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PubMed2000

Detection of Treponema denticola in endodontic infections by 16S rRNA gene-directed polymerase chain reaction.

A 16S rDNA-based polymerase chain reaction (PCR) method was used to detect the occurrence of Treponema denticola in root canal infections. Samples were collected from 21 single-root teeth having carious lesions, necrotic pulps and radiographic evidences of periradicular bone loss. DNA extracted from the samples was amplified using the PCR assay, which yielded specific fragment of T. denticola 16S rDNA. T. denticola was detected in 11 of 21 cases (52.4%), regardless of the presence or absence of symptoms. Since this spirochete was found in a relatively high percentage of the endodontic infections examined and because it is a pathogenic microorganism involved in periodontal diseases, there are reasons to believe that T. denticola can also participate in the pathogenesis of periradicular lesions of endodontic origin.

Adult↗
PubMed2001

Effects of the oral spirochete Treponema denticola on interleukin-8 expression from epithelial cells.

This communication demonstrates that the interaction of the oral spirochete Treponema denticola 35405 with KB epithelial cells does not lead to the induction of interleukin-8 production as occurs with a variety of other bacteria. Utilizing the dentilisin protease mutant K1 of T. denticola, this property was demonstrated to be primarily a function of the expression of the protease by strain 35405.

Bacterial Proteins↗
PubMed1999

Inability of intact cells of Treponema denticola to degrade human serum proteins IgA, IgG and albumin.

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.

Bacteroides↗
PubMed2002

Treponema denticola may stimulate both epithelial proliferation and apoptosis through MAP kinase signal pathways.

Mitogen-activated protein kinases (MAP kinases) play a key role in the regulation of cell survival and death. Effects of Treponema denticola ATCC 35405 on ERK, p38 and JNK MAP kinases, and cell behavior was studied using non-keratinizing periodontal ligament epithelial cells (PLE) in vitro. Compared to Chinese hamster ovary cells, human cervix adenocarcinoma cells, human osteosacroma cells and human gingival fibroblasts, PLE cells were much more resistant to T. denticola-induced reduction in cell viability, assayed by tetrazolium and crystal violet assays. A low dose of 5 x 10(7) T. denticola cells/ml increased DNA synthesis ([3H]thymidine uptake) in PLE cells but at higher concentrations DNA synthesis was decreased. TUNEL staining analysis showed that about 50% of epithelial cells in onolayers died through apoptosis when exposed to a high dose of 10(11) T. denticola/ml for 24 h. Morphological light and electron microscopic analysis supported the idea that both apoptotic and necrotic cell death took place. Rounding, membrane damage, fragmentation and detachment were observed in selective cells of both mono- and multilayered PLE cultures challenged with T. denticola. Western blot analysis using MAP kinase phosphospecific antibodies showed that T. denticola strongly but transiently activated ERK1 and ERK2, signals mediating cell proliferation, and JNK and p38, kinases mediating apoptosis. While a specific inhibitor of the ERK MAP kinase pathway prevented the T. denticola stimulation of cell proliferation, inhibitor of p38 increased the cell numbers in T. denticola-treated cultures. The results suggest that T. denticola activates epithelial cell MAP kinase signal pathways controlling cell proliferation and cell survival. In addition, T. denticola exerts cytotoxic effects that appear to predominate at higher bacterial concentrations.

Adenocarcinoma↗
PubMed1999

Treponema pallidum TroA is a periplasmic zinc-binding protein with a helical backbone.

The crystal structure of recombinant TroA, a zinc-binding protein component of an ATP-binding cassette transport system in Treponema pallidum, was determined at a resolution of 1.8 A. The organization of the protein is largely similar to other periplasmic ligand-binding proteins (PLBP), in that two independent globular domains interact with each other to create a zinc-binding cleft between them. The structure has one bound zinc pentavalently coordinated to residues from both domains. Unlike previous PLBP structures that have an interdomain hinge composed of beta-strands, the N- and C-domains of TroA are linked by a single long backbone helix. This unique backbone helical conformation was possibly adopted to limit the hinge motion associated with ligand exchange.

ATP-Binding Cassette Transporters↗
PubMed1980

Abrogation of genetically controlled resistance of mice to Treponema pallidum by irradiation.

Infection with Treponema pallidum, the causative agent of human syphilis, gives rise to a complex immune response involving both humoral and cellular components. The exact nature of this response and how it relates to the disease process is a matter of considerable speculation. In recent years, studies have been directed towards defining the role of cell-mediated immunity (CMI) in syphilis. These have been conducted mainly in vitro because the general unavailability of inbred rabbits, the principal animals for experimental syphilis research, has limited the application of in vivo procedures. A prime deterrent to using mice for the study of syphilis has been their failure to exhibit pathology, even in the face of a persistent infection. We report here that on intradermal (i.d.) infection, transient primary lesions, characteristic of those seen in naturally acquired human syphilis, can be produced regularly in some strains of mice but not others, indicating a genetic basis for host susceptibility. Strains of mice which normally fail to develop lesions, do so after exposure to ionising radiation. Evidence is presented for a multiple role of the immune response during local infection.

Animals↗
PubMed2006

In situ structure of the complete Treponema primitia flagellar motor.

The bacterial flagellar motor is an amazing nanomachine: built from approximately 25 different proteins, it uses an electrochemical ion gradient to drive rotation at speeds of up to 300 Hz (refs 1, 2). The flagellar motor consists of a fixed, membrane-embedded, torque-generating stator and a typically bidirectional, spinning rotor that changes direction in response to chemotactic signals. Most structural analyses so far have targeted the purified rotor, and hence little is known about the stator and its interactions. Here we show, using electron cryotomography of whole cells, the in situ structure of the complete flagellar motor from the spirochaete Treponema primitia at 7 nm resolution. Twenty individual motor particles were computationally extracted from the reconstructions, aligned and then averaged. The stator assembly, revealed for the first time, possessed 16-fold symmetry and was connected directly to the rotor, C ring and a novel P-ring-like structure. The unusually large size of the motor suggested mechanisms for increasing torque and supported models wherein critical interactions occur atop the C ring, where our data suggest that both the carboxy-terminal and middle domains of FliG are found.

Cryoelectron Microscopy↗
PubMed2003

Treponema denticola cystalysin exhibits significant alanine racemase activity accompanied by transamination: mechanistic implications.

To obtain information on the reaction specificity of cystalysin from the spirochaete bacterium Treponema denticola, the interaction with L- and D-alanine has been investigated. Binding of both alanine enantiomers leads to the appearance of an external aldimine absorbing at 429 nm and of a band absorbing at 498 nm, indicative of a quinonoid species. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The steady-state kinetic parameters for racemization, k (cat) and K (m), for L-alanine are 1.05+/-0.03 s(-1) and 10+/-1 mM respectively, whereas those for D-alanine are 1.4+/-0.1 s(-1) and 10+/-1 mM. During the reaction of cystalysin with L- or D-alanine, a time-dependent loss of beta-elimination activity occurs concomitantly with the conversion of the pyridoxal 5'-phosphate (PLP) coenzyme into pyridoxamine 5'-phosphate (PMP). The catalytic efficiency of the half-transamination of L-alanine is found to be 5.3x10(-5) mM(-1) x s(-1), 5-fold higher when compared with that of D-alanine. The partition ratio between racemization and half-transamination reactions is 2.3x10(3) for L-alanine and 1.4x10(4) for D-alanine. The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with p K values of 6.5-6.6, which must be unprotonated for catalysis. Addition of pyruvate converts the PMP form of the enzyme back into the PLP form and causes the concomitant recovery of beta-elimination activity. In contrast with other PLP enzymes studied so far, but similar to alanine racemases, the apoform of the enzyme abstracted tritium from C4' of both (4' S)- and (4' R)-[4'-(3)H]PMP in the presence of pyruvate. Together with molecular modelling of the putative binding sites of L- and D-alanine at the active site of the enzyme, the implications of these studies for the mechanisms of the side reactions catalysed by cystalysin are discussed.

Alanine↗
PubMed1998

Cytokine gene expression in skin of susceptible guinea-pig infected with Treponema pallidum.

Using a semi-quantitative multiplex reverse transcription-polymerase chain reaction assay, we examined cytokine mRNA expression for interleukin-1alpha (IL-1alpha), IL-2, IL-10, IL-12p40, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) in skin samples obtained from C4-deficient (C4D) guinea-pigs inoculated intradermally with virulent Treponema pallidum (VTP). Controls included unmanipulated animals, guinea-pigs injected with T. pallidum-free rabbit inflammatory testicular fluid (ITF) alone, or mixed with heat-killed organisms (HKTP). The expression of IL-1alpha, IL-12p40, and TNF-alpha mRNA [T helper type 1 (Th1)] remained within the normal range in both infected and control animals throughout the experimental period. However, a significant increase (P<0.05) in IL-10 mRNA (Th2) was found exclusively in the VTP-inoculated animals from 3 to 30 days post-infection. Another unique characteristic of the inflammatory response in infected guinea-pigs was the appearance, between 11 and 30 days post-inoculation, of a substantial number of eosinophils in addition to infiltrating mononuclear cells. The results showed a local Th2 response which is consistent with an inadequate immune response. This is reflected by the lengthy and incomplete clearance of the pathogen from the local site of entry and the chronic infection of distant organs.

Animals↗
PubMed2001

Polymerase chain reaction detection of Treponema denticola in endodontic infections within root canals.

AIM: A 16S rDNA-based polymerase chain reaction (PCR) method was used to detect the occurrence of Treponema denticola in root canals. METHODOLOGY: Samples were collected from 54 single-rooted teeth having carious lesions and necrotic pulps. DNA extracted from the samples was amplified using the PCR assay, which yielded a specific fragment of T. denticola 16S rDNA. RESULTS: T. denticola was detected in 10 of 29 asymptomatic cases (34.5%), eight of 15 root canals that were tender to percussion (53.3%), and five of 10 teeth with acute periradicular abscess (50%). In general, T. denticola was found in 23 of 54 cases (42.6%). CONCLUSIONS: Findings suggest that T. denticola can be involved in the pathogenesis of periradicular lesions of endodontic origin.

Adolescent↗
PubMed2003

Structural elucidation of polysaccharide part of glycoconjugate from Treponema medium ATCC 700293.

Glycoconjugates are distributed on the cell surfaces of some small-sized treponemes and have been reported to be completely different from lipopolysaccharides. We separated a glycoconjugate fraction from Treponema medium ATCC 700293, a medium-sized oral spirochete, to assess its immunobiological activities and elucidate the chemical structure of its polysaccharide part using phenol/water extraction, hydrophobic chromatography, and gel filtration. The glycoconjugate showed negligible or weak endotoxic and immunobiological properties. The chemical structure of the polysaccharide part was shown by two-dimensional NMR and MALDI-TOF-MS to be a tetrasaccharide backbone with two amino acids: [-->4)beta-d-GlcpNAc3NAcA(1-->4)beta-d-ManpNAc3NAOrn(1-->3)beta-d-GlcpNAc(1-->3)alpha-D-Fucp4NAsp(1-->] where GlcNAc3NAcA is 2,3-diacetamido-2,3-dideoxyglucuronic acid, ManNAc3NAOrn is Ndelta-(2-acetamido-3-amino-2,3-dideoxymannuronyl)ornithine, and Fuc4NAsp is 4-(alpha-aspartyl)amino-4,6-dideoxygalactose.

Carbohydrate Conformation↗
PubMed2002

Rapid seroconversion to Treponema pallidum and HIV positivity in a patient with retinal vasculitis.

A patient with idiopathic retinal vasculitis was found to have rapid conversion of serological tests for Treponema pallidum and HIV. The diagnosis was confirmed by Venereal Disease Research Laboratory (VDRL) testing and dark ground illumination for T. pallidum, and enzyme-linked immunosorbent assay (ELISA) and Western blot assay tests for HIV. Following treatment with intravenous penicillin, the fundus lesions resolved and visual acuity recovered from inaccurate light projection to 6/24 in the right eye and from counting fingers close to face to 2/60 in the left eye. Serological tests for T. pallidum in patients with concurrent HIV infection may be unpredictable. Hence, it is important to repeat these tests even in the early treatment phase of patients with retinal vasculitis who have shown initial seronegativity. This enables earlier diagnosis and initiation of specific treatment.

Adult↗
PubMed2002

Prevalence of circulating Treponema pallidum DNA and RNA in blood donors with confirmed-positive syphilis tests.

BACKGROUND: Although there have been no well-documented cases of transfusion-transmitted syphilis in more than 30 years, serologic tests for syphilis (STS) continues to be required for donated blood. Previously, the methods for detecting viable spirochetes were dependent on the use of the rabbit infectivity test. DNA PCR and RNA RT-PCR were applied to assess the presence of Treponema pallidum DNA or RNA in blood donors with confirmed-positive results in STS. STUDY DESIGN AND METHODS: This case series describes T. pallidum DNA and RNA testing of platelet concentrates prepared from blood donors with reactive results in an automated treponemal screening test and positive test results in the fluorescent treponomal antibody absorption test. The first DNA test was specific for the T. palladium polA gene. The second DNA test was a multiplex PCR using a T. pallidum 47-kDa gene target. The RT-PCR for RNA used T. pallidum 16S rRNA as a template for production of a cDNA target. RESULTS: One hundred sixty-nine samples (including rapid plasma reagin [RPR]+ and RPR-) tested for T. pallidum DNA and/or RNA were negative. CONCLUSIONS: A lack of demonstrable T. pallidum DNA or RNA suggests that blood donors with confirmed-positive results in STS are unlikely to have circulating T. pallidum in their blood and that that their blood is unlikely to be infectious for syphilis.

Adult↗
PubMed1991

Identification of spirochetes related to Treponema pallidum in necrotizing ulcerative gingivitis and chronic periodontitis.

BACKGROUND: Spirochetes are commonly associated with periodontal disease, but it is not known whether these treponemes are pathogenic or merely opportunistic. We sought to determine whether spirochetes present in periodontal disease share antigens thought to be unique to spirochetes that are known pathogens. METHODS: We examined dental plaque from 24 healthy subjects, from ulcerative sites in 17 patients with ulcerative gingivitis, and from areas of involvement in 19 patients with chronic periodontitis, using an immunocyto-chemical technique with monoclonal antibodies against pathogen-specific determinants on 47-kd and 37-kd molecules from Treponema pallidum subspecies pallidum. Serum was tested against T. pallidum by immunoblotting and by serologic assays for syphilis. RESULTS: Spirochetes with a pathogen-specific epitope on a 47-kd molecule were not found in plaque samples from any of the 24 healthy subjects, but they were identified in plaque samples from 11 of 17 patients with ulcerative gingivitis (P less than 0.001) and from 10 of 19 patients with periodontitis (P less than 0.01). Monoclonal antibodies directed against a 37-kd molecule reacted with spirochetes in plaque samples from 1 of 14 controls, from all 11 patients with gingivitis from whom samples could be obtained (P less than 0.001), and from 14 of 19 patients with periodontitis (P less than 0.001). Five of 18 normal subjects had IgG against 47-kd and 37-kd molecules, but none had IgG against 14-kd or 12-kd molecules from T. pallidum subspecies pallidum. Among 19 patients with ulcerative gingivitis, IgG was identified against 47-kd molecules in 15, against 37-kd molecules in 12, against 14-kd molecules in 4, and against 12-kd molecules in 15. CONCLUSIONS: The spirochetes found in dental plaque from patients with ulcerative gingivitis or chronic periodontitis have antigens that are thought to be unique to pathogenic treponemes. This close antigenic relation suggests that T. pallidum or a closely related organism may be involved in the pathogenesis of periodontal disease.

Adult↗
PubMed2001

"Red complex" (Bacteroides forsythus, Porphyromonas gingivalis, and Treponema denticola) in endodontic infections: a molecular approach.

OBJECTIVE: The "red complex," composed of Bacteroides forsythus, Porphyromonas gingivalis, and Treponema denticola, is implicated in severe forms of periodontal diseases. The purpose of this study was to assess the occurrence of the red complex in root canal infections through the use of a sensitive technique-the 16S rDNA-directed polymerase chain reaction (PCR). STUDY DESIGN: Samples were obtained from 50 necrotic pulps with periradicular pathosis. Ten cases were diagnosed as acute periradicular abscesses. DNA was extracted from the samples and analyzed with a PCR-based identification assay. RESULTS: At least 1 member of the red complex was found in 33 of 50 cases. T denticola, P gingivalis, and B forsythus were detected in 44%, 30%, and 26% of the cases, respectively. The red complex was found in 4 of 50 cases. No particular signs or symptoms were associated with the presence of these bacterial species. CONCLUSIONS: Despite what is indicated in reports with respect to marginal periodontitis, red complex bacteria-either singularly or collectively-was not associated with any particular pattern of clinical symptoms. However, because the bacterial species from the red complex are recognized oral pathogens, their occurrence in root canal infections suggests that they may play a role in the pathogenesis of periradicular diseases.

Adolescent↗
PubMed1994

The 47-kDa major lipoprotein immunogen of Treponema pallidum is a penicillin-binding protein with carboxypeptidase activity.

The recent model of Treponema pallidum molecular architecture proposes that the vast majority of the bacterium's integral membrane proteins are lipoprotein immunogens anchored in the cytoplasmic membrane while the outer membrane contains only a limited number of surface-exposed transmembrane proteins. This unique model explains, in part, the organism's remarkable ability to evade host immune defenses and establish persistent infection. Our strategy for refining this model involves demonstrating that the physiological functions of treponemal membrane proteins are consistent with their proposed cellular locations. In this study, we used an ampicillin-digoxigenin conjugate to demonstrate by chemiluminescence that the 47-kDa lipoprotein immunogen of T. pallidum (Tpp47) is a penicillin-binding protein. Reexamination of the Tpp47 primary sequence revealed the three amino acid motifs characteristic of penicillin-binding proteins. A recombinant, nonlipidated, soluble form of Tpp47 was used to demonstrate that Tpp47 is a zinc-dependent carboxypeptidase. Escherichia coli expressing Tpp47 was characterized by cell wall abnormalities consistent with altered peptidoglycan biosynthesis. Though the inability to cultivate T. pallidum in vitro and the lack of genetic exchange systems continue to impede treponemal research, this study advances strategies for utilizing E. coli molecular genetics as a means of elucidating the complex relationships between syphilis pathogenesis and T. pallidum membrane biology.

Amino Acid Sequence↗
PubMed1999

Characterization of a manganese-dependent regulatory protein, TroR, from Treponema pallidum.

Genome sequence analysis of Treponema pallidum, the causative agent of syphilis, suggests that this bacterium has a limited iron requirement with few, if any, proteins that require iron. Instead, T. pallidum may use manganese-dependent enzymes for metabolic pathways. This strategy apparently alleviates the necessity of T. pallidum to acquire iron from the host, thus overcoming iron limitation, which is a primary host defense. Interestingly, a putative metal-dependent regulatory protein, TroR, which has homology with the diphtheria toxin regulatory protein, DtxR, from Corynebacterium diphtheriae was identified from T. pallidum. We describe here the characterization of TroR, a regulatory protein. Mobility-shift DNA binding and DNase I footprint assays indicated that purified TroR bound to a 22-nt region of dyad symmetry that overlaps the -10 region of the promoter of the tro operon, which contains the genes for a putative metal transport system, the glycolytic enzyme phosphoglycerate mutase, and TroR. Unlike other metal-dependent regulatory proteins like diphtheria toxin regulatory protein and the ferric ion uptake regulator, Fur, which can be activated by divalent metals such as Fe(2+), Mn(2+), Co(2+), Ni(2+), and Zn(2+), TroR is activated only by Mn(2+). The TroR-Mn(2+) complex binds its target sequence and blocks transcription of the troPO/lacZ fusion, suggesting that TroR acts as a metal-dependent repressor in vivo. In addition, TroR exists as a dimer in both its inactive (metal free) and active states as indicated by chemical crosslinking experiments. Based on these data, we propose that TroR represents a unique regulatory system for controlling gene expression in T. pallidum in response to Mn(2+).

Bacterial Proteins↗
PubMed1998

Evidence for a new type of outer membrane lipid in oral spirochete Treponema denticola. Functioning permeation barrier without lipopolysaccharides.

A new class of outer membrane lipid (OML) was isolated from the oral spirochete Treponema denticola strain ATCC 33521 using a phenol/chloroform/light petroleum procedure normally applied for lipopolysaccharide extraction. In addition to chemical analysis, Fourier transform infrared (FTIR) spectroscopy was applied to compare the biophysical properties of OML with lipopolysaccharides (LPS) and lipoteichoic acids (LTA). Isolated OML fractions represent 1.4% of the total dry cell weight, are about 4 kDa in size, and contain 6% amino sugars, 8% neutral sugars, 14% phosphate, 35% carbazol-positive compounds, and 11% fatty acids (containing iso- and anteiso-fatty acyl chains). Rare for outer membrane lipids, OML contains no significant amount of 3-deoxy-D-manno-octulosonic acids, heptoses, and beta-hydroxy fatty acids. The fatty acyl chain composition, being similar to that of the cytoplasmic membrane, is quite heterogeneous with anteiso-pentadecanoic acid (12%), palmitic acid (51%), and iso-palmitic acid (19%) as the predominant fatty acids present. Findings of a glycerol-hexose unit and two glycerol-hexadecanoic acid fragments indicate a glycolipid membrane anchor typically found in LTA. There was also no evidence for the presence of a sphingosine-based lipid structure. The results of FTIR measurements strongly suggest that the reconstituted lipid forms normal bilayer structures (vesicles) expressing a high membrane state of order with a distinct phase transition as typical for isolated LPS. However, in contrast to LPS, OML of T. denticola has a lower Tm near 22 degreesC and a lower cooperativity of the phase transition. The results suggest a different kind of permeation barrier that is built up by this particular OML of T. denticola, which is quite different from LPS normally essential for Gram-negative bacteria.

Cell Membrane Permeability↗