Distribution and characterization of plasmids in oral anaerobic spirochetes

Fifteen oral spirochete strains belonging to the species Treponema denticola, Treponema vencentii and Treponema socranskii as well as 9 fresh clinical isolates were screened for the presence of extrachromosomal plasmid DNA by a modified alkaline lysis procedure. A 2.6–kb plasmid was detected in both T. denticola ATCC 33520 and T. denticola e'. The 2.6–kb plasmid from T. denticola e' was shown to be similar to pTDl, previously reported by Ivic et al. in T. denticola ATCC 33520 on the basis of molecular weight, restriction endonuclease profile and DNA:DNA hybridization. T. denticola ATCC 33520 and T. denticola e'share 65% DNA homology and belong to different serological groups. This dissimilarity has been reconfirmed by specific immunofluorescence using polyclonal and monoclonal antibodies. A plasmid-free T. denticola ATCC 33520 was identified. Comparative studies have shown no antigenic, morphological, or genetic differences between the plasmid-bearing and the plasmid-free strain. In addition, screening of fresh clinical isolates of spirochetes revealed the presence of a 4.2–kb plasmid in 4 of these strains.     

Multiple forms of the major phenylalanine specific protease in Treponema denticola

The 160, 190 and 270 kDa outer sheath proteases of Treponema denticola ATCC 35404 were found to be multiple forms of the major 91 kDa phenylalanine protease (PAP) by immunoblotting using anti-91 kDa specific antibodies. Multiple forms of the phenylalanine protease were also found in 2 other T. denticola strains studied, ATCC 33520 and the clinical isolate GM-1. Protein, proteolytic and Western blot analyses using antibodies against the PAP and the major outer sheath protein (MSP) indicated that the 190 and 270 kDa proteases were protein complexes formed by the MSP and the PAP. These complexes dissociated by storage in 0.3% or higher SDS concentrations. The purified PAP was found to completely degrade keratin, but was unable to degrade native actin either in its monomeric or polymerized form. The association of the MSP adhesin with a protease capable of degrading host native proteins may benefit the obtention of protein-based nutrients necessary to support the growth of these treponemes. These complexes may also play a role in the structural organization of T. denticola outer sheath.     

Involvement of treponemal surface-located protein and carbohydrate moieties in the attachment of Treponema denticola ATCC 33520 to cultured rat palatal epithelial cells

We studied the nature of attachment of Treponema denticola ATCC 33520 to a microscopically distinct population of rounded rat palatal epithelial cells. The motility of the freshly harvested spirochetes appeared not to be a prerequisite for attachment. Treatment of T. denticola ATCC 33520 with proteinase-K, heat, glutaraldehyde, formaldehyde and periodate oxidation decreased the attachment to the rounded rat palatal epithelial cells, indicating the involvement of protein and carbohydrate moieties. Trypsin treatment had no effect on the attachment. The attachment of T. denticola ATCC 33520 was decreased after treatment with native non-immune rabbit serum, native polyclonal rabbit serum, D-mannose, N-acetyl-D-galactosamine and sialic acid. The results indicate that the attachment of T. denticola ATCC 33520 to rounded rat palatal epithelial cells is mediated by trypsin-resistant adhesin(s) of protein and carbohydrate nature, with affinity for D-mannose, N-acetyl-D-galactosamine and sialic acid.     

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 (E_h) 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 H? 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.     

A common antigen of Treponema denticola and other Treponema species detected by monoclonal antibody

Murine monoclonal antibodies against Treponema denticola were produced. One monoclonal antibody (MSA257) reacted with 34,000 dalton antigens of all T. denticola strains, including ATCC strains and our isolates. This monoclonal antibody also reacted with antigens of other treponema strains tested.     

Homology among Treponema denticola plasmids

Three of 16 isolates of Treponema denticola were found to contain small (2.0–2.7 kb) cryptic plasmids. These were pTD1 from T. denticola ATCC 33520, pTD2 from strain T32A, and pTD3 from strain D3A1. These plasmids were characterized by restriction mapping and cloned into E. coli plasmid pUC19. Extensive homology between these plasmids was revealed by Southern blot, and single-stranded DNA regions were found by neutral Southern blots and S₁ nuclease mapping. These plasmids were not found in serovars usually associated with human periodontal disease nor are they universal in all T. denticola strains and serovars.     

口腔螺旋体的胶原结合蛋白

目的 研究口腔螺旋体胶原结合蛋白的致病作用。方法 用免疫印迹、免疫金标电 体外粘附实验等方法分析了口腔螺旋体胶原结合蛋白的性质及其在菌细胞表面的定位。结果 Ｔｒｅｐｏｎｅｍａ ｄｅｎｔｉｃｏｌａ菌细胞外膜上有２７０００Ⅴ型,Ｔｒｅｐｏｎｅｍａ ｓｏｃｒａｎｓｋｉｉ菌有９５０００Ⅳ型和１１０００Ⅰ型,Ｔｒｅｐｏｎｅｍａ ｍｅｄｉｕｍ有９５０００Ⅳ型胶原结合蛋白;Ｔｄ和Ｔｓ可粘附于Ⅳ型胶原表面,而Ｔｍ     

Effects of a mixed infection with Porphyromonas gingivalis and Treponema denticola on abscess formation and immune responses in mice

Porphyromonas gingivalis and Treponema denticola have been found together in lesions of human periodontitis. We examined the ability of a mixed infection by both bacteria to synergistically form abscesses and disturb immune responses in mice. Absorbance of an invasive P. gingivalis 16-1 strain grown in tryptic soy broth and T. denticola ATCC 33520 strain grown in TYGVS medium were adjusted. BALB/c mice were injected with 200 microliters of the cell suspension at a site on the lateral dorsal area. The sizes of the subsequent subcutaneous abscesses were measured with a caliper gauge, and the area was expressed in square mm. Mixed infections by P. gingivalis and T.denticola produced larger abscesses than those formed after mono-infections by either P. gingivalis or T.denticola. The abscesses caused by mixed infection reached their maxima on the 6th day and maintained that size for the subsequent 5 days. The delayed type hypersensitivities against extracted antigens of P.gingivalis in mixed infection mice were significantly lower than those in the mono-infected mice. However, the IgG response to sonicated antigen of P.gingivalis did not differ between the two groups. The sizes of the abscesses caused by mixed infections in mice immunized with whole cells of P.gingivalis 16-1 were compared to those caused in sham-immunized mice. The average size of the abscess caused by mixed infection in immunized mice did not differ from that in sham-immunized mice, but many of the abscesses in immunized mice ruptured on the 4th or 5th day, followed by recovery in two weeks. These results suggest that mixed infection with P.gingivalis and T.denticola attenuates protective immune responses.     

Identity of 1:2:1 and 2:4:2 subgingival spirochetes by DNA hybridization

A group of 1:2:1 and 2:4:2 subgingival spirochetes, well characterized by transmission electron microscopy, biochemical tests, cellular fatty acid and carbohydrate analyses, and ribotyping, was recently suggested to represent new treponemal species. The present study used DNA hybridization to examine this possibility. When DNA of a representative strain (no. 16) of the 8 1:2:1 spirochetes examined was labeled by iodination, it showed, after SI nuclease treatment, from 58 to 104% (average 76%) homology with DNA from the 1:2:1 spirochetes. 94% homology with DNA from the type strain of Treponema socranskii and of T. socranskii subsp. socranskii , i.e., ATCC 35536T, and 62% homology with DNA from T. socranskii subsp. buccale , strain ATCC 35534^T. Similarly treated DNA from a representative strain (no. 3) of 8 2:4:2 spirochetes exhibited from 90 to 105% (average 97%) homology with DNA from the 2:4:2 spirochetes, and 85% and 87% homology, respectively, with DNA from Treponema denticola strains ATCC 33520 and FDC T1. There was a negligible degree of homology between the 1:2:1 and 2:4:2 spirochetes. Thus, all the 2:4:2 spirochetes belonged to T. denticola . 1:2:1 strains with DNA homology levels >70% (5 strains) belonged to T. socranskii or T. socranskii subsp. socranskii , while those with homology levels from 58 to 63% (3 strains) most likely belonged to other subspecies of T. socranskii .     

Cloning and expression of protease genes from Treponema denticola in Escherichia coli

A gene bank of random fragments of chromosomal DNA from Treponema denticola ATCC 33520 was constructed in the bacteriophage vector lambda L47.1. The gene bank was plated on Escherichia coli C600 and screened for the presence of plaques in which enzyme activity was expressed, using overlays of fluorogenic synthetic substrates and a two-step procedure in which immunological screening was followed by enzyme assays of immunopositive lysates. Recombinants expressing trypsin-like, chymotrypsin-like and proline iminopeptidase activities were found. The gene for the trypsin-like activity was subcloned into plasmid pJDC9 and maintained in E. coli.     

Resistance to human beta-defensins is common among oral treponemes

BACKGROUND/AIMS: Oral treponemes are implicated in the pathogenesis of periodontal disease. We have previously shown that Treponema denticola ATCC type strains and strain GM-1 are resistant to killing by human beta-defensins (hbetaD)-1 and -2. We hypothesize that resistance to beta-defensins is a common feature of oral treponemes, which allows colonization and persistence in the oral cavity. In this study, we tested additional isolates of T. denticola, as well as six other species of treponemes, for resistance to hbetaD-1, -2 and -3. We also examined the four ATCC strains of T. denticola and strain GM-1 for resistance to hbetaD-3. METHODS: Resistance was determined by motility and Alamar Blue assays for metabolic activity. RESULTS: All T. denticola strains tested were resistant to hbetaD-1, -2 and -3, with the exception of strain Ambigua, which was sensitive to hbetaD-2 and -3. All other treponemes except Treponema vincentii were resistant to hbetaD-1. Treponema pectinovorum was sensitive to hbetaD-2, while T. vincentii, T. pectinovorum and Treponema maltophilum were sensitive to hbetaD-3. Escherichia coli was used as a control organism and was killed by all three defensins. CONCLUSION: Resistance to the constitutively expressed hbetaD-1 may assist treponemes in initial colonization of epithelial surfaces, while resistance to the inducible hbetaD-2 and -3 would allow some treponemes to survive in active periodontal lesions.     

Isolation and characterization of a 53 kDa major cell envelope protein antigen from Treponema denticola ATCC 35405

A major cell envelope protein was purified from the cell envelope fraction of Treponema denticola ATCC 35405 by ion exchange chromatography after extraction with Zwittergent 3–14. Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis showed a relative molecular mass of 53 kDa for this protein with a pI of 6.3–6.8. Amino acid analysis revealed that this protein contained high proportions of hydrophobic amino acids (40.4%), and no cysteine could be detected. The N-terminus of the protein was blocked to Edman degradation. Rabbit antiserum raised against the purified 53 kDa protein reacted with the outer envelope of the T. denticola cell surface as confirmed by immunoelectron microscopy. This rabbit antiserum reacted with 4 of the 11 strains of treponemes tested in this study. Sera from 9 to 18 periodontitis patients reacted strongly with this 53 kDa cell envelope protein of T. denticola as determined by immunoblotting analysis.     

Binding of hemin and Congo red by oral hemolytic spirochetes

Colony-forming units or cells in suspension of oral anaerobic spirochetes ( Treponema denticola, Treponema vincentii and Treponema socranskii ) bind hemin and Congo red. Hemin or Congo red binds to a hydrophobia polypeptide receptor that is located in the outer membrane of the bacterial cells and it has a relative molecular mass of 47 kDa. These oral spirochetes also lyse sheep erythrocytes to produce beta-hemolytic zones around colony-forming units. The oral spirochetes may acquire iron for growth when they lyse erythrocytes and bind heme from which they may sequester and transport iron into the cells.     

Human serum antibodies recognize Treponema denticola Msp and PrtP protease complex proteins

BACKGROUND/AIMS: Treponema denticola outer membrane proteins are postulated to have key roles in microbe-host interactions in periodontitis. Because there are no reports of in vivo expression of these putative virulence factors, we examined several T. denticola strains to determine whether sera from human subjects recognized specific T. denticola outer membrane proteins. METHODS: Soluble extracts were prepared from exponential phase cultures of T. denticola strains representing three serotypes, from defined T. denticola mutants defective in Msp (major surface protein) or PrtP lipoprotein protease complex (CTLP; dentilisin), and Escherichia coli strains expressing distinctly different T. denticola Msp. Extracts were subjected to Western immunoassays using archived human serum samples. RESULTS: Human serum antibodies (immunoglobulin G class) recognized multiple protein bands in T. denticola strains. In the parent strain ATCC 35405, these included bands at 72-, 53-, 40-, and 30-kDa. Bands corresponding to Msp and the PrtP protease complex proteins were absent in isogenic msp and protease complex mutants, respectively. Individual human sera showed specificity for one or more Msp types. CONCLUSIONS: This is the first definitive report of human serum antibody responses to specific T. denticola antigens. T. denticola Msp and the proteins comprising the PrtP lipoprotein protease complex are expressed in vivo and are immunogenic in humans. Human antibody recognition of Msp exhibits strain specificity and is consistent with strain serotyping. These results demonstrate the utility of T. denticola isogenic mutants in characterizing host immune responses to periodontal pathogens.     

Steroid 5alpha-reductase activity of Treponema denticola

INTRODUCTION: Previously we have shown that reference and freshly isolated Treponema denticola cultures possess 5alpha-reductase (5alpha-R) and 3beta- and 17beta-hydroxysteroid dehydrogenase activity. A gene matching the 3-oxo-5alpha-steroid 4-dehydrogenase family protein (gene ID: 2739284; locus tag: TDE2697) has been identified in T. denticola ATCC 35405. The aim of the work presented here was to optimize assay conditions and determine steroid substrate specificities for the 5alpha-R activity of T. denticola ATCC 33520. METHODS: 5alpha-R activity of cell-free preparations was assayed with radioactive steroid substrates. 5alpha-R-reduced products were identified using thin-layer chromatography and a radioisotope scanner. Assay conditions were optimized for co-factor, buffer and pH requirements. Apparent substrate specificities were determined for progesterone, 4-androstenedione, testosterone and corticosterone. The time-course for metabolism of radiolabelled progesterone and cholesterol substrates was investigated with anaerobic cultures. RESULTS: The optimum pH for 5alpha-R was 5.5 and the preferred co-factor was NADPH. The order of the steroids with respect to their 5alpha-R substrate specificities was (in descending order): progesterone, 4-androstenedione, testosterone and corticosterone. There are at least two intermediates in the synthesis of 5alpha-dihydrocholesterol from cholesterol. CONCLUSION: These results suggest that the 3-oxo-5alpha-steroid 4-dehydrogenase family protein gene of T. denticola codes for a functional protein that resembles mammalian 5alpha-R isoenzyme 2 with regard to co-factor requirement and pH optimum.     

Analysis of the genetic control of antibody response to Actinobacillus actinomycetemcomitans by immunoblotting in inbred strains of mice

Genetic regulation of the immune response may be involved in the onset and progression of an early-onset type of periodontitis. We analyzed the genetic control of the primary antibody response to Actinobacillus actinomycetemcomitans in inbred strains of mice using an immunoblot technique. Mice of 5 independent inbred strains, 6 H-2 congenic strains and 4 B10 intra-H-2 recombinant strains were immunized with sonicated extracts of A. actinomycelemcomitans . On the seventh day their sera were examined for reactivity to the antigenic components of this organism. Western blot analysis clearly distinguished 2 different groups of antigens, one consisting of common antigens (molecular weights, 28, 34, 36 and 40 kDa) that reacted with sera from all strains and one consisting of specific antigens (molecular weights 31, 65 and 69 kDa) that reacted only with sera from distinct strains. Blot analysis of sera from H-2 congenic strains demonstrated that the reactivity to the second group of antigens was regulated by the H-2 complex. In B10 intra-H-2 recombinant strains, only the 1-A^b allotype strains produced immunoglobulin G antibody that reacted to the 65 kDa antigen. This evidence indicates that the primary immune response to the A. actinomycetemcomitans antigen with a molecular weight of 65 kDa is controlled by genes in the 1-A subregion of the H-2 complex. This 65 kDa antigen was also highly reactive with some human sera from early-onset periodontitis patients. Further analysis of this antigen is required.     

Use of randomly cloned DMA fragments for the identification of oral spirochetes

DNA probes were produced for the detection and identification of 4 cultivable species of oral spirochetes, Treponema denticola, Treponema socranskii, Treponema vincentii and Treponema pectinovorum. To obtain probe sequences, chromosomal DNA, isolated from representative strains within each species, was cloned in Escherichia coli K-12. Cloned DNA fragments were screened for the ability to hybridize to DNA only from homologous strains. Several such fragments were identified and shown to be specific when tested against a series of DNAs from gram-negative and gram-positive oral bacteria. The selected probe sequences were semi-conserved within strains of T. denticola and T. socranskii such that restriction fragment length polymorphism (RFLP) was observed. In the case of T. socranskii, RFLP was useful in distinguishing between the 3 known subspecies. Chromosomal DNA fragments from 2 strains of T. vincentii failed to cross-hybridize, under stringent conditions, to genomic DNA from each of these strains. The hybridization probes were suitable for the identification of clinical isolates of T. denticola and could be used to detect the presence of individual Treponema species in mixed cultures. On this basis, the probes were used successfully to detect T. denticola in uncultured plaque samples.     

Identification of the major surface protein antigens of Porphyromonas gingivalis using IgG antibody reactivity of periodontal case-control serum

The identity of the major surface antigens of Porphyromonas gingivalis was investigated. Outer membranes of P. gingivalis strains W83, W50, 381 and NCTC 11843 were prepared following inactivation of the trypsin-like enzyme activity. Three proteins, molecular weight 115, 55 and 40 kDa, were major components of the outer membranes of strains W83 and W50 and were also present in strains 381 and NCTC 11834. Two proteins, 55 and 47 kDa, were released from the cells during the sonication step of the outer membrane preparation procedure. Immunoblots using preparations of P. gingivalis W83 and serum from a case-control study of adult periodontitis demonstrated higher mean antibody reactivity in the case population to all the major proteins except for the 115 kDa outer membrane protein, which was recognized equally well by both populations. We conclude that the 55, 47 and 40 kDa proteins are important surface antigens of P. gingivalis. Characterization of the structure and function of these components should lead to an improved understanding of the host-parasite interactions in adult periodontitis.     

Comparative study of six random oral spirochete isolates

Six anaerobic oral spirochetes designated strains a, b, c, d, e, and e' were isolated randomly from periodontal pockets and were maintained in pure culture. The strains were found to belong to the genus Treponema . All strains were similar in cultural, morphological, biochemical, and physiological properties with the exception of the following features: strains c and d were more fastidious nutritionally for sodium bicarbonate as a medium supplement than the other strains; they differed in the number of axial fibrils; and they were also serologically distinct from strains a, b, e, and e' by agglutination tests and fluorescent immunoassays. However, DNA homology studies indicated that all the isolates were strains of Treponema denticola with divergent phenotypic and serological properties. Serovar a and serovar c, representing two different serogroups, have been deposited in the American Type Culture Collection with the accession numbers ATCC 35405 and ATCC 35404, respectively.     

Amended biochemical characteristics and phylogenetic position of Treponema medium

Umemoto et al. (1997, Int J Syst Bacteriol 47, pp. 67-72) proposed spirochete strain G7201, isolated from the periodontal pocket of an adult patient, as a new species, Treponema medium. They deposited this strain in the American Type Culture Collection (ATCC) as type strain ATCC 700293T. Recently, ATCC suggested that there is a discrepancy between the previous report and the results obtained by ATCC in biochemical tests on T. medium ATCC 700293T. In this study, we re-examined and verified the biochemical characteristics of T. medium. The fermentation pattern of carbohydrates of T. medium resembled that of Treponema vincentii and Treponema denticola, but T. medium was clearly differentiated from T. vincentii in the production of indole, and from T. denticola in the hydrolysis of esculin. Also, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profile analysis and phylogenetic comparison of 16S rDNA sequences revealed that T. medium is clearly differentiated from any established treponemal species, which supports the validity of the proposal of Treponema medium as a new species.