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IgA nephropathy (IgAN) has been considered to have a relationship with infection in the tonsil, because IgAN patients often manifest macro hematuria just after tonsillitis. In terms of oral-area infection, the red complex of periodontal bacteria ( Porphyromonas gingivalis ( P. gingivalis ), Treponema denticol (T. denticola) and Tannerella forsythia (T. forsythia) ) is important, but the relationship between these bacteria and IgAN remains unknown. In this study, the prevalence of the red complex of periodontal bacteria in tonsil was compared between IgAN and tonsillitis patients. The pathogenicity of IgAN induced by P. gingivalis was confirmed by the mice model treated with this bacterium. The prevalence of P. gingivalis and T. forsythia in IgAN patients was significantly higher than that in tonsillitis patients ( p < 0.001 and p < 0.05, respectively). A total of 92% of tonsillitis patients were free from red complex bacteria, while only 48% of IgAN patients had any of these bacteria. Nasal administration of P. gingivalis in mice caused mesangial proliferation ( p < 0.05 at days 28a nd 42; p < 0.01 at days 14 and 56) and IgA deposition ( p < 0.001 at day 42 and 56 after administration). Scanning-electron-microscopic observation revealed that a high-density Electron-Dense Deposit was widely distributed in the mesangial region in the mice kidneys treated with P. gingivalis . These findings suggest that P. gingivalis is involved in the pathogenesis of IgAN.

Tannerella forsythia is a Gram-negative oral pathogen known to possess an O-glycosylation system responsible for targeting multiple proteins associated with virulence at the three-residue motif (D)(S/T)(A/I/L/V/M/T). Multiple proteins have been identified to be decorated with a decasaccharide glycan composed of a poorly defined core plus a partially characterized species-specific section. To date, glycosylation studies have focused mainly on the two S-layer glycoproteins, TfsA and TfsB, so the true extent of glycosylation within this species has not been fully explored. In the present study, we characterize the glycoproteome of T. forsythia by employing FAIMS-based glycopeptide enrichment of a cell membrane fraction. We demonstrate that at least 13 glycans are utilized within the T. forsythia glycoproteome, varying with respect to the presence of the three terminal sugars and the presence of fucose and digitoxose residues at the reducing end. To improve the localization of glycosylation events and enhance the detection of glycopeptides, we utilized trifluoromethanesulfonic acid treatment to allow the selective chemical cleavage of glycans. Reducing the chemical complexity of glycopeptides dramatically improved the number of glycopeptides identified and our ability to localize glycosylation sites by ETD fragmentation, leading to the identification of 312 putative glycosylation sites in 145 glycoproteins. Glycosylation site analysis revealed that glycosylation occurs on a much broader motif than initially reported, with glycosylation found at (D)(S/T)(A/I/L/V/M/T/S/C/G/F). The prevalence of this broader glycosylation motif in the genome suggests the existence of hundreds of potential O-glycoproteins in this organism. IMPORTANCE Tannerella forsythia is an oral pathogen associated with severe forms of periodontal disease characterized by destruction of the tooth's supporting tissues, including the bone. The bacterium releases a variety of proteins associated with virulence on the surface of outer membrane vesicles. There is evidence that these proteins are modified by glycosylation, and this modification is essential for virulence in producing disease. We have utilized novel techniques coupled with mass spectrometry to identify over 13 glycans and 312 putative glycosylation sites in 145 glycoproteins within T. forsythia . Glycosylation site analysis revealed that this modification occurs on a much broader motif than initially reported such that there is a high prevalence of potential glycoproteins in this organism that may help to explain its role in periodontal disease.

Background: High levels of periodontopathic bacteria as well as Streptococcus anginosus were detected in cancer tissue from patients with esophageal cancer. An association between oral infectious bacteria and esophageal cancer has been reported., Methods: Characteristics of the oral microbiota and periodontal conditions were studied as clinicopathologic factors in patients with esophageal cancer. The study included 61 patients with esophageal cancer and 62 matched individuals without any cancers. Samples of subgingival dental plaque and unstimulated saliva were collected to evaluate the prevalence and abundance of the following oral bacteria using a real-time polymerase chain reaction assay: Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema denticola, and S. anginosus., Results: In the cancer group, the prevalence of all bacteria, with the exception of F. nucleatum, in dental plaque; the prevalence of A. actinomycetemcomitans in saliva; the abundance of all bacteria, with the exception of F. nucleatum and P. intermedia, in dental plaque; and the abundance of A. actinomycetemcomitans and S. anginosus in saliva were significantly higher. Furthermore, a logistic regression analysis suggested that the prevalence of T. forsythia and S. anginosus in dental plaque and of A. actinomycetemcomitans in saliva, as well as a drinking habit, were associated with a high risk of esophageal cancer, with a high odds ratio., Conclusions: The current findings have potential implications for the early diagnosis of esophageal cancer., (© 2020 American Cancer Society.)

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded β-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases., Competing Interests: Conflict of interest N. T. is employed by PerioTrap Pharmaceuticals GmbH, of which J.-U. R., M. B., S. S., and H.-U. D. are shareholders., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Recent advances in the next-generation sequencing (NGS) allowed the metagenomic analyses of DNA from many different environments and sources, including thousands of years old skeletal remains. It has been shown that most of the DNA extracted from ancient samples is microbial. There are several reports demonstrating that the considerable fraction of extracted DNA belonged to the bacteria accompanying the studied individuals before their death., Results: In this study we scanned 344 microbiomes from 1000- and 2000- year-old human teeth. The datasets originated from our previous studies on human ancient DNA (aDNA) and on microbial DNA accompanying human remains. We previously noticed that in many samples infection-related species have been identified, among them Tannerella forsythia, one of the most prevalent oral human pathogens. Samples containing sufficient amount of T. forsythia aDNA for a complete genome assembly were selected for thorough analyses. We confirmed that the T. forsythia-containing samples have higher amounts of the periodontitis-associated species than the control samples. Despites, other pathogens-derived aDNA was found in the tested samples it was too fragmented and damaged to allow any reasonable reconstruction of these bacteria genomes. The anthropological examination of ancient skulls from which the T. forsythia-containing samples were obtained revealed the pathogenic alveolar bone loss in tooth areas characteristic for advanced periodontitis. Finally, we analyzed the genetic material of ancient T. forsythia strains. As a result, we assembled four ancient T. forsythia genomes - one 2000- and three 1000- year-old. Their comparison with contemporary T. forsythia genomes revealed a lower genetic diversity within the four ancient strains than within contemporary strains. We also investigated the genes of T. forsythia virulence factors and found that several of them (KLIKK protease and bspA genes) differ significantly between ancient and modern bacteria., Conclusions: In summary, we showed that NGS screening of the ancient human microbiome is a valid approach for the identification of disease-associated microbes. Following this protocol, we provided a new set of information on the emergence, evolution and virulence factors of T. forsythia, the member of the oral dysbiotic microbiome.

Periodontal disease is a microbially-mediated inflammatory disease of tooth-supporting tissues that leads to bone and tissue loss around teeth. Although bacterially-mediated mechanisms of alveolar bone destruction have been widely studied, the effects of a polymicrobial infection on the periodontal ligament and microbiome/virome have not been well explored. Therefore, the current investigation introduced a new mouse model of periodontal disease to examine the effects of a polymicrobial infection on periodontal ligament (PDL) properties, changes in bone loss, the host immune response, and the microbiome/virome using shotgun sequencing. Periodontal pathogens, namely Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum were used as the polymicrobial oral inoculum in BALB/cByJ mice. The polymicrobial infection triggered significant alveolar bone loss, a heightened antibody response, an elevated cytokine immune response, a significant shift in viral diversity and virome composition, and a widening of the PDL space; the latter two findings have not been previously reported in periodontal disease models. Changes in the PDL space were present at sites far away from the site of insult, indicating that the polymicrobial radius of effect extends beyond the bone loss areas and site of initial infection and wider than previously appreciated. Associations were found between bone loss, specific viral and bacterial species, immune genes, and PDL space changes. These findings may have significant implications for the pathogenesis of periodontal disease and biomechanical properties of the periodontium. This new polymicrobial mouse model of periodontal disease in a common mouse strain is useful for evaluating the features of periodontal disease.

Background: Tannerella forsythia is a bacterial pathogen implicated in periodontal disease. Numerous virulence-associated T. forsythia genes have been described, however, it is necessary to expand the knowledge on T. forsythia's genome structure and genetic repertoire to further elucidate its role within pathogenesis. Tannerella sp. BU063, a putative periodontal health-associated sister taxon and closest known relative to T. forsythia is available for comparative analyses. In the past, strain confusion involving the T. forsythia reference type strain ATCC 43037 led to discrepancies between results obtained from in silico analyses and wet-lab experimentation., Results: We generated a substantially improved genome assembly of T. forsythia ATCC 43037 covering 99% of the genome in three sequences. Using annotated genomes of ten Tannerella strains we established a soft core genome encompassing 2108 genes, based on orthologs present in > = 80% of the strains analysed. We used a set of known and hypothetical virulence factors for comparisons in pathogenic strains and the putative periodontal health-associated isolate Tannerella sp. BU063 to identify candidate genes promoting T. forsythia's pathogenesis. Searching for pathogenicity islands we detected 38 candidate regions in the T. forsythia genome. Only four of these regions corresponded to previously described pathogenicity islands. While the general protein O-glycosylation gene cluster of T. forsythia ATCC 43037 has been described previously, genes required for the initiation of glycan synthesis are yet to be discovered. We found six putative glycosylation loci which were only partially conserved in other bacteria. Lastly, we performed a comparative analysis of translational bias in T. forsythia and Tannerella sp. BU063 and detected highly biased genes., Conclusions: We provide resources and important information on the genomes of Tannerella strains. Comparative analyses enabled us to assess the suitability of T. forsythia virulence factors as therapeutic targets and to suggest novel putative virulence factors. Further, we report on gene loci that should be addressed in the context of elucidating T. forsythia's protein O-glycosylation pathway. In summary, our work paves the way for further molecular dissection of T. forsythia biology in general and virulence of this species in particular.

Interleukin-24 is a pleiotropic immunoregulatory cytokine and a member of the IL-20R subfamily of the IL-10 family. The aim of this study was to investigate the regulation of IL-24 in the human oral keratinocyte cell line HOK-16B following infection with Tannerella forsythia, a major periodontal pathogen. T. forsythia induced the expression of IL-24 mRNA and the secretion of glycosylated IL-24 in HOK-16B cells. Glycosylation of IL-24 is linked to its solubility and bioavailability. T. forsythia-stimulated reactive oxygen species (ROS) induced the expression of IL-24, which was regulated by IL-6. The ROS inhibitor N-acetylcysteine and MAPK inhibitors significantly reduced the expression of IL-6 and IL-24 induced by T. forsythia. Recombinant human IL-24 significantly enhanced the expression of IL-1α, IL-8, CXCL10, and MCP-1 in HOK-16B cells. Together, these results indicate that ROS, MAPKs, and IL-6 comprise the axis of IL-24 expression in HOK-16B cells stimulated with T. forsythia. Thus, IL-24 may be involved in inflammation in oral keratinocytes., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by degradation of complement system proteins and by inactivation of the antimicrobial peptide LL-37 by proteolytic cleavage. This makes karilysin a highly interesting virulence factor to study in the framework of drug development and diagnostics. However, to date the presence of karilysin in clinical samples has not been demonstrated due to the lack of specific probes. In the present work, a high titer and stable affinity-purified avian IgY antibody against karilysin was developed. By surface plasmon resonance imaging the IgY affinity was found to be in the low nanomolar range. The antibody could be used to detect karilysin in saliva samples by immuno-blotting and was specific when tested towards human MMP-3. Furthermore, an avian IgY-based immunoassay was developed, which demonstrated low intra- and interday assay variability (CV's below 10%). Application of the immunoassay on a well-characterized set of saliva samples from adolescents with or without signs of periodontitis showed that it was possible to detect karilysin in saliva. A significant difference in karilysin concentration was found between saliva from participants with signs of periodontitis and saliva from healthy controls (p = .0024). The median of karilysin levels among periodontitis cases was 957 pg/ml (IQR, 499-2132 pg/ml) and the median for controls was 569 pg/ml (IQR, 210-1343 pg/ml). Collectively our data confirm the presence of karilysin in clinical samples. The described IgY-based immunoassay may prove useful as part of protein-based biomarker screenings in the clinic or in point-of care settings., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aim: To determine if bacteria associated with persistent apical periodontitis induce species-specific pro-inflammatory cytokine responses in macrophages, and the effects of this species-specific microenvironment on osteogenic differentiation., Methodology: Macrophages were exposed to Enterococcus faecalis, Streptococcus oralis, Streptococcus mitis, Fusobacterium nucleatum, Treponema denticola or Tannerella forsythia, and levels of TNF-α and IL-1β elicited were determined by immunoassay. Following treatment of MG-63 pre-osteoblasts with conditioned media from bacteria-exposed macrophages, osteogenic differentiation and viability of osteoblasts were analyzed by Alizarin Red Staining and MTS assay, respectively. Statistical analysis was carried out by one-way anova with the Tukey post-hoc test. Differences were considered to be significant if P < 0.05., Results: Macrophages exposed to Gram-positive bacteria did not produce significant amounts of cytokines. F. nucleatum-challenged macrophages produced up to four-fold more TNF-α and IL-1β compared to T. denticola or T. forsythia. Only conditioned media from macrophages treated with Gram-negative bacteria decreased mineralization and viability of osteoblasts., Conclusions: Gram-positive bacteria did not impact osteogenic differentiation and appeared innocuous. Gram-negative bacteria, in particular F. nucleatum elicited an enhanced pro-inflammatory response in macrophages, inhibited osteogenic differentiation and reduced cell viability. The findings suggest that the presence of this organism could potentially increase the severity of persistent apical periodontitis., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Porphyromonas gingivalis is considered the principal etiologic agent and keystone pathogen of chronic periodontitis. As an auxotrophic bacterium, it must acquire heme to survive and multiply at the infection site. P. gingivalis HmuY is the first member of a novel family of hemophore-like proteins. Bacterial heme-binding proteins usually use histidine-methionine or histidine-tyrosine residues to ligate heme-iron, whereas P. gingivalis HmuY uses two histidine residues. We hypothesized that other 'red complex' members, i.e. Tannerella forsythia and Treponema denticola might utilize similar heme uptake mechanisms to the P. gingivalis HmuY. Comparative and phylogenetic analyses suggested differentiation of HmuY homologs and low conservation of heme-coordinating histidine residues present in HmuY. The homologs were subjected to duplication before divergence of Bacteroidetes lineages, which could facilitate evolution of functional diversification. We found that T. denticola does not code an HmuY homolog. T. forsythia protein, termed as Tfo, binds heme, but preferentially in the ferrous form, and sequesters heme from the albumin-heme complex under reducing conditions. In agreement with that, the 3D structure of Tfo differs from that of HmuY in the folding of heme-binding pocket, containing two methionine residues instead of two histidine residues coordinating heme in HmuY. Heme binding to apo-HmuY is accompanied by movement of the loop carrying the His 166 residue, closing the heme-binding pocket. Molecular dynamics simulations (MD) demonstrated that this conformational change also occurs in Tfo. In conclusion, our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme-binding properties., (© 2018 The Author(s).)

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.

The periodontal pathogen Tannerella forsythia has the unique ability to produce methylglyoxal (MGO), an electrophilic compound which can covalently modify amino acid side chains and generate inflammatory adducts known as advanced glycation endproducts (AGEs). In periodontitis, concentrations of MGO in gingival-crevicular fluid are increased and are correlated with the T. forsythia load. However, the source of MGO and the extent to which MGO may contribute to periodontal inflammation has not been fully explored. In this study we identified a functional homolog of the enzyme methylglyoxal synthase (MgsA) involved in the production of MGO in T. forsythia. While wild-type T.forsythia produced a significant amount of MGO in the medium, a mutant lacking this homolog produced little to no MGO. Furthermore, compared with the spent medium of the T. forsythia parental strain, the spent medium of the T. forsythia mgsA-deletion strain induced significantly lower nuclear factor-kappa B activity as well as proinflammogenic and pro-osteoclastogenic cytokines from THP-1 monocytes. The ability of T. forsythia to induce protein glycation endproducts via MGO was confirmed by an electrophoresis-based collagen chain mobility shift assay. Together these data demonstrated that T. forsythia produces MGO, which may contribute to inflammation via the generation of AGEs and thus act as a potential virulence factor of the bacterium., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Porphyromonas gingivalis and Tannerella forsythia have been thought to be associated with periodontitis; however comprehensive histopathological localization of bacteria in affected human periodontal tissues is not well documented. In the present study, we examined formalin-fixed paraffin-embedded gingival and subgingival granulation tissues from 71 patients with chronic periodontitis and 11 patients with aggressive periodontitis, using immunohistochemistry with novel monoclonal antibodies specific to P. gingivalis or T. forsythia, together with quantitative real-time polymerase chain reaction for each bacterial DNA. Immunohistochemisty revealed both bacterial species extracellularly, as aggregates or within bacterial plaque, and intracellularly in stromal inflammatory cells, squamous epithelium, and capillary endothelium of granulation tissue. Combined analysis with the results from polymerase chain reaction suggested that localization and density of T. forsythia is closely associated with those of P. gingivalis, and that bacterial density is a factor responsible for the cell-invasiveness and tissue-invasiveness of these periodontal bacteria. Detection of these bacteria in the capillary endothelium in some samples suggested possible bacterial translocation into the systemic circulation from inflamed gingival and subgingival granulation tissues. Immunohistochemistry with the novel antibodies showed high specificity and sensitivity, and can be used to locate these periodontal bacteria in routinely-used formalin-fixed paraffin-embedded human tissue sections from systemic locations.

Porphyromonas gingivalis and Tannerella forsythia secrete proteases, gingipains and KLIKK-proteases. In addition, T. forsythia produces a serpin (miropin) with broad inhibitory spectrum. The aim of this pilot study was to determine the level of expression of miropin and individual proteases in vivo in periodontal and peri-implant health and disease conditions. Biofilm and gingival crevicular fluid (GCF)/ peri-implant sulcular fluid (PISF) samples were taken from healthy tooth and implant sites (n = 10), gingivitis and mucositis sites (n = 12), and periodontitis and peri-implantitis sites (n = 10). Concentration of interleukin-8 (IL-8), IL-1β and IL-10 in GCF was determined by enzyme-linked immunosorbent assay. Loads of P. gingivalis and T. forsythia and the presence of proteases and miropin genes were assessed in biofilm by quantitative PCR, whereas gene expression was estimated by quantitative RT-PCR. The presence of P. gingivalis and T. forsythia, as well as the level of IL-8 and IL-1β, were associated with disease severity in the periodontal and peri-implant tissues. In biofilm samples harboring T. forsythia, genes encoding proteases were found to be present at 72.4% for karilysin and 100% for other KLIKK-protease genes and miropin. At the same time, detectable mRNA expression of individual genes ranged from 20.7% to 58.6% of samples (for forsylisin and miropsin-1, respectively). In comparison with the T. forsythia proteases, miropin and the gingipains were highly expressed. The level of expression of gingipains was associated with those of miropin and certain T. forsythia proteases around teeth but not implants. Cumulatively, KLIKK-proteases and especially miropin, might play a role in pathogenesis of both periodontal and peri-implant diseases., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The oral pathogen Tannerella forsythia possesses a unique surface (S-) layer with a complex O-glycan containing a bacterial sialic acid mimic in the form of either pseudaminic acid or legionaminic acid at its terminal position. We hypothesize that different T. forsythia strains employ these stereoisomeric sugar acids for interacting with the immune system and resident host tissues in the periodontium. Here, we show how T. forsythia strains ATCC 43037 and UB4 displaying pseudaminic acid and legionaminic acid, respectively, and selected cell surface mutants of these strains modulate the immune response in monocytes and human oral keratinocytes (HOK) using a multiplex immunoassay. When challenged with T. forsythia, monocytes secrete proinflammatory cytokines, chemokines and vascular endothelial growth factor (VEGF) with the release of interleukin-1β (IL-1β) and IL-7 being differentially regulated by the two T. forsythia wild-type strains. Truncation of the bacteria's O-glycan leads to significant reduction of IL-1β and regulates macrophage inflammatory protein-1. HOK infected with T. forsythia produce IL-1Ra, chemokines and VEGF. Although the two wild-type strains elicit preferential immune responses for IL-8, both truncation of the O-glycan and deletion of the S-layer result in significantly increased release of IL-8, granulocyte-macrophage colony-stimulating factor and monocyte chemoattractant protein-1. Through immunofluorescence and confocal laser scanning microscopy of infected HOK we additionally show that T. forsythia is highly invasive and tends to localize to the perinuclear region. This indicates, that the T. forsythia S-layer and attached sugars, particularly pseudaminic acid in ATCC 43037, contribute to dampening the response of epithelial tissues to initial infection and hence play a pivotal role in orchestrating the bacterium's virulence., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Tannerella forsythia is a Gram-negative oral pathogen strongly associated with periodontitis. This bacterium has an absolute requirement for exogenous N-acetylmuramic acid (MurNAc), an amino sugar that forms the repeating disaccharide unit with amino sugar N-acetylglucosamine (GlcNAc) of the peptidoglycan backbone. In silico genome analysis indicates that T. forsythia lacks the key biosynthetic enzymes needed for the de novo synthesis of MurNAc, and so relies on alternative ways to meet its requirement for peptidoglycan biosynthesis. In the subgingival niche, the bacterium can acquire MurNAc and peptidoglycan fragments (muropeptides) released by the cohabiting bacteria during their cell wall breakdown associated with cell division. Tannerella forsythia is able to also use host sialic acid (Neu5Ac) in lieu of MurNAc or muropeptides for its survival during the biofilm growth. Evidence suggests that the bacterium might be able to shunt sialic acid into a metabolic pathway leading to peptidoglycan synthesis. In this review, we explore the mechanisms by which T. forsythia is able to scavenge MurNAc, muropeptide and sialic acid for its peptidoglycan synthesis, and the impact of these scavenging activities on pathogenesis., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Bacterial sialidases cleave terminal sialic acid from a variety of host glycoproteins, and contribute to survival and growth of many human-dwelling bacterial species, including various pathogens. Tannerella forsythia , an oral, Gram-negative, fastidious anaerobe, is a key organism in periodontal disease and possesses a dedicated sialic acid utilisation and scavenging ( nan ) operon, including NanH sialidase. Here, we describe biochemical characterisation of recombinant NanH, including its action on host-relevant sialoglycans such as sialyl Lewis A and sialyl Lewis X (SLe A/X ), and on human cell-attached sialic acids directly, uncovering that it is a highly active broad specificity sialidase. Furthermore, the N-terminal domain of NanH was hypothesised and proved to be capable of binding to a range of sialoglycans and non-sialylated derivatives with K d in the micromolar range, as determined by steady-state tryptophan fluorescence spectroscopy, but it has no catalytic activity in isolation from the active site. We consider this domain to represent the founding member of a novel subfamily of carbohydrate-binding module (CBM), involved in glycosidase-ligand binding. In addition, we created a catalytically inactive version of the NanH enzyme (FRIP → YMAP) that retained its ability to bind sialic acid-containing ligands and revealed for the first time that binding activity of a CBM is enhanced by association with the catalytic domain. Finally, we investigated the importance of Lewis-type sialoglycans on T. forsythia -host interactions, showing that nanomolar amounts of SLe A/X were capable of reducing invasion of oral epithelial cells by T. forsythia , suggesting that these are key ligands for bacterial-cellular interactions during periodontal disease., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Tannerella forsythia is a major periodontal pathogen, and T. forsythia GroEL is a molecular chaperone homologous to human heat-shock protein 60. Interleukin-17 (IL-17) has been implicated in the pathogenesis of periodontitis and several systemic diseases. This study investigated the potential of T. forsythia GroEL to induce inflammatory bone resorption and examined the cooperative effect of IL-17 and T. forsythia GroEL on inflammatory responses. Human gingival fibroblasts (HGFs) and periodontal ligament (PDL) fibroblasts were stimulated with T. forsythia GroEL and/or IL-17. Gene expression of IL-6, IL-8, and cyclooxygenase-2 (COX-2) and concentrations of IL-6, IL-8, and prostaglandin E 2 (PGE 2 ) were measured by real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. After stimulation of MG63 cells with T. forsythia GroEL and/or IL-17, gene expression of osteoprotegerin (OPG) was examined. After subcutaneous injection of T. forsythia GroEL and/or IL-17 above the calvaria of BALB/c mice, calvarial bone resorption was assessed by micro-computed tomography and histological examination. Tannerella forsythia GroEL induced IL-6 and IL-8 production in HGFs and PDL cells, and IL-17 further promoted IL-6 and IL-8 production. Both T. forsythia GroEL and IL-17 synergistically increased PGE 2 production and inhibited OPG gene expression. Calvarial bone resorption was induced by T. forsythia GroEL injection, and simultaneous injection of T. forsythia GroEL and IL-17 further increased bone resorption. These results suggest that T. forsythia GroEL is a novel virulence factor that can contribute to inflammatory bone resorption caused by T. forsythia and synergizes with IL-17 to exacerbate inflammation and bone resorption., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Tannerella forsythia is an anaerobic, Gram-negative periodontal pathogen. A unique O-linked oligosaccharide decorates the bacterium's cell surface proteins and was shown to modulate the host immune response. In our study, we investigated the biosynthesis of the nonulosonic acid (NulO) present at the terminal position of this glycan. A bioinformatic analysis of T. forsythia genomes revealed a gene locus for the synthesis of pseudaminic acid (Pse) in the type strain ATCC 43037 while strains FDC 92A2 and UB4 possess a locus for the synthesis of legionaminic acid (Leg) instead. In contrast to the NulO in ATCC 43037, which has been previously identified as a Pse derivative (5-N-acetimidoyl-7-N-glyceroyl-3,5,7,9-tetradeoxy-l-glycero-l-manno-NulO), glycan analysis of strain UB4 performed in this study indicated a 350-Da, possibly N-glycolyl Leg (3,5,7,9-tetradeoxy-d-glycero-d-galacto-NulO) derivative with unknown C5,7 N-acyl moieties. We have expressed, purified and characterized enzymes of both NulO pathways to confirm these genes' functions. Using capillary electrophoresis (CE), CE-mass spectrometry and NMR spectroscopy, our studies revealed that Pse biosynthesis in ATCC 43037 essentially follows the UDP-sugar route described in Helicobacter pylori, while the pathway in strain FDC 92A2 corresponds to Leg biosynthesis in Campylobacter jejuni involving GDP-sugar intermediates. To demonstrate that the NulO biosynthesis enzymes are functional in vivo, we created knockout mutants resulting in glycans lacking the respective NulO. Compared to the wild-type strains, the mutants exhibited significantly reduced biofilm formation on mucin-coated surfaces, suggestive of their involvement in host-pathogen interactions or host survival. This study contributes to understanding possible biological roles of bacterial NulOs., (© The Author 2017. Published by Oxford University Press.)

The oral pathogen Tannerella forsythia is implicated in the development of periodontitis, a common inflammatory disease that leads to the destruction of the gum and tooth supporting tissues, often leading to tooth loss. T. forsythia is a unique Gram-negative organism endowed with an elaborate protein O-glycosylation system that allows the bacterium to express a glycosylated surface (S)-layer comprising two high molecular weight glycoproteins modified with O-linked oligosaccharides. The T. forsythia S-layer has been implicated in the modulation of cytokine responses of antigen presenting cells, such as macrophages, that play a significant role during inflammation associated with periodontitis. The macrophage-inducible C-type lectin receptor (Mincle) is an FcRγ-coupled pathogen recognition receptor that recognizes a wide variety of sugar containing ligands from fungal and bacterial pathogens. In this study, we aimed to determine if Mincle might be involved in the recognition of T. forsythia S-layer and modulation of cytokine response of macrophages against the bacterium. Binding studies using recombinant Mincle-Fc fusion protein indicated a specific Ca2+-dependent binding of Mincle to T. forsythia S-layer. Subsequent experiments with Mincle-expressing and Mincle-knockdown macrophages revealed a role for Mincle/S-layer interaction in the induction of both pro- and anti-inflammatory cytokine secretion in macrophages stimulated with T. forsythia as well as its S-layer. Together, these studies revealed Mincle as an important macrophage receptor involved in the modulation of cytokine responses of macrophages against T. forsythia, and thus may play a critical role in orchestrating the host immune response against the bacterium.

Background: Peri-implantitis is a complex polymicrobial biofilm-induced inflammatory osteolytic gingival infection that results in orofacial implant failures. To the best knowledge of the authors, there are no preclinical in vivo studies in implant dentistry that have investigated the inflammatory response to known microbial biofilms observed in humans. The aim of this study is to develop a novel peri-implant rat model using an established model of polymicrobial periodontitis., Methods: Wistar rats were used for the study of experimental peri-implantitis. One month after extraction of maxillary first molars, a titanium mini-implant was inserted. Two months after implant healing, implants were uncovered, and abutment fixing was done using cyanoacrylate to prevent abutment loosening. Rats were separated into two groups (group A: polymicrobial-infected and group B: sham-infected). One week after healing of abutments, rats were infected with Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia for 12 weeks. Bacterial colonization, bone resorption, and implant inflammation were evaluated by polymerase chain reaction (PCR), microcomputed tomography, and histology, respectively., Results: Three rats with four implants in the infection group and two rats with three implants in the sham-infection group were analyzed. PCR analysis revealed presence of bacterial genomic DNA, and infection elicited significant immunoglobulin (Ig)G and IgM antibody responses, indicating bacterial colonization/infection around implants. Infection induced an enhanced mean distance from implant platform to the first bone-to-implant contact, extensive peri-implantitis with advanced bone resorption, and extensive inflammation with granulation tissue and polymorphonuclear leukocytes., Conclusions: To the best knowledge of the authors, this is the first study to develop a novel rat model of polymicrobial peri-implantitis. With modifications to improve implant retention it could offer significant advantages for studies of initiation and progression of peri-implantitis.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Periodontitis is characterized by inflammation associated with the colonization of different oral pathogens. We here aimed to investigate how bacteria and host cells shape their environment in order to limit inflammation and tissue damage in the presence of the pathogen. Human dental follicle stem cells (hDFSCs) were co-cultured with gram-negative P. intermedia and T. forsythia and were quantified for adherence and internalization as well as migration and interleukin secretion. To delineate hDFSC-specific effects, gingival epithelial cells (Ca9-22) were used as controls. Direct effects of hDFSCs on neutrophils (PMN) after interaction with bacteria were analyzed via chemotactic attraction, phagocytic activity and NET formation. We show that P. intermedia and T. forsythia adhere to and internalize into hDFSCs. This infection decreased the migratory capacity of the hDFSCs by 50%, did not disturb hDFSC differentiation potential and provoked an increase in IL-6 and IL-8 secretion while leaving IL-10 levels unaltered. These environmental modulations correlated with reduced PMN chemotaxis, phagocytic activity and NET formation. Our results suggest that P. intermedia and T. forsythia infected hDFSCs maintain their stem cell functionality, reduce PMN-induced tissue and bone degradation via suppression of PMN-activity, and at the same time allow for the survival of the oral pathogens.

This study aimed to investigate the mechanism by which periodontitis affects the inflammatory response and systemic insulin resistance in the white adipose and liver tissues in an obese rat model. The obese model was generated by feeding rats a high fat diet. The periodontitis model was induced by ligatures and injection of "red complex", which consisted of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, for two weeks. When compared with rats without periodontitis, fasting glucose levels and homeostasis model assessment index were significantly increased in rats with periodontitis, suggesting that periodontitis promotes the development of insulin resistance in obese rats. Gene and protein expression analysis in white adipose and liver tissue revealed that experimental periodontitis stimulated the expression of inflammatory cytokines, such as tumor necrosis factors-alpha, interleukin-1 beta, toll-like receptor 2 and toll-like receptor 4. Signals associated with inflammation and insulin resistance, including nuclear factor- B, c-Jun amino-terminal kinase and extracellular-signal regulated kinase were significantly activated in the white adipose tissue from obese rats with periodontitis compared to obese rats without periodontitis. Taken together, these findings suggest that periodontitis plays an important role in aggravating the development of local white adipose inflammation and systemic insulin resistance in rat models., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Despite significant advances in recent years in culture-independent molecular microbiology methods, the detailed study of individual bacterial species still relies on having pure cultures in the laboratory. Yet, more than a third of the approximately 700 bacterial taxa found in the human oral cavity are as yet uncultivated in vitro. One such taxon, Tannerella sp. HOT-286 (phylotype BU063), is the focus of much interest since it is associated with periodontal health, while Tannerella forsythia, its closest phylogenetic neighbor, is strongly associated with periodontal disease. HOT-286, however, has remained uncultivated despite the efforts of several research groups, spanning over a decade. The aim of this study was to cultivate Tannerella sp. HOT-286. A heavily diluted sample of subgingival plaque was inoculated onto culture plates supplemented with siderophores (pyoverdines-Fe complex or desferricoprogen) or a neat plaque suspension. After 8 d of anaerobic incubation, microcolonies and colonies showing satellitism were passaged onto fresh culture plates cross-streaked with potential helper strains or onto cellulose-acetate membranes placed over lawn cultures of helper strains. Subcultured colonies were identified by 16S rRNA gene sequencing, and purity was confirmed by sequencing 20 clones per library prepared from a single colony. Three colonies of interest (derived from pyoverdines- and plaque-supplemented plates) were identified as Tannerella sp. HOT-286. The isolates were found to be incapable of independent growth, requiring helpers such as Propionibacterium acnes and Prevotella intermedia for stimulation, with best growth on membranes over "helper" lawns. A representative isolate was subjected to phenotypic characterization and found to produce a range of glycosidic and proteolytic enzymes. Further comparison of this novel "periodontal health-associated" taxon with T. forsythia will be valuable in investigating virulence factors of the latter and possible health benefits of the former., Competing Interests: The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article., (© International & American Associations for Dental Research 2016.)

Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. Although the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, six being classical pathogens and four putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone-marrow-derived macrophages (BMDM) from wild-type (WT) and Toll-like receptor (TLR)-specific and MyD88 knockouts (KOs), we demonstrated that heat-killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. Campylobacter concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1-specific KOs and NOD2-expressing human embryonic kidney cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2 stimulatory activity. These studies allowed us to provide important evidence on newly identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855)., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Although enolases are cytosolic enzymes involved in the glycolytic pathway, they can also be secreted or expressed on the surface of a variety of eukaryotic cells and bacteria. Surface-exposed enolases of eukaryotes and bacteria can function as plasminogen receptors. Furthermore, antibodies raised against bacterial enolases can react with host enolases, suggesting molecular mimicry between bacterial and host enzymes. In this study, we analyzed an enolase of the major periodontopathogen Tannerella forsythia, which is either secreted or present on the cell surface, via matrix-assisted laser desorption ionization time-of-flight mass spectrometry and immunofluorescence, respectively. The T. forsythia enolase retained the enzymatic activity converting 2-phosphoglycerate to phosphoenolpyruvate and showed plasminogen binding and activating ability, which resulted in the degradation of fibronectin secreted from human gingival fibroblasts. In addition, it induced proinflammatory cytokine production, including interleukin-1β (IL-1β), IL-6, IL-8, and tumour necrosis factor-α (TNF-a) in the human THP-1 monocytic cell line. Taken together, our results demonstrate that T. forsythia enolase plays a role in pathogenesis in the host by plasminogen activation and proinflammatory cytokine induction, which has the potential to exaggerate inflammation in periodontitis., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Here we report a case of generalized aggressive periodontitis treated with periodontal therapy including adjunct antimicrobial therapy and periodontal surgery. The patient was a 22-year-old woman who presented with the chief complaint of gingival recession. Baseline examination revealed generalized plaque deposition and gingival inflammation. Thirty-nine percent of the sites had a probing depth (PD) of 4-6 mm and 2% a PD of ≥7 mm; 63% exhibited bleeding on probing (BOP). Radiographic examination revealed vertical bone loss in the molars and horizontal bone loss in other teeth. Microbiological examination of subgingival plaque revealed the presence of Aggregatibacter actinomycetemcomitans and Tannerella forsythia. Oral health-related quality of life was assessed as a measure of patient-reported outcome. Based on a clinical diagnosis of generalized aggressive periodontitis, initial periodontal therapy and adjunct antimicrobial therapy were implemented. After reducing inflammation and subgingival bacteria, open flap debridement was performed for teeth with a PD of ≥4 mm. Reevaluation showed no sites with a PD of ≥5 mm, a minimal level of BOP, and a marked reduction in the level of the targeted periodontal pathogens. The patient's oral health-related quality of life was slightly worsened during supportive periodontal therapy (SPT). Implementation of adjunct antimicrobial therapy targeting periodontal pathogens and subsequent periodontal surgery resulted in improvement in periodontal and microbiological parameters. This improvement has been adequately maintained over a 2-year period. However, additional care is necessary to further improve the patient's oral health-related quality of life during SPT.