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PubMed1996

Development of a gene transfer system in Treponema denticola by electroporation.

Treponema denticola is strongly implicated in the etiology of periodontal diseases. However, genetic transformation of this organism has not been reported. We now demonstrate a gene transfer system in T. denticola by electroporation using a broad host plasmid pKT210 as a shuttle vector. Plasmid extraction, Southern blot hybridization as well as the polymerase chain reaction indicated the presence of the plasmid in T. denticola transformants. The restriction patterns of plasmid pKT210 rescued from the T. denticola transformants in Escherichea coli suggested that some of the rescued plasmids were identical to the original pKT210, but some of them had been modified. This transformation system could be a potentially useful tool for genetic manipulation of oral spirochetes.

DNA, Bacterial↗
PubMed1997

Characterization of protease activities in Capnocytophaga spp., Porphyromonas gingivalis, Prevotella spp., Treponema denticola and Actinobacillus actinomycetemcomitans.

Protease activities in cell sonicates of defined bacterial strains were examined using peptide substrates and class-specific inhibitors. Capnocytophaga spp. all produced serine dipeptidyl peptidase activity and arginine/lysine, elastase- and chymotrypsin-like enzymes with some metalloprotease characteristics. The elastase-like activity was strongest in Capnocytophaga sputigena, but the others were greatest in Capnocytophaga gingivalis. The latter also had a separate arginine-specific enzyme which appeared not to be present in the other two species. Porphyromonas gingivalis showed serine dipeptidyl peptidase activity and very strong arginine and lysine cysteine protease activities. Prevotella spp. had inhibitor-resistant dipeptidyl peptidase activity and arginine cysteine protease activity that was much weaker but biochemically similar to P. gingivalis. Treponema denticola possessed a strong trypsin-like serine protease activity as well as very weak dipeptidyl peptidase and chymotrypsin-like activities that were sensitive to some cysteine protease reagents. Actinobacillus actinomycetemcomitans showed a novel alanine- and lysine-specific activity, but its nature was unclear.

Aggregatibacter actinomycetemcomitans↗
PubMed1997

Environmental effects on the growth and proteolysis of Treponema denticola ATCC 33520.

The effect of pH, redox potential, O2 and H2 on the growth and proteolytic activity of Treponema denticola ATCC 33520 was studied in a chemostat at different growth rates. The peptidase and protease activities were estimated using different amido-methyl coumarin derivatives and azocasein. The maximum growth rate of T. denticola ATCC 33520 was 0.14 h-1. Reduction of the growth rate of T. denticola by 50-60% gave: an increase in cell mass of 150-200%, a higher acetogenesis and a shift of the pH optimum. The protease and phenylalanine peptidase activities seemed to be of greater importance for the growth of T. denticola ATCC 33520 than the rather low arginine and proline peptidase activities. The redox potential (Eh) played a secondary role. At microaerophilic conditions with 1-5% O2, the cultures maintained a redox potential below -311 mV and an optimal acetogenesis. The presence of H2 induced a marked growth stimulation of T. denticola ATCC 33520. It is concluded that the cell mass and proteolytic activity of T. denticola ATCC 33520 are modulated by the growth rate and the pH and to a lesser extend by the redox potential and presence of O2. Stagnation of the exudate-flow influences these factors and will lead to an increase of the spirochetal population and proteolysis in the periodontal pocket.

Culture Media↗
PubMed1998

Virulence of a polymicrobic complex, Treponema denticola and Porphyromonas gingivalis, in a murine model.

The effect of a polymicrobic infection employing Treponema denticola and Porphyromonas gingivalis in the murine lesion model was used to determine the synergistic virulence of these two periodontopathic bacteria. At high doses of P. gingivalis W50, addition of T. denticola in the infection mixture had no effect on the formation and size of the spreading lesion caused by this microorganism. However, at low P. gingivalis challenge doses, T. denticola significantly enhanced the virulence of P. gingivalis compared with monoinfection of this microorganism. A potential role of the trypsin-like protease enzyme activity of P. gingivalis in this synergistic virulence was tested using P. gingivalis mutants deficient (i.e., BEI) or devoid (i.e., NG4B19) of this protease activity. These findings demonstrated that T. denticola-P. gingivalis complexes exhibit enhanced virulence in this model and that even using a polymicrobic challenge infection, the trypsin-like protease activity was important to P. gingivalis virulence expression.

Abscess↗
PubMed2006

Association of Eubacterium nodatum and Treponema denticola with human periodontitis lesions.

BACKGROUND: The purpose of the present investigation was to compare the levels, proportions and percentage of sites colonized by 40 bacterial species in subgingival plaque samples from periodontally healthy subjects and patients with chronic periodontitis to seek possible pathogens other than the consensus pathogens Porphyromonas gingivalis and Tannerella forsythia. METHOD: Subgingival plaque samples were taken from the mesial aspect of each tooth in 635 subjects with chronic periodontitis and 189 periodontally healthy subjects. The samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization (total samples = 21,832). Mean counts, % DNA probe counts and percentage of sites colonized at >10(5) were determined for each species in each subject and then averaged in each clinical group. Significance of difference between groups was determined using the Mann-Whitney test. Association between combinations of species and periodontal status was examined by stepwise logistic regression analysis. Analyses were repeated using a subset of subjects from both clinical groups who had proportions of P. gingivalis plus T. forsythia less than the median (4.42%) found in periodontally healthy subjects. All analyses were adjusted for multiple comparisons. RESULTS: For the 824 subjects the consensus pathogens P. gingivalis and T. forsythia as well as Eubacterium nodatum and Treponema denticola had significantly higher mean counts, proportions and percentage of sites colonized in samples from subjects with periodontitis than from periodontally healthy subjects. There were significantly more Capnocytophaga gingivalis, Streptococcus gordonii and Veillonella parvula in periodontally healthy subjects. E. nodatum, T. denticola, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp. vincentii all had higher counts and proportions in diseased than healthy subjects who had low proportions of P. gingivalis and T. forsythia. Logistic regression analysis indicated that the same species groups were associated with disease status after adjusting for the proportions of the other species. CONCLUSIONS: This investigation confirmed the strong association of P. gingivalis and T. forsythia with chronic periodontitis and emphasized a strong association of E. nodatum and T. denticola with periodontitis whether in the presence or absence of high levels of the consensus pathogens. Other species, including S. oralis, Eikenella corrodens, S. intermedius and F. nucleatum ssp. vincentii, were associated with disease when P. gingivalis and T. forsythia were present in low proportions.

Adult↗
PubMed2004

The major outer sheath protein of Treponema denticola inhibits the binding step of collagen phagocytosis in fibroblasts.

Bacterial infections contribfute to the chronicity of connective tissue lesions in part by perturbing extracellular matrix remodelling processes. We examined a novel mechanism by which the major outer sheath protein (Msp) of the spirochaete Treponema denticola disrupts matrix remodelling mediated by intracellular digestion of collagen. The initial collagen-binding step of phagocytosis was examined in human gingival fibroblasts and Rat-2 fibroblasts. Cells were pretreated with Msp or vehicle, and binding of collagen-coated beads was measured by flow cytometry. Exposure to Msp induced a dose- and time-dependent decrease in cells that bound collagen beads; the inhibition of binding was reversed by absorption with anti-Msp antibodies. Msp-treated fibroblasts remained viable but underwent actin reorganization, including the assembly of a dense meshwork of subcortical actin filaments. Shear force assays showed that Msp abrogated collagen-binding interactions in the minimal affinity range required for stable adhesion. Fluorescence microscopy and immunoblotting showed equivalent amounts of beta1 integrin associated with collagen beads bound to Msp- and vehicle-treated cells. Photobleaching experiments found a similar percentage mobile fraction of beta1 integrins recovered in bleached areas of the plasma membrane. In contrast, Msp-induced inhibition of collagen binding was reversed by beta1 integrin affinity-activating antibodies and by latrunculin B, which prevented subcortical actin assembly. We conclude that native Msp of T. denticola inhibits the binding step of collagen phagocytosis in fibroblasts by inducing subcortical actin filament assembly and restricting affinity modulation of beta1 integrins. We suggest that, like Msp, bacterial toxins that target the cytoskeleton may also perturb the signalling networks required for cellular engagement of matrix ligands.

Actins↗
PubMed2006

Specific and sensitive diagnosis of syphilis using a real-time PCR for Treponema pallidum.

A real-time PCR assay with a Taqman probe was developed that targeted the polA gene of Treponema pallidum. The test was validated using an analytical panel (n = 140) and a clinical panel of genital samples (n = 112) from patients attending a sexually transmitted infections clinic. High sensitivities and specificities of 94-100% were achieved using two real-time PCR platforms, the Rotor-Gene and the iCycler. The assay can be completed within 2 h, enabling reporting in <8 h. This fast and robust assay is suitable for implementation in routine laboratories for diagnosing primary syphilis.

DNA Polymerase III↗
PubMed1993

Characterization of the serum requirement for macrophage-mediated killing of Treponema pallidum ssp. pallidum: relationship to the development of opsonizing antibodies.

Serum pools were collected from rabbits bled at various times after intra-testicular infection with Treponema pallidum ssp. pallidum. These were tested for their ability to opsonize T. pallidum and promote killing of the organisms by macrophages. Compared to normal sera, significant opsonization was first seen on day 10 of infection as measured by both ingestion (P < 0.001) and macrophage-mediated killing (P = 0.006); significant levels of functional antibodies persisted through 300 days of infection. Although opsonic activity peaked early in infection, antibodies that promoted optimal macrophage-mediated killing developed much later, suggesting that these two functions may represent activities of antibodies with differing specificities or affinities. The initial development of antibodies that augment both phagocytosis and killing corresponds with the in vivo clearance of treponemes from the primary site of infection. These observations support the hypothesis that macrophages are the major effector mechanism for elimination of T. pallidum during early syphilis infection.

Animals↗
PubMed1991

Circulating immune complexes in experimental syphilis and their relation to immunological response against Treponema pallidum.

It was found that circulating immune complexes (CIC) were formed in rabbits at different times after infection with Treponema pallidum. The CIC which appeared at the beginning of the disease were short-lived (2-6 weeks) but those appearing later than 20 weeks after infection remained for 10-25 weeks. CIC contained both IgM and IgG classes of immunoglobulin. The antibodies present in CIC were found to be specific and nonspecific for T. pallidum. The presence of CIC led to a marked decline of treponemal antibodies in rabbit sera. The cell-mediated immune response measured by the macrophage migration inhibition (MMI) test at the beginning of the disease (up to 12 weeks) was not decreased. However, when syphilis lasted for more than 14 weeks and when CIC were formed mainly from IgG, a distinct decrease in the ability of lymphocytes to cause MMI was observed. These findings strongly suggest that IgG-complexes suppress the immunological responsiveness of lymphocytes against T. pallidum which in turn facilitates the multiplication of treponemes in the host.

Animals↗
PubMed1996

Expression and sequence analysis of a Treponema pallidum gene, tpn38(b), encoding an exported protein with homology to T. pallidum and Borrelia burgdorferi proteins.

An Escherichia coli clone containing recombinant plasmid C19 was identified from a Treponema pallidum genomic DNA library by in situ immunoassay. E. coli maxicells containing pC19 synthesized a treponemal protein doublet of 39.2 and 38.2 kDa, designated TpN38(b). Pulse-chase and protein processing studies showed that TpN38(b) is synthesized with a cleavable amino-terminal signal peptide. A 2.0-kb fragment of pC19 containing the tpn38(b) gene was subcloned and sequenced. The tpn38(b) gene is 1029 nucleotides long and encodes a protein of 343 amino acids with a calculated molecular mass of 37.9 kDa. The deduced amino acid sequence of TpN38(b) has homology with the T. pallidum TpN35 lipoprotein and the Borrelia burgdorferi BmpA, BmpB, BmpC, and BmpD proteins.

Amino Acid Sequence↗
PubMed1997

Identification and characterization of the gyrB gene from Treponema pallidum subsp. pallidum.

The nucleotide sequence of a DNA gyrase B subunit gene (gyrB) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The organization of genes flanking the gyrB gene is unique in comparison to that of other bacteria. The gyrB gene encodes a 637 amino acid protein whose deduced sequence has a high degree of homology with type-II topoisomerase ATPase subunits (GyrB and ParE). Five type-II topoisomerase motifs, an ATP-binding site (Walker A), and amino acid residues that putatively interact with ATP, are highly conserved in the T. pallidum GyrB protein.

Adenosine Triphosphate↗
PubMed1997

Identification of the Treponema pallidum subsp. pallidum glycerophosphodiester phosphodiesterase homologue.

To identify potential opsonic targets of Treponema pallidum subsp. pallidum, a treponemal genomic expression library was constructed and differentially screened with opsonic and non-opsonic T. pallidum antisera. This method identified an immunoreactive clone containing an open reading frame encoding a 356 residue protein. Nucleotide sequence analysis demonstrated the translated protein to be a homologue of glycerophosphodiester phosphodiesterase, a glycerol metabolizing enzyme previously identified in Haemophilus influenzae, Escherichia coli, Bacillus subtilis and Borrelia hermsii. Sequence alignment analyses revealed the T. pallidum and H. influenzae enzymes share a high degree of amino acid sequence similarity (72%), suggesting that in T. pallidum this molecule may be surface exposed and involved in IgD binding as is the case with its counterpart in H. influenzae.

Amino Acid Sequence↗
PubMed1997

Identification and characterization of a Treponema pallidum subsp. pallidum gene encoding a DNA adenine methyltransferase.

The nucleotide sequence of a DNA adenine methyltransferase gene (dam) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The dam gene encodes a 303 amino acid protein whose deduced sequence has significant homology with DNA (N6-adenine) methyltransferases. T. pallidum Dam can be assigned to group alpha DNA amino methyltransferases based on the order of nine conserved motifs that are present in the protein. Digests of T. pallidum chromosomal DNA performed with isoschizomer restriction endonucleases (Sau3AI, DpnI, and MboI) confirmed the presence of methylated adenine residues in GATC sequences (Dam+ phenotype).

Amino Acid Sequence↗
PubMed1997

Identification, sequence, and expression of Treponema denticola mcpA, a putative chemoreceptor gene.

The nucleotide sequence of a methyl-accepting chemotaxis protein gene, mcpA, from Treponema denticola has been determined. The mcpA gene encodes a 729-amino acid protein whose deduced amino acid sequence has significant homology with several bacterial MCPs. T. denticola McpA contains two N-terminal transmembrane regions and two C-terminal putative methylation sequences that are separated by a highly conserved signaling domain. The organization of these structural features is characteristic of MCPs. The observed molecular mass of the in vitro synthesized McpA (76.0 kDa) correlates with the predicted molecular mass of the protein (80.1 kDa).

Amino Acid Sequence↗
PubMed1998

Mutagenesis of outer membrane virulence determinants of the oral spirochete Treponema denticola.

The pore-forming major surface protein (Msp) and the chymotrypsin-like protease (CTLP) of Treponema denticola ATCC 35405 have been implicated in periodontal pathogenicity. Allelic replacement mutations were constructed at two sites in the msp gene and at one site in the CTLP locus. All mutant strains lacked the Msp hexagonal array outer membrane ultrastructure. Strain CKE, in which the locus encoding CTLP was disrupted, produced no CTLP and had aberrant Msp expression. The msp mutant MHE lacked Msp, and produced increased levels of CTLP. In contrast, msp mutant MPE made small amounts of a truncated Msp, but produced no CTLP. Interactions between Msp and CTLP in transport or assembly of the outer membrane complex are proposed.

Alleles↗
PubMed1998

Effect of temperature and viscosity on the motility of the spirochete Treponema denticola.

Treponema denticola is an oral spirochete associated with periodontal diseases. Because bacterial motility is likely to be a potential virulence factor, we investigated the effect of viscosity and temperature on cell speed. In agreement with the work of others, translational motility was a function of the macroscopic viscosity of the medium. In addition, we found that although the speed of spirochetes was slow at 25 degrees C (4 microns s-1), it increased quite markedly at 35 degrees C (19 microns s-1). The results indicate that both viscosity and temperature are critical factors in T. denticola translational motility.

Culture Media↗
PubMed1999

Molecular pathogenesis of the cell surface proteins and lipids from Treponema denticola.

Treponema denticola, frequently isolated from the human oral cavity, is thought to be a major pathogen of human periodontal disease. Recent developments in molecular analysis have clarified the surface structure of this microorganism and the characteristics of its pathogenic factors. Structural analysis of the outer sheath showed T. denticola to have a new type of outer membrane lipid. Limited exposure of the major outer sheath protein is suggested by electron-microscopic analysis. A protease-deficient mutant has revealed the roles of the protease in the organization of the outer sheath material and in T. denticola pathogenicity. The surface features that contribute to the pathogenicity of T. denticola in periodontal disease are gradually being elucidated, and are reviewed.

Bacterial Adhesion↗
PubMed2001

Biochemical characterization of an active pyrophosphate-dependent phosphofructokinase from Treponema pallidum.

An active pyrophosphate-dependent phosphofructokinase containing a six residue polyhistidine tag has been cloned from Treponema pallidum, and characterized biochemically. The phosphofructokinase has pH optima for activity of 8.0 for both the forward and reverse reactions. The apparent K(m) for pyrophosphate was 0.042 mM (V(max) of 141 U mg(-1) protein) and for fructose-6-phosphate, 0.529 mM. The apparent K(m) for the reverse reaction for fructose-1,6-diphosphate was 0.267 mM (V(max) of 42.4 U mg(-1) protein). The enzyme appears to be both a dimer and non-allosteric.

Binding, Competitive↗