths were observed around ZrO(2) abutments after 3 month.

Mixture of periodontopathogenic bacteria influences interaction with KB cells.

INTRODUCTION: The purpose of this study was to investigate the adhesion and invasion of periodontopathogenic bacteria in varied mixed infections and the release of interleukins from an epithelial cell line (KB cells). METHODS: KB cells were co-cultured with Porphyromonas gingivalis ATCC 33277 and M5-1-2, Tannerella forsythia ATCC 43037, Treponema denticola ATCC 35405 and Fusobacterium nucleatum ATCC 25586 in single and mixed infections. The numbers of adherent and internalized bacteria were determined up to 18 h after bacterial exposure. Additionally, the mRNA expression and concentrations of released interleukin (IL)-6 and IL-8 were measured. RESULTS: All periodontopathogenic bacteria adhered and internalized in different numbers to KB cells, but individually without any evidence of co-aggregation also to F. nucleatum. High levels of epithelial mRNA of IL-6 and IL-8 were detectable after all bacterial challenges. After the mixed infection of P. gingivalis ATCC 33277 and F. nucleatum ATCC 25586 the highest levels of released interleukins were found. No IL-6 and IL-8 were detectable after the mixed infection of P. gingivalis M5-1-2 and F. nucleatum ATCC 25586 and the fourfold infection of P. gingivalis ATCC 33277, T. denticola ATCC 35405, T. forsythia ATCC 43037 and F. nucleatum ATCC 25586. CONCLUSION: Anaerobic periodontopathogenic bacteria promote the release of IL-6 and IL-8 by epithelial cells. Despite a continuous epithelial expression of IL-8 mRNA by all bacterial infections these effects are temporary because of the time-dependent degradation of cytokines by bacterial proteases. Mixed infections have a stronger virulence potential than single bacteria. Further research is necessary to evaluate the role of mixed infections and biofilms in the pathogenesis of periodontitis.

Treponema denticola suppresses expression of human beta-defensin-2 in gingival epithelial cells through inhibition of TNFalpha production and TLR2 activation.

We previously reported that Treponema denticola, a periodontal pathogen, suppressed the expression of human beta-defensins (HBDs) and IL-8 in human gingival epithelial cells. To clarify the receptor(s) involved in the suppression of HBD-2, immortalized gingival epithelial (HOK-16B) cells were infected with live or heat-killed T. denticola for 24 h, and the expression of HBD-2 was examined by real-time RT-PCR. Live T. denticola, but not heat-killed bacteria, suppressed the expression of HBD-2 about 40%. Time courses of suppression revealed that T. denticola suppressed HBD-2 expression only at late time points, which was accompanied with the suppression of TNFalpha production. Neutralization of TNFalpha with an antibody abrogated the suppressive effect of T. denticola on HBD-2. Accordingly, heat-killed T. denticola did not suppress TNFalpha production. Knock-down of toll-like receptor (TLR) 2 via RNA interference reversed the suppressive effect of T. denticola on the expression of HBD-3, but not on the production of TNFalpha. Collectively, T. denticola suppresses the expression of HBD-2 in gingival epithelial cells by inhibiting the TLR2 axis and TNFalpha production, which may contribute to the pathogenesis of periodontitis by T. denticola.

Persistence of extracrevicular bacterial reservoirs after treatment of aggressive periodontitis.

BACKGROUND: The purpose of this study was to test the hypothesis that periodontal pathogens associated with aggressive periodontitis persist in extracrevicular locations following scaling and root planing, systemic antibiotics, and antimicrobial rinses. METHODS: Eighteen patients with aggressive periodontitis received a clinical examination during which samples of subgingival plaque and buccal epithelial cells were obtained. Treatment consisted of full-mouth root planing, systemic antibiotics, and chlorhexidine rinses. Clinical measurements and sampling were repeated at 3 and 6 months. Quantitative polymerase chain reaction determined the number of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia (previously T. forsythensis), and Treponema denticola in the plaque. Fluorescence in situ hybridization and confocal microscopy determined the extent of intracellular invasion in epithelial cells. RESULTS: Clinical measurements improved significantly following treatment. All bacterial species except P. gingivalis were significantly reduced in plaque between baseline and 3 months. However, all species showed a trend to repopulate between 3 and 6 months. This increase was statistically significant for log T. denticola counts. All species were detected intracellularly. The percentage of cells infected intracellularly was not affected by therapy. CONCLUSIONS: The 6-month increasing trend in the levels of plaque bacteria suggests that subgingival recolonization was occurring. Because the presence of these species within epithelial cells was not altered after treatment, it is plausible that recolonization may occur from the oral mucosa. Systemic antibiotics and topical chlorhexidine did not reduce the percentage of invaded epithelial cells. These data support the hypothesis that extracrevicular reservoirs of bacteria exist, which might contribute to recurrent or refractory disease in some patients.

Novel mechanism for conditional aerobic growth of the anaerobic bacterium Treponema denticola.

Treponema denticola, a periodontal pathogen, has recently been shown to exhibit properties of a facultative anaerobic spirochete, in contrast to its previous recognition as an obligate anaerobic bacterium. In this study, the capacity and possible mechanism of T. denticola survival and growth under aerobic conditions were investigated. Factors detrimental to the growth of T. denticola ATCC 33405, such as oxygen concentration and hydrogen sulfide (H(2)S) levels as well as the enzyme activities of gamma-glutamyltransferase, cysteinylglycinase, and cystalysin associated with the cells were monitored. The results demonstrated that T. denticola grew only at deeper levels of broth (>or=3 ml in a 10-ml tube), high inoculation ratios (>or=20% of culture in medium), and short cultivation times (

The chymotrypsin-like protease complex of Treponema denticola ATCC 35405 mediates fibrinogen adherence and degradation.

Treponema denticola is an anaerobic spirochete strongly associated with human periodontal disease. T. denticola bacteria interact with a range of host tissue proteins, including fibronectin, laminin, and fibrinogen. The latter localizes in the extracellular matrix where tissue damage has occurred, and interactions with fibrinogen may play a key role in T. denticola colonization of the damaged sites. T. denticola ATCC 35405 showed saturable binding of fluid-phase fibrinogen to the cell surface and saturable adherence to immobilized fibrinogen. Levels of fibrinogen binding were enhanced in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride. The Aalpha and Bbeta chains of fibrinogen, but not the gamma chains, were specifically recognized by T. denticola. Following fibrinogen affinity chromatography analysis of cell surface extracts, a major fibrinogen-binding component (polypeptide molecular mass, approximately 100 kDa), which also degraded fibrinogen, was purified. Upon heating at 100 degrees C, the polypeptide was dissociated into three components (apparent molecular masses, 80, 48, and 45 kDa) that did not individually bind or degrade fibrinogen. The native 100-kDa polypeptide complex was identified as chymotrypsin-like protease (CTLP), or dentilisin. In an isogenic CTLP(-) mutant strain, CKE, chymotrypsin-like activity was reduced >90% compared to that in the wild type and fibrinogen binding and hydrolysis were ablated. Isogenic mutant strain MHE, deficient in the production of Msp (major surface protein), showed levels of CTLP reduced 40% relative to those in the wild type and exhibited correspondingly reduced levels of fibrinogen binding and proteolysis. Thrombin clotting times in the presence of wild-type T. denticola cells, but not strain CKE (CTLP(-)) cells, were extended. These results suggest that interactions of T. denticola with fibrinogen, which may promote colonization and modulate hemostasis, are mediated principally by CTLP.

Synergistic biofilm formation by Treponema denticola and Porphyromonas gingivalis.

Biofilm formation is an important step in the etiology of periodontal diseases. In this study, in vitro biofilm formation by Treponema denticola and Porphyromonas gingivalis 381 displayed synergistic effects. Confocal microscopy demonstrated that P. gingivalis attaches to the substratum first as a primary colonizer followed by coaggregation with T. denticola to form a mixed biofilm. The T. denticola flagella mutant as well as the cytoplasmic filament mutant were shown to be essential for biofilm formation as well as coaggregation with P. gingivalis. The major fimbriae and Arg-gingipain B of P. gingivalis also play important roles in biofilm formation with T. denticola.