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Periodontal disease is driven by dysbiosis in the oral microbiome, resulting in over-representation of species that induce the release of pro-inflammatory cytokines, chemokines, and tissue-remodeling matrix metalloproteinases (MMPs) in the periodontium. These chronic tissue-destructive inflammatory responses result in gradual loss of tooth-supporting alveolar bone. The oral spirochete Treponema denticola, is consistently found at significantly elevated levels in periodontal lesions. Host-expressed Toll-Like Receptor 2 (TLR2) senses a variety of bacterial ligands, including acylated lipopolysaccharides and lipoproteins. T. denticola dentilisin, a surface-expressed protease complex comprised of three lipoproteins has been implicated as a virulence factor in periodontal disease, primarily due to its proteolytic activity. While the role of acylated bacterial components in induction of inflammation is well-studied, little attention has been given to the potential role of the acylated nature of dentilisin. The purpose of this study was to test the hypothesis that T. denticola dentilisin activates a TLR2-dependent mechanism, leading to upregulation of tissue-destructive genes in periodontal tissue. RNA-sequencing of periodontal ligament cells challenged with T. denticola bacteria revealed significant upregulation of genes associated with extracellular matrix organization and degradation including potentially tissue-specific inducible MMPs that may play novel roles in modulating host immune responses that have yet to be characterized within the context of oral disease. The Gram-negative oral commensal, Veillonella parvula, failed to upregulate these same MMPs. Dentilisin-induced upregulation of MMPs was mediated via TLR2 and MyD88 activation, since knockdown of expression of either abrogated these effects. Challenge with purified dentilisin upregulated the same MMPs while a dentilisin-deficient T. denticola mutant had no effect. Finally, T. denticola-mediated activation of TLR2/MyD88 lead to the nuclear translocation of the transcription factor Sp1, which was shown to be a critical regulator of all T. denticola-dependent MMP expression. Taken together, these data suggest that T. denticola dentilisin stimulates tissue-destructive cellular processes in a TLR2/MyD88/Sp1-dependent fashion., Competing Interests: The authors have declared that no competing interests exist.

Accumulation of amyloid-β (Aβ) in the brain is a central component of pathology in Alzheimer's disease. A growing volume of evidence demonstrates close associations between periodontal pathogens including Porphyromonas gingivalis (P. gingivalis) and Treponema denticola (T. denticola) and AD. However, the effect and mechanisms of T. denticola on accumulation of Aβ remain to be unclear. In this study, we demonstrated that T. denticola was able to enter the brain and act directly on nerve cells resulting in intra- and extracellular Aβ 1-40 and Aβ 1-42 accumulation in the hippocampus of C57BL/6 mice by selectively activating both β-secretase and γ-secretase. Furthermore, both KMI1303, an inhibitor of β-secretase, as well as DAPT, an inhibitor of γ- secretase, were found to be able to inhibit the effect of T. denticola on Aβ accumulation in N2a neuronal cells. Overall, it is concluded that T. denticola increases the expression of Aβ 1-42 and Aβ 1-40 by its regulation on beta-site amyloid precursor protein cleaving enzyme-1 and presenilin 1.

Objective: In the gingival crevice, the interaction between epithelial cells and periodontopathic bacteria is important for the development of periodontitis. Treponema denticola is a major pathogen of chronic periodontitis and possesses several virulence factors, such as major surface protein (Msp) and prolyl-phenylalanine-specific protease (dentilisin). Here, we investigated the behaviours of epithelial cells infected with T. denticola by measuring the expression of interleukin (IL)-1β, IL-6, β defensin 2 (BD-2) and heat-shock protein 70 (HSP70)., Methods: Epithelial cells were infected with T. denticola wild-type strain, Msp-deficient mutant or dentilisin-deficient mutant, and the expression levels of the above targets were analysed by polymerase chain reaction., Results: Infection with T. denticola wild-type strain and mutants induced the production of IL-6 and HSP70. The level of BD-2 induced by T. denticola wild-type strain at 24 hr was significantly higher than that of the dentilisin-deficient mutant. The level of IL-1β mRNA in the wild-type strain and dentilisin-deficient mutant was slightly lower than that in the uninfected control., Conclusion: These results suggest that the levels of BD-2 were affected by Msp and dentilisin. This effect may contribute to the disruption of the response of epithelial cells to eradicate T. denticola., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Spirochetes are suspected to be linked to the genesis of neurological diseases, including neurosyphillis or neurodegeneration (ND). Impaired iron homeostasis has been implicated in loss of function in several enzymes requiring iron as a cofactor, formation of toxic oxidative species, inflammation and elevated production of beta-amyloid proteins. This review proposes to discuss the link that may exist between the involvement of Treponema spp. in the genesis or worsening of ND, and iron dyshomeostasis. Proteins secreted by Treponema can act directly on iron metabolism, with hemin binding ability (HbpA and HbpB) and iron reductase able to reduce the central ferric iron of hemin, iron-containing proteins (rubredoxin, neelaredoxin, desulfoferrodoxin metalloproteins, bacterioferritins etc). Treponema can also interact with cellular compounds, especially plasma proteins involved in iron metabolism, contributing to the virulence of the syphilis spirochetes (e.g. treponemal motility and survival). Fibronectin, transferrin and lactoferrin were also shown to be receptors for treponemal adherence to host cells and extracellular matrix. Association between Treponema and iron binding proteins results in iron accumulation and sequestration by Treponema from host macromolecules during systemic and mucosal infections., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Treponema pallidum subsp. pallidum membrane proteins are considered as potent inducers in the initiation and development of inflammation. In the present study, the mechanism that leads to the production of interleukin 6 (IL-6), one of the key proinflammatory cytokines, by human monocytic THP-1 cells when these cells are treated with T. pallidum flagellin FlaA2 was investigated. Stimulation with flagellin FlaA2 can induce IL-6 expression in human monocytes and augment the phosphorylation of ERK, p38, and NF-κB, but has no effect on the phosphorylation of JNK. Likewise, FlaA2-induced IL-6 production was found to be attenuated by inhibitors for ERK, p38, and NF-κB, but not by JNK inhibitor. Immunofluorescence analysis showed that flagellin FlaA2 could stimulate the translocation of IκBα from the cytosol to the nucleus, and this phenomenon could be inhibited by the specific inhibitor BAY11-7082. FlaA2-induced IL-6 expression was also proved to be abrogated by transfection with dominant negative (DN) plasmid of MyD88. We further demonstrated that transfection with DN-TLR2 was sufficient to attenuate IL-6 expression and the phosphorylation of ERK, p38, and IκBα. These results suggest that flagellin FlaA2 induces IL-6 production via signaling pathways involving TLR2, MyD88, ERK, p38, and NF-κB in monocytes, which could contribute to the pathogenesis of T. pallidum., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

While FbpA, a family of bacterial fibronectin (FN) binding proteins has been studied in several gram-positive bacteria, the gram-negative Treponema denticola, an anaerobic periodontal pathogen, also has an overlooked fbp gene (tde1579). In this research, we confirm that recombinant Fbp protein (rFbp) of T. denticola binds human FN with a Kdapp of 1.5 × 10(-7) M and blocks the binding of T. denticola to FN in a concentration-dependent manner to a level of 42%. The fbp gene was expressed in T. denticola. To reveal the roles of fbp in T. denticola pathogenesis, an fbp isogenic mutant was constructed. The fbp mutant had 51% reduced binding ability to human gingival fibroblasts (hGF). When hGF were challenged with T. denticola, the fbp mutant caused less cell morphology change, had 50% reduced cytotoxicity to hGF, and had less influence on the growth of hGF cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The purpose of this study was to investigate the mechanism(s) of interleukin (IL)-8 suppression by Treponema denticola, one of the major periodontal pathogens, in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with wild-type (WT), dentilisin-deficient (K1) or flagellin-deficient (flgE) T. denticola in the presence or absence of 2% human serum for 24 h. The levels of IL-8 expression were measured with real-time reverse transcription PCR and ELISA. In the absence of human serum, the WT and flgE, but not K1, substantially reduced not only the levels of IL-8 protein but also of IL-8 mRNA. Such downregulation of IL-8 mRNA was independent of bacterial invasion. Degradation of cytokine mixture by the WT, K1 and flgE revealed dentilisin-dependent preferential degradation of tumor necrosis factor (TNF)-α, an IL-8-inducing cytokine. WT and flgE significantly decreased the levels of TNFα secreted by HOK-16B cells, suggesting modulation of IL-8 through dentilisin-mediated degradation of TNFα. The addition of human serum to the culture potentiated the suppressive effect of T. denticola, resulting in substantial reductions of IL-8 and TNFα levels, even by K1. The serum-dependent effects of T. denticola were attributed to its ability to suppress the accumulation of intracellular reactive-oxygen species (ROS), a group of ubiquitous signaling molecules. Pretreatment with an antioxidant suppressed TNFα-induced IL-8 expression, confirming the role of ROS in TNFα signaling. Collectively, T. denticola targeted a key inflammatory cytokine and its signaling molecule to modulate the host innate immune response, which provides a new insight into modulation of host immunity by a periodontal pathogen.

Inflammatory pseudotumor of lymph nodes (IPT-LN) represents an unusual cause of lymphadenitis of unknown etiology. Upon the observation of a case of IPT-LN associated with Treponema pallidum (Tp) infection, we analyzed a series of 9 IPT-LN and 9 extranodal IPT (spleen, 4 cases; lung, orbit, gut, skin, and liver) for the presence of Tp, using a polyclonal antibody anti-Tp. At the time of biopsy, none of the patients was suspected for luetic infection, nor specific serologic tests were available. IPT-LN areas extensively involved the nodal parenchyma in 4 cases, whereas they were focal in the remaining 5 cases. Capsular thickening and inflammation (6/9), venulitis (3/9), small granulomas (3/9), and follicular hyperplasia (7/9) were observed in the associated lymphoid parenchyma. Tp were detected in 4/9 cases of LN-IPT and in none of the extranodal IPT. Tp were extremely abundant within the IPT areas and in the perivascular tissues in the surrounding parenchyma, whereas they were scattered within the capsule. In Tp+ cases, marked follicular hyperplasia was the single distinctively associated feature. Double immunostains revealed that Tp were predominantly contained in the cytoplasm of CD11c+ CD163+ macrophages, some of which co-expressed HLA-DR. In addition, scattered S100+ interdigitating dendritic cells also showed intracytoplasmic Tp. This study shows that a significant number of IPT-LN is associated with Tp infection. A spirochetal etiology can be suspected in cases of IPT-LN, independently from the extension of the lesions, especially when pronounced follicular hyperplasia is found. Infection by Tp of macrophages and dendritic cells are in keeping with in vitro data and indicate that immune mediated mechanisms may be involved in the pathogenesis of the lesions.

The tprK gene in Treponema pallidum undergoes antigenic variation. In all T. pallidum isolates examined to date, except the Nichols type strain, heterogeneous tprK sequences have been identified. This heterogeneity is localized to seven variable (V) regions, and tprK sequence diversity accumulates with serial passage in naïve rabbits. The T. pallidum Nichols genome described a single tprK sequence, and after decades of independent passage, only minor tprK sequence diversity is seen among the Nichols strains from different laboratories. We hypothesized that T. pallidum Nichols is capable of only limited tprK diversification. To address this hypothesis, we passaged the T. pallidum Nichols strain in naïve rabbits at the peak of infection (rapid passage) or after the adaptive immune response had cleared most organisms in vivo (slow passage). After 22 rapid passages (9- to 10-day intervals), no tprK V region sequence changes were observed. In contrast, after two slow passages (30- to 35-day intervals), three V regions had sequences that were completely different from that of the original inoculum. New sequences were observed in all seven V regions by the fifth slow passage. In contrast to the rapid-passaged Nichols strain, rapid-passaged Chicago C, a clonal strain isolated from the highly diverse parent Chicago strain, developed significant tprK diversification. These findings suggest that tprK variation can occur, but at a lower rate, in Nichols and that immune pressure may be required for accumulation of bacteria with diverse tprK sequences. Adaptation to growth in rabbits may explain the limited repertoire of V region sequences seen in the Nichols strain.

Treponema denticola has been shown to be associated with periodontitis in man and animals. The organism ferments amino acids and thrives on the proteins in the periodontal pocket. Accordingly, T. denticola possesses various proteolytic enzymes, including a chymotrypsin-like protease, capable of hydrolyzing a whole range of proteins, including immunoglobulins. Yet, it is not clear whether the intact cells of T. denticola can degrade immunoglobulins and albumin. The purpose of this study was to clarify this point. Three strains of T. denticola were cultured in liquid medium, and cells were harvested by centrifugation. Protein degradation in cell suspensions was assayed by capillary electrophoresis and immunonephelometry. None of the T. denticola strains appeared to be able to degrade IgA, IgG, or albumin, while a strain of P. gingivalis completely hydrolyzed these proteins. The findings suggest that, in the periodontal pocket, T. denticola depends on proteinases from other bacteria for utilization of the available serum proteins. This is in accordance with clinical data showing a close relationship between T. denticola and strongly proteolytic bacteria, such as Porphyromonas gingivalis and Bacteroides forsythus.

The therapeutic effects of subcutaneously administered sedecamycin on experimental Treponema hyodysenteriae infection in mice were evaluated. Sedecamycin was more active than tiamulin and lincomycin. The efficacy of sedecamycin upon subcutaneous administration was similar to that upon oral administration. Sedecamycin given subcutaneously provided similar degrees of protection in bile duct-ligated and intact mice. Pharmacokinetic studies utilizing a liquid chromatographic technique were carried out to determine the concentration of sedecamycin in the cecum, the site of T. hyodysenteriae infection in mice. Little sedecamycin was found; however, lankacidinol, a major metabolite of sedecamycin, was found in the cecal contents of intact mice after subcutaneous or oral administration of sedecamycin. Lankacidinol was also found in the cecal contents of bile duct-ligated mice, although the concentration found after subcutaneous administration of sedecamycin was much lower than that found after subcutaneous or oral administration to intact mice. These results indicate that sedecamycin is excreted directly into the intestinal tract as an active metabolite by a route other than the bile duct. It is suggested that this intestinal excretion plays an important role in the efficacy of subcutaneously administered sedecamycin against cecal infection of mice by T. hyodysenteriae.

The redox potential of (Eh) +111 +/- 25 mV was measured in the large intestine of newly born piglets. In the post-weaning period the Eh values decreased significantly to -173 +/- 27 mV and remained at this level also in the healthy sows (-214 +/- 55 mV). The Eh value recorded in dysenteric pigs was -188 +/- 5 mV, and this was not statistically significant in relation to the healthy weaned piglets. The Eh level measured in the blood agar prepared with cysteine and covered by a thick growth of the strain Treponema hyodysenteriae, which had been incubated in an anaerobic medium for five days, was -218 +/- 18 mV. The Eh of piglets after weaning was not the decisive condition for the development of dysentery. However, it can be assumed that the impossibility of eliciting dysentery in microbe-free and gnotobiotic pigs is associated with a relatively high redox potential of a microbially unpopulated or insufficiently populated intestine.

Net electrolyte and water transport and unidirectional Na+ fluxes were examined in ligated colonic loops of clinically normal pigs and in pigs with swine dysentery (etiologic agent Treponema hyodysenteriae) in the presence or absence of theophylline. In normal pigs, theophylline abolished net Na+ absorption via a reduction in the lumen-to-blood flux, decreased Cl- absorption, and increased HCO3- accumulation in the lumen. In infected pigs, all net ion transport was abolished, with the addition of theophylline producing little effect. The absence of net Na+ absorption in infected pigs was also the result of a decreased lumen-to-blood flux. Seemingly, colonic malabsorption may be the primary transport alteration in swine dysentery. Concentrations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured in samples of colonic mucosa from normal and infected pigs after in vitro exposure to a Ringer's solution containing 0 or 20 mM theophylline. Basal values of cAMP or cGMP did not increase in infected colonic mucosa. There was a diminished capacity of the infected mucosa to respond to theophylline. Alterations in ion transport in conjunction with measurements of cAMP and cGMP indicated that the pathogenic mechanism(s) in swine dysentery were not similar to those of Salmonella, Shigella, Vibrio cholerae, or Escherichia coli diarrhea.

Glucose metabolism and the mechanisms of NADH oxidation by Treponema hyodysenteriae were studied. Under an N2 atmosphere, washed cell suspensions of the spirochete consumed glucose and produced acetate, butyrate, H2, and CO2. Approximately twice as much H2 as CO2 was produced. Determinations of radioactivity in products of [14C]glucose and [14C]pyruvate metabolism and analyses of enzyme activities in cell lysates revealed that glucose was catabolized to pyruvate via the Embden-Meyerhof-Parnas pathway. The results of pyruvate exchange reactions with NaH14CO3 and Na14COOH demonstrated that pyruvate was converted to acetyl coenzyme A (acetyl-CoA), H2, and CO2 by a clostridium-type phosphoroclastic mechanism. NADH:ferredoxin oxidoreductase and hydrogenase activities were present in cell lysates and produced H2 from NADH oxidation. Phosphotransacetylase and acetate kinase catalyzed the formation of acetate from acetyl-CoA. Butyrate was formed from acetyl-CoA via a pathway that involved 3-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, butyryl-CoA dehydrogenase, and butyryl-CoA transferase. T. hyodysenteriae cell suspensions generated less H2 and butyrate under 10% O2-90% N2 than under 100% N2. Cell lysates contained NADH oxidase, NADH peroxidase, and superoxide dismutase activities. These findings indicated there are three major mechanisms that T. hyodysenteriae cells use to recycle NADH generated from the Embden-Meyerhof-Parnas pathway--enzymes in the pathway from acetyl-CoA to butyrate, NADH:ferredoxin oxidoreductase, and NADH oxidase. Versatility in methods of NADH oxidation and an ability to metabolize oxygen could benefit T. hyodysenteriae cells in the colonization of tissues of the swine large bowel.