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PubMed1988

Identification of the major antigens of Treponema hyodysenteriae and comparison with those of Treponema innocens.

Eleven strains of Treponema hyodysenteriae isolated from pigs with swine dysentery were examined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. T. hyodysenteriae strains formed a homogeneous group with respect to sodium dodecyl sulfate-soluble proteins. However, immunoblotting with antiserum from rabbits immunized with T. hyodysenteriae CN8368 revealed heterogeneity among the lipopolysaccharide complexes of different strains. Polypeptides of molecular weights between 30,000 and 36,000 were the predominant T. hyodysenteriae polypeptides detected by porcine immune serum. In contrast, Treponema innocens did not form a homogeneous group with respect to sodium dodecyl sulfate-soluble proteins. Adsorption studies and immunoblotting identified polypeptide antigens present on cells of T. hyodysenteriae which were not detected on cells of T. innocens. These unique antigens may play a role in the virulence of T. hyodysenteriae.

Animals↗
PubMed1989

Comparison of the biological responses induced by lipopolysaccharide and endotoxin of Treponema hyodysenteriae and Treponema innocens.

The chemical composition and classical biologic activities of lipopolysaccharide (LPS; phenol-water) and endotoxin (butanol-water) preparations from virulent Treponema hyodysenteriae and avirulent Treponema innocens were examined. The LPS and endotoxin preparations from T. hyodysenteriae B204 contained approximately 80.9 and 35.2% hexose, 0.12 and 0.45% thiobarbituric acid-reactive compound, and less than 1 and 11.3% protein, respectively. The LPS and endotoxin preparations of T. innocens B1555a contained approximately 56.3 and 37.8% hexose, 0.45 and 0.4% thiobarbituric acid-reactive compound, and less than 1 and 26% protein, respectively. A silver-stained 7.5 to 15% sodium dodecyl sulfate-polyacrylamide gel showed four bands for the T. hyodysenteriae preparations, while the T. innocens preparations failed to resolve into discrete bands on electrophoresis. We determined by the Limulus amebocyte lysate assay that the treponemal preparations had comparable amounts of endotoxin activity when Escherichia coli LPS was used as a standard. The 50% lethal doses of LPS and endotoxin from T. hyodysenteriae for BALB/cByJ mice were 380 and 80 micrograms, respectively. The treponemal preparations were poor adjuvants, failed to induce a dermal Shwartzman reaction, and were not pyrogenic. The treponemal LPS preparations, unlike the endotoxin preparations, were not mitogenic for murine spleen cells. Differences in virulence between the two treponemal species could not be associated with the biologic activities of the respective LPS or endotoxin moieties, but the endotoxin preparations were consistently more active than the purified LPS preparations.

Adjuvants, Immunologic↗
PubMed2000

Cytokine responses to treponema pectinovorum and treponema denticola in human gingival fibroblasts.

Human gingival fibroblasts were challenged with Treponema pectinovorum and Treponema denticola to test three specific hypotheses: (i) these treponemes induce different cytokine profiles from the fibroblasts, (ii) differences in cytokine profiles are observed after challenge with live versus killed treponemes, and (iii) differences in cytokine profiles are noted from different gingival fibroblast cell lines when challenged with these treponemes. Three normal gingival fibroblast cell cultures were challenged with T. pectinovorum and T. denticola strains, and the supernatants were analyzed for cytokine production (i.e., interleukin-1alpha [IL-1alpha], IL-1beta, IL-6, IL-8, IL-10, gamma interferon, macrophage chemotactic protein 1 [MCP-1], platelet-derived growth factor, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor). Unstimulated fibroblast cell lines produced IL-6, IL-8, and MCP-1. T. pectinovorum routinely elicited the greatest production of these cytokines from the fibroblast cell lines, increasing 10- to 50-fold over basal production. While T. denticola also induced IL-6 and IL-8 production, these levels were generally lower than those elicited by challenge with T. pectinovorum. MCP-1 levels were significantly lower after T. denticola challenge, and the kinetics suggested that this microorganism actually inhibited basal production by the fibroblasts. No basal or stimulated production of the other cytokines was observed. Significant differences were noted in the responsiveness of the various cell lines with respect to the two species of treponemes and the individual cytokines produced. Finally, dead T. pectinovorum generally induced a twofold-greater level of IL-6 and IL-8 than the live bacteria. These results supported the idea that different species of oral treponemes can elicit proinflammatory cytokine production by gingival cells and that this stimulation did not require live microorganisms. Importantly, a unique difference was noted in the ability of T. pectinovorum to induce a robust MCP-1 production, while T. denticola appeared to inhibit this activity of the fibroblasts. While the general cytokine profiles of the fibroblast cell cultures were similar, significant differences were noted in the quantity of individual cytokines produced, which could relate to individual patient variation in local inflammatory responses in the periodontium.

Chemokine CCL2↗
PubMed2005

Upregulation of intercellular adhesion molecule 1 and proinflammatory cytokines by the major surface proteins of Treponema maltophilum and Treponema lecithinolyticum, the phylogenetic group IV oral spirochetes associated with periodontitis and endodontic infections.

Treponema maltophilum and Treponema lecithinolyticum belong to the group IV oral spirochetes and are associated with endodontic infections, as well as periodontitis. Recently, the genes encoding the major surface proteins (Msps) of these bacteria (MspA and MspTL, respectively) were cloned and sequenced. The amino acid sequences of these proteins showed significant similarity. In this study we analyzed the functional role of these homologous proteins in human monocytic THP-1 cells and primary cultured periodontal ligament (PDL) cells using recombinant proteins. The complete genes encoding MspA and MspTL without the signal sequence were cloned into Escherichia coli by using the expression vector pQE-30. Fusion proteins tagged with N-terminal hexahistidine (recombinant MspA [rMspA] and rMspTL) were obtained, and any possible contamination of the recombinant proteins with E. coli endotoxin was removed by using polymyxin B-agarose. Flow cytometry showed that rMspA and rMspTL upregulated the expression of intercellular adhesion molecule 1 (ICAM-1) in both THP-1 and PDL cells. Expression of proinflammatory cytokines, such as interleukin-6 (IL-6) and IL-8, was also induced significantly in both cell types by the Msps, as determined by reverse transcription-PCR and an enzyme-linked immunosorbent assay, whereas IL-1beta synthesis could be detected only in the THP-1 cells. The upregulation of ICAM-1, IL-6, and IL-8 was completely inhibited by pretreating the cells with an NF-kappaB activation inhibitor, l-1-tosylamido-2-phenylethyl chloromethyl ketone. This suggests involvement of NF-kappaB activation. The increased ICAM-1 and IL-8 expression in the THP-1 cells obtained with rMsps was not inhibited in the presence of the IL-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1. Our results show that the Msps of the group IV oral spirochetes may play an important role in amplifying the local immune response by continuous inflammatory cell recruitment and retention at an infection site by stimulation of expression of ICAM-1 and proinflammatory cytokines.

Cell Line↗
PubMed1980

Susceptibility of rabbits venereally infected with Treponema paraluis-cuniculi to superinfections with Treponema pallidum.

Three female rabbits, venereally infected with Treponema paraluis-cuniculi between five and eight months previously, had treponeme-containing genital lesions and positive results to serological tests for syphilis. These rabbits and four normal female rabbits were challenged with Treponema pallidum intradermally on the shaved back with triplicate doses of 10(4), 10(3), 10(2), and 10 treponemes (all at different sites). Significantly fewer syphilitic lesions developed in the rabbits previously infected with T paraluis-cuniculi (4/36) compared with the control rabbits (37/48), and the mean size of the lesions (diameter of induration) was significantly less (4 mm compared with 10 mm). It appears that previous venereal infection of female rabbits with T paraluis-cuniculi induced a level of cross-immunity against infection with T pallidum. Protection was however not complete.

Animals↗
PubMed1981

Sequential changes in susceptibility to Treponema pallidum of rabbits previously infected with Treponema paraluis-cuniculi.

Rabbits immunised with virulent Treponema paraluis-cuniculi were challenged intradermally with graded doses of Treponema pallidum at three, five, seven, 12, and 30 months to ascertain the level of protection to T pallidum at various intervals after immunisation.Rabbits challenged at three months after immunisation showed no protection against T pallidum and developed syphilitic lesions significantly faster than the control rabbits, which suggests that the former rabbits were immunosuppressed. Some protection was evident at five and seven months after immunisation, as fewer inoculation sites developed syphilitic lesions with challenges of 10(3), 10(2), and 10 T pallidum and lesions developed significantly slower with 10(6) challenge. Two rabbits showed significant protection at 12 months after immunisation but a third, presumably still immunosuppressed, developed lesions significantly faster than the control rabbits after challenge. At 30 months after immunisation one rabbit was completely protected and developed no lesions after challenge; the other rabbit showed only partial protection against challenge with 10(4), 10(3), and 10(2) but complete protection against challenge with 10 T pallidum.T paraluis-cuniculi appeared to induce a state of immunosuppression by three months after infection; in one rabbit this may have been 12 months. In most immunised rabbits, however, limited cross-protection to low challenge doses of T pallidum developed by five months and was also detectable at seven and 12 months. Only one rabbit was completely resistant to challenge with 10(4)T pallidum after 30 months and another was only partly immune. Thus, T paraluis-cuniculi infection does not produce a rapid pronounced cross-immunity to T pallidum in rabbits, which may thus limit its usefulness as a vaccine against syphilis.

Animals↗
PubMed1981

Treponema-specific and antilipoidal 19S(IgM) antibodies in penicillin-treated and untreated rabbits after infection with Treponema pallidum.

The titres of treponema-specific and antilipoidal 19S(IgG) antibodies were determined in rabbits infected intratesticularly with Treponema pallidum. One group of rabbits was treated with penicillin the other served as control. Using different serological tests it was shown that 19S(IgM) antibodies were still detectable eight months after infection at about the same titres in both groups. In contrast, 19S(IgM) antibody titres in patients with syphilis became undetectable within three to six months after penicillin treatment. It is suggested therefore that the rabbit is not a reliable model for studying the effect of penicillin in human T pallidum infections.

Animals↗
PubMed1983

Albumin requirement of Treponema denticola and Treponema vincentii.

Treponema denticola and Treponema vincentii were found to require albumin, oleic acid, and thiamine pyrophosphate (TPP) for growth. Previous studies indicated that commercial human alpha globulin, which is 50% albumin, was the only serum fraction that supported growth of these two oral treponemes. The alpha-globulin proteins were separated from the contaminating albumin with Affi-Gel Blue affinity chromatography. Both the albumin fraction and one of the alpha-globulin fractions were required for growth of T. denticola. Oleic acid was supplied by the alpha-globulin fraction and the albumin functioned as a chelator to maintain a low level of free fatty acid in the medium. Purified serum albumin (bovine or human) could substitute for the alpha-globulin fraction that contained albumin. Optimal growth of T. denticola and T. vincentii was in a medium supplemented with 0.4% (w/v) delipified albumin, 0.08 mg/mL of sodium oleate, and 25 micrograms/mL of TPP. Serum albumin tightly bound TPP (0.5 microgram of TPP per milligram of albumin). Optimal growth of T. denticola was only in an albumin-oleate supplemented medium with sufficient TPP to saturate the albumin binding sites and provide excess free TPP. Albumin bound long-chain fatty acids and thus detoxified the medium. Neither starch- nor charcoal-treated Tween 80 (polysorbitan monooleate) replaced albumin for optimal growth. Short-chain fatty acids supported only limited growth of T. denticola when added to a medium with TPP or to a medium that contained 0.4% delipified albumin and TPP.

Animals↗
PubMed1990

Minimal inhibitory concentrations of five antimicrobials against Treponema hyodysenteriae and Treponema innocens.

The minimal inhibitory concentrations of carbadox, dimetridazole, lincomycin, ronidazole, and tiamulin against isolates of Treponema hyodysenteriae and Treponema innocens were determined by an agar-dilution method. The results obtained indicated that tiamulin was the most effective antimicrobial in vitro against T. hyodysenteriae, followed by carbadox. Dimetridazole, lincomycin, and ronidazole had poor efficacy in vitro against the T. hyodysenteriae isolates. Isolates of T. innocens were more sensitive to the various antimicrobials. Carbadox and tiamulin were the most effective in vitro, followed by ronidazole, dimetridazole, and lincomycin.

Animals↗
PubMed2001

Treponema socranskii, Treponema denticola, and Porphyromonas gingivalis are associated with severity of periodontal tissue destruction.

BACKGROUND: The aim of the present study was to identify Treponema socranskii in addition to Treponema denticola and Porphyromonas gingivalis by polymerase chain reaction (PCR), and to clarify the relationship between the presence of these microorganisms and the severity of clinical periodontal parameters. METHODS: Saliva and subgingival plaque collected from 123 subjects (38 aggressive periodontitis patients, 65 chronic periodontitis patients, 20 healthy patients) were subjected to PCR to detect the aforementioned 3 microorganisms. RESULTS: Detection frequencies of T. socranskii, T. denticola, and P. gingivalis in plaque samples from aggressive periodontitis patients (71.1%, 73.7%, 84.2%, respectively) and chronic periodontitis patients (89.2%, 93.8%, 95.3%) were much higher than those from healthy subjects (30%, 5.0%, 10.0%). In aggressive and chronic periodontitis patients, these 3 species of bacteria were detected frequently at sites that showed deep periodontal pockets and severe attachment loss. The percentage of these bacteria-positive sites increased as the gingival index score of chronic periodontitis patients increased. T. socranskii was frequently detected together with T. denticola or P. gingivalis at the same sites, and coexistence of these microorganisms was frequently observed in deep periodontal pockets of aggressive periodontitis patients. CONCLUSIONS: T. socranskii, T. denticola, and P. gingivalis were frequently detected in periodontitis patients by PCR. The prevalence of these 3 microorganisms was correlated with various clinical parameters. Taken together, our findings suggest that T. socranskii, T. denticola, and P. gingivalis are associated with the severity of periodontal tissue destruction.

Adolescent↗
PubMed1990

[Chemical composition and biological activities of lipopolysaccharides extracted from Treponema denticola and Treponema vincentii].

Lipopolysaccharides (LPSs) were isolated from Treponema denticola (T. denticola) and Treponema vincentii (T. vincentii) by the phenol/water (PW) and the phenol/chloroform/petroleum-ether (PCP) procedures. 1) T. denticola PW-LPS (LPS isolated by the PW procedure), PCP-sup-LPS (LPS isolated by the PCP procedure in the supernates of ultracentrifugation), PCP-ppt-LPS (precipitated LPS isolated by the PCP procedure, obtained after ultracentrifugation), and T. vincentii PCP-ppt-LPS were composed of carbohydrate, hexosamine, protein, fatty acid, and phosphorus. T. vincentii PW-LPS was contained major amount of carbohydrates and small amount of fatty acids. 2-keto-3-deoxyoctonic acid (KDD) was not detected in these LPSs. 2) The major fatty acids of T. denticola PW-LPS and PCP-sup-LPS were palmitic, stearic, oleic, and linoleic acids. The major fatty acids of T. vincentii PCP-ppt-LPS were palmitic, stearic, myristic, oleic, and linoleic acids. Hydroxy fatty acids were not detected. 3) Glucose, galactose, and mannose were comprised in T. denticola PW-LPS. Glucose, galactose, and arabinose were comprised in T. denticola PCP-sup-LPS. Glucose and galactose were comprised in T. vincentii PCP-ppt-LPS. 4) The Limulus amoebocyte lysate (LAL) clotting activity of T. denticola PW-LPS was 1/10, as compared with that of Escherichia coli (E. coli) UKT-B LPS standard. The LAL clotting activities of T. denticola PCP-sup-LPS, T. vincentii PW-LPS, and T. vincentii PCP-ppt-LPS were 1/100, as compared with that of E. coli LPS standard. 5) Five hundred micrograms/kg of T. denticola PCP-sup-LPS was pyrogenic in rabbits. Two thousand micrograms/kg of T. vincentii PCP-ppt-LPS was pyrogenic in rabbits. 6) T. denticola PCP-sup-LPS and T. vincentii PCP-ppt-LPS were capable of increasing or decreasing the release of lysosomal enzymes from human polymorphonuclear leukocytes.

Endotoxins↗
PubMed1983

Effect of local and systemic macrophage activation in hamsters on infection with Treponema pertenue and Treponema pallidum Bosnia A.

The role of nonspecific macrophage activation in the destruction of treponemes needs to be defined. Studies have been hindered by an inability to confirm that macrophages have enhanced bactericidal activity at the site of treponemal infection. We show that subcutaneous and intravenous vaccination with BCG (Mycobacterium bovis) induces macrophage activation in hamsters, as determined by an enhanced ability to suppress the growth of Listeria monocytogenes in the livers, spleens, and inguinal lymph nodes. However, hamsters challenged in the inguinal region with Treponema pertenue during periods of enhanced microbial resistance (3 to 8 weeks after BCG vaccination) developed lesions faster and with more necrosis. Increased numbers of treponemes were recovered from the regional lymph nodes of BCG-vaccinated hamsters than from nonvaccinated controls, although the differences were not statistically significant. No pathological differences were detected in BCG-vaccinated and non-vaccinated hamsters challenged with Treponema pallidum Bosnia A. These studies demonstrate that BCG vaccination influences the pathogenesis of some treponemal diseases without inducing macrophage-mediated treponemicidal activity.

Animals↗
PubMed1985

Identification of glycosylated protein antigens of Treponema pallidum and Treponema phagedenis.

Comparison of autoradiographies of intrinsically [35S] methionine and [14C] glucosamine labeled Treponema pallidum (Nichols strain) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed four glycosylated proteins with molecular weights 30,500, 33,000, 35,000, and 59,000. T. phagedenis (biotype Reiter) was comparatively investigated and showed only two glycosylated proteins with molecular weights 33,000 and 34,000. The at the first time in treponemes identified glycosylated proteins could be precipitated with homologous human antibodies and characterized as antigens. By comparison with 125I surface labeling of T. pallidum and T. phagedenis it is suggested that the glycosylated protein antigens are localized on the surface of these treponemes.

Antigens, Bacterial↗
PubMed1979

Comparative behavior of virulent strains of Treponema pallidum and Treponema pertenue in gradient cultures of various mammalian cells.

Two strains of virulent Treponema pallidum and two of virulent T. pertenue were investigated for their ability to attach to and survive in gradient cultures of five different mammalian cells under aerobic conditions. The strains of T. pallidum studied were the high-rabbit-passage Nichols and the low-rabbit-passage KKJ. The former was known to readily attach to cottontail rabbit epithelial cells (Sf1Ep) and to survive in the virulent state for up to 21 days. We therefore compared attachment of the other virulent treponemes with that of T. pallidum (Nichols). The KKJ strain of T. pallidum behaved in a fashion similar to T. pallidum (Nichols) in all of the cultures. Both strains exhibited preferential attachment to cells of Sf1Ep and those derived from the ear of a nude athymic (nu/nu) mouse. In these cultures, we observed a consistent three- to fivefold increase in attached treponemes up to 12 days after initial inoculation. The strains of T. pertenue were the human-derived Gauthier and cynocephalus-derived FB. These two strains of T. pertenue also attached to cells of all five types of cultures, but in smaller numbers than were seen with T. pallidum and equally to all of the cultures. Neither preferential attachment to Sf1Ep and nude mouse ear cells nor increased attachment with time was seen.

Animals↗
PubMed1984

Molecular characterization of glycoprotein antigens on surface of Treponema pallidum: comparison with nonpathogenic Treponema phagedenis biotype Reiter.

Four glycoproteins of Treponema pallidum were identified by intrinsic [14C]glucosamine labeling. Only two glycoproteins were demonstrated in T. phagedenis biotype Reiter with the same technique. Glycoproteins of both treponemes were characterized as antigens and shown to be localized within the outer membranes of the microorganisms.

Antigens, Bacterial↗
PubMed1987

Activation of the classical and alternative pathways of complement by Treponema pallidum subsp. pallidum and Treponema vincentii.

Both in vivo and in vitro studies have indicated that complement plays an important role in the syphilitic immune responses. Few quantitative data are available concerning activation of the classical pathway by Treponema pallidum subsp. pallidum, and no information is available on treponemal activation of the alternative pathway. Activation of both pathways was compared by using T. pallidum subsp. pallidum and the nonpathogen T. vincentii. With rabbit and human sources of complement, both organisms rapidly activated the classical pathway, as shown by hemolysis of sensitized sheep erythrocytes and by the generation of soluble C4a. With human sources of complement, both organisms also activated the alternative pathway, as shown by hemolysis of rabbit erythrocytes and by the generation of soluble C3a in the presence of magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). During incubation, organisms remained actively mobile and did not lyse, indicating that activation was a function of complement reactivity with the intact outer treponemal surface. In addition, freshly harvested T. pallidum subsp. pallidum immediately activated both pathways of complement; preincubation of organisms did not enhance complement reactivity. T. vincentii was a more potent activator of this pathway. T. pallidum subsp. pallidum contained almost four times as much surface sialic acid as T. vincentii did. When sialic acid was enzymatically removed from T. pallidum subsp. pallidum, enhanced activation of the alternative pathway was detected. It is proposed that T. pallidum subsp. pallidum retards complement-mediated damage by the alternative pathway through surface-associated sialic acid. This may be an important virulence determinant that enables these organisms to readily disseminate through the bloodstream to infect other tissues.

Animals↗
PubMed1999

Development of a system for expressing heterologous genes in the oral spirochete Treponema denticola and its use in expression of the Treponema pallidum flaA gene.

The present communication describes the construction of a new Escherichia coli-Treponema denticola shuttle vector based on the naturally occurring spirochete plasmid pTS1 and the expression of the heterologous T. pallidum flaA gene from the plasmid in T. denticola. This new shuttle vector system should prove useful in characterizing virulence factors from unculturable pathogenic spirochetes.

Flagellin↗
PubMed1978

Genetics of Treponema: relationship between Treponema pallidum and five cultivable treponemes.

Three genetically distinct groups of treponemes have been identified by saturation reassociation assays using 125I-labeled treponemal DNAs. The three groups are (i) virulent Treponema pallidum (Nichols strain), (ii) T. phagedenis and its biotypes Reiter and Kazan 5, and (iii) T. refringens biotypes Nichols and Noguchi. There is no detectable DNA sequence homology (less than 5%) among the three groups. The groups have distinct guanine + cytosine contents: 52.4 to 53.7% for T. pallidum, 41.5% for T. refringens, and 38 to 39% for T. phagedenis.

Base Sequence↗