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Combined ultrastructural and molecular studies have revealed that the syphilis and Lyme-disease spirochetes, Treponema pallidum and Borrelia burgdorferi, have distinctive molecular architectures. Both organisms persist in their hosts and have strategies for immune evasion that include the use of rare, poorly immunogenic surface-exposed proteins as potential virulence determinants.
Recent studies suggest that the tip-oriented adhesion of Treponema denticola is a consequence of polar clustering of its adhesins on contact with the substratum and is compatible with viability. Such adhesion to cells stimulates a host of cytoskeletal and volume regulatory changes. These studies may give insight into mechanisms of colonization and cytopathogenicity of other pathogenic spirochetes.
Cystalysin, the key virulence factor in the bacterium Treponema denticola responsible for periodontis, is a pyridoxal 5'-phosphate (PLP) enzyme which catalyzes, in addition to alpha,beta-elimination of L-cysteine, racemization and transamination of both enantiomers of alanine. In this paper several indicators have been used as probes of the different conformational status of T. denticola cystalysin in the holo and apo form. Compared to holoenzyme, the apoenzyme displays an altered reactivity of cysteine residues, a significantly different pI, and a differential susceptibility to proteinase K. The site of cleavage that is accessible in apocystalysin and masked in holocystalysin has been identified by mass spectrometry as the peptide bond between Phe 360 and Gly 361. This cleavage results in the loss of the C-terminal fragment corresponding to a molecular mass of 4289.21+/-0.1Da. The major fragment of cleaved enzyme retains its dimeric structure, binds the coenzyme with an affinity approximately 5000-fold lower than that of uncleaved holoenzyme, and in the reconstituted form is able to form the external aldimine with substrates. Although the break causes the loss of lyase, racemase and transaminase activities of D-alanine, it does not abolish the transaminase activity of L-alanine. Possible mechanistic and physiological implications are proposed.
Methyl mercaptan is derived from l-methionine by the action of l-methionine-alpha-deamino-gamma-mercaptomethane lyase (METase) and is a major component of oral malodor. This compound is highly toxic and is thought to play an important role in periodontal disease. We found that Treponema denticola, a member of the subgingival biofilm at periodontal disease sites, produced a large amount of methyl mercaptan even at low concentration of l-methionine. METase activity in a cell-free extract from T. denticola was detected by two-dimensional electrophoresis under non-denaturing conditions, and the protein spot that exhibited high METase activity was identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The identified gene produced a METase with a K(m) value for l-methionine (0.55mM) that is much lower than those of METases previously identified in the other organisms. This result suggests that T. denticola is an important producer of methyl mercaptan in the subgingival biofilm.
Superoxide reductases (SORs) are superoxide (O2-)-detoxifying enzymes that catalyse the reduction of O2- into hydrogen peroxide. Three different classes of SOR have been reported on the basis of the presence or not of an additional N-terminal domain. They all share a similar active site, with an unusual non-heme Fe atom coordinated by four equatorial histidines and one axial cysteine residues. Crucial catalytic reaction intermediates of SOR are purported to be Fe(3+)-(hydro)peroxo species. Using resonance Raman spectroscopy, we compared the vibrational properties of the Fe3+ active site of two different classes of SOR, from Desulfoarculus baarsii and Treponema pallidum, along with their ferrocyanide and their peroxo complexes. In both species, rapid treatment with H2O2 results in the stabilization of a side-on high spin Fe(3+)-(eta(2)-OO) peroxo species. Comparison of these two peroxo species reveals significant differences in vibrational frequencies and bond strengths of the Fe-O2 (weaker) and O-O (stronger) bonds for the T. pallidum enzyme. Thus, the two peroxo adducts in these two SORs have different stabilities which are also seen to be correlated with differences in the Fe-S coordination strengths as gauged by the Fe-S vibrational frequencies. This was interpreted from structural variations in the two active sites, resulting in differences in the electron donating properties of the trans cysteine ligand. Our results suggest that the structural differences observed in the active site of different classes of SORs should be a determining factor for the rate of release of the iron-peroxo intermediate during enzymatic turnover.
Polymerase chain reaction (PCR) was used to detect the 47-kDa immunogen gene of Treponema pallidum in peripheral blood mononuclear cells (PBMCs), skin lesions, and serum, but less consistently in purified granulocytes or ejaculates of patients with manifest and latent syphilis. Therefore, skin lesions and PBMCs may serve as the most reliable sources for a PCR-based diagnosis of syphilis.
We have recently shown that a monoclonal antibody, designated M131, that binds a surface phosphorylcholine epitope on Treponema pallidum possesses complement-dependent killing activity and confers partial protection in rabbits following passive immunization (Blanco et al., 2005, Infect. Immun. 73:3083-3095). In this study, the protective potential of M131 was further tested using the rabbit skin protection assay of Titus and Weiser. Both M131 and infection-derived immune rabbit serum resulted in significant lesion delays corresponding to at least a 90% reduction of the treponemal challenge inoculum. The skin protection assay provides a way to assess the protective potential of specific immunogens while using far less antibody than in passive immunization protocols.
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.
The aim of this study was to investigate the presence of strict anaerobes such as Filifactor alocis, Tannerella forsythia, and Treponema denticola in primary and secondary root-infected canals with periapical lesions by molecular analysis and the association of these species with specific endodontic signs and symptoms. Microbial samples were taken from 100 root canals, 50 with necrotic pulp tissues (NPT, primary infection), and 50 with failed endodontic treatment (FET, secondary infection). DNA was extracted from the samples, which were analyzed for the presence of three endodontic pathogens using species-specific primers and PCR. F. alocis were isolated from 23 canals with NPT and 12 canals with FET; T. forsythia from 12 canals with NPT and three canals with FET; T. denticola from 19 canals with NPT and 12 canals with TEP. Suggested associations were found between primary infection and the presence of F. alocis and T. forsythia (both p < 0.05). In particular, associations were found between: pain and F. alocis; swelling and F. alocis; tenderness to percussion and T. forsythia; mobility and T. forsythia and T. denticola; wet canals and F. alocis, T. forsythia, and T. denticola; purulent exsudate and F. alocis, T. forsythia and T. denticola; abscess and F. alocis, T. forsythia, and T. denticola (all p < 0.05). The findings of this study indicated that F. alocis, T. forsythia, and T. denticola seem to be associated with endodontic signs and symptoms. Additionally, F. alocis and T. forsythia were detected more frequently in teeth with necrotic pulp than in teeth with failing endodontic treatment.
Periodontopathic anaerobes such as Porphyromonas gingivalis are frequently found in aspiration pneumonia and lung abscesses. However, defense mechanisms and responses to these bacterial infections in the lung in vivo remain poorly understood. The coexistence of P. gingivalis with Treponema denticola has been found at higher levels and proportions in periodontally diseased sites. We hypothesized that mixed infections with P. gingivalis and T. denticola can cause severe respiratory disease. In the present study, inflammatory responses to mono- and mixed inoculations with P. gingivalis and T. denticola in the bronchoalveolar lavage (BAL) fluid were investigated. Acute pneumonia and lung abscesses in mice with the mixed infection resulted in a 40% mortality rate within 72 h, compared with only 10% mortality for the respective monoinfections. Pulmonary clearance of P. gingivalis was delayed in the mice with mixed infections with P. gingivalis and T. denticola. Tumor necrosis factor alpha (TNFalpha) interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) levels from BAL fluid of mice with mixed infections at 24 h after inoculation were significantly higher than those after P. gingivalis monoinfection (TNFalpha: P < 0.05, Il-1beta: P < 0.001, IL-6: P < 0.05). The chemokine KC level from BAL fluid of mice at 48 h (P < 0.05) and 72 h after mixed infection was also significantly increased when compared with that after P. gingivalis monoinfection (P < 0.001). The present study demonstrates that a mixed infection of P. gingivalis with T. denticola in mouse causes a marked bronchopneumonia and lung abscess in the mouse model.
A 12-membered Treponema pallidum repeat (Tpr) protein family has been identified in T. pallidum subsp. pallidum, the causative agent of syphilis. The subfamily I Tpr proteins (C, D, F, and I) possess conserved sequence at the N- and C-termini and central regions that differentiate the members. These proteins may be important in the immune response during syphilis infection and in protective immunity. Strong antibody responses have been observed toward some of the subfamily I Tpr proteins during infection with different syphilis isolates. Some sequence variation has also been identified in one subfamily I Tpr member, TprD, among T. pallidum subsp. pallidum isolates. In this study, we examined sequences in the remaining subfamily I Tpr proteins among strains. Both TprF and TprI were conserved among T. pallidum subsp. pallidum isolates. While some heterogeneity was identified in TprC. We further examined the immune response and protective capacity of TprF protein in this paper. We demonstrate that the N-terminal conserved region of the subfamily I Tpr proteins elicits strong antibody and T-cell responses during infection, and immunization with this region attenuates syphilitic lesion development upon infectious challenge.
Accumulated evidence points to Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as three major etiologic agents of chronic periodontitis. Epithelial cells and macrophages play a major role in the host response to periodontopathogens, and the secretion of inflammatory mediators and matrix metalloproteinases (MMPs) by these host cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of a macrophage/epithelial cell co-culture model following mono or mixed infections with the above three periodontopathogens. An in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 monocytic cell line) was challenged with whole cells or lipopolysaccharides (LPS) of P. gingivalis, T. denticola, and T. forsythia, individually and in combination. Following stimulation, the production of interleukin-1 beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), regulated on activation normal T cell expressed and secreted (RANTES), prostaglandin E2 (PGE2), and MMP-9 were quantified by enzyme-linked immunoassays. We observed that mono or mixed infections of the co-culture model induced the secretion of IL-1beta, IL-6, IL-8, PGE2, and MMP-9. P. gingivalis and T. forsythia induced an increase in RANTES secretion, whereas T. denticola alone or in combination resulted in a significant decrease in RANTES levels. All LPS challenges induced an increase in chemokine, MMP-9, and PGE2 production. No synergistic effect on the production of cytokines, chemokines, PGE2, and MMP-9 was observed for any of the bacterial or LPS mixtures tested. This study supports the view that P. gingivalis, T. denticola, and T. forsythia may induce high levels of pro-inflammatory mediators and MMP-9 in periodontal lesions, thus contributing to the progression of periodontitis.
Treponema denticola is a dominant microorganism in human periodontal lesions. One of the major virulence factors of this microorganism is its chymotrypsin-like surface protease, dentilisin. The purpose of this study was to evaluate the effect of dentilisin on human polymorphonuclear leukocytes (PMNs). We used chemiluminescence to assess production of O(-)(2) by PMNs against T. denticola ATCC 35405 and dentilisin-deficient mutant K1. T. denticola ATCC 35405 induced production of O(-)(2), whereas dentilisin-deficient K1 did not. We found that chymostatin, a protease inhibitor, strongly reduced the ability of T. denticola ATCC 35405 to induce production of, O(-)(2), whereas K1 was relatively unaffected. We also used Immunoblot and ELISA to evaluate the activation of complement by this microorganism in relation to PMNs. T. denticola ATCC 35405 hydrolyzed the alpha-chain of C3, producing iC3b. Furthermore, strain ATCC 35405 induced a larger release of MMP-9 from PMNs than strain K1. Dentilisin activated PMNs via complement pathways and may play a role in establishing periodontal lesions.
Periodontitis are mixed bacterial infections leading to destruction of tooth-supporting tissues, including periodontal ligament and alveolar bone. Among over 500 bacterial species living in the oral cavity, a bacterial complex named "red complex" and made of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia has been strongly related to advanced periodontal lesions. While periodontopathogenic bacteria are the primary etiologic factor of periodontitis, tissue destruction essentially results from the host immune response to the bacterial challenge. Members of the red complex are Gram negative anaerobic bacteria expressing numerous virulence factors allowing bacteria to colonize the subgingival sites, to disturb the host defense system, to invade and destroy periodontal tissue as well as to promote the immunodestructive host response. This article reviews current knowledge of the pathogenic mechanisms of bacteria of the red complex leading to tissue and alveolar bone destruction observed during periodontitis.
LL-37 displays potent broad-spectrum activity against a number of pathogenic bacteria and is the only cathelicidin thus far identified in humans. In this study, we examined the capacity of human LL-37 and the similar CAP-18-derived peptide from rabbits to exert antimicrobial activity against the causative agent of syphilis, Treponema pallidum. We found that both peptides, as well as a truncated version of human LL-37 that contains its bactericidal domain, could exert rapid, but salt-sensitive antimicrobial activity against T. pallidum. Infectivity of T. pallidum in a rabbit model could effectively be blocked with the synthetic truncated LL-37-derived peptide WS22-N-amide.
Treponema denticola is an oral anaerobic spirochete implicated in periodontal diseases. The chymotrypsin-like protease, dentilisin (PrtP), has been suggested to be an important virulence factor of T. denticola. In this study, we examined the role of dentilisin in T. denticola epithelial monolayer penetration by comparing the wild type and prtP mutant. Wild-type T. denticola can disrupt transepithelial resistance (TER) and substantially penetrate the HEp-2 cell layer. The prtP mutant altered the monolayer only slightly and penetrated the Hep-2 layer in very low numbers. The membrane fraction of wild-type T. denticola is able to complement the prtP mutant in monolayer penetration, while the comparable fraction from the mutant has no such effect. Immunofluorescence studies suggested that wild-type T. denticola altered the TER by likely degrading the tight junctional proteins such as ZO-1. Cytotoxicity was not a major factor in the disruption of TER. The outer membrane vesicles (OMVs) of wild-type T. denticola also disrupted epithelial barrier function and penetrated the epithelial layers. Taken together, these results suggest that T. denticola penetrates the epithelial cell monolayers by altering cellular tight junctions.
The aim was to investigate whether reference cultures and fresh isolates of Treponema denticola are able to 5alpha-reduce and further metabolise testosterone, 4-androstenedione, progesterone, corticosterone, cortisol or cholesterol. Two reference and five freshly isolated cultures of T. denticola were incubated with either radiolabeled or unlabeled steroid substrates; in the first case products were identified by thin layer chromatography and in the latter by gas chromatography-mass spectroscopy. All the substrates were 5alpha-reduced. Both reference cultures and fresh isolates of T. denticola presented 3beta- and 17beta-hydroxy steroid dehydrogenase activity. It was concluded that T. denticola was capable of steroid metabolism and hypotheses are discussed regarding the in vivo function of this metabolism including, T. denticola utilising host supplied steroids as growth factors and T. denticola steroid metabolism acting as a virulence factor.
Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2*- to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi-molecular reaction of the ferrous center with O2, with a rate constant of 6 x 10 (8) M(-1) s(-1). A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s(-1). This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron-peroxide species.