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.     

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.     

Role of Aggregatibacter actinomycetemcomitans in glutathione catabolism

Introduction:  Our previous studies demonstrated that three enzymes, γ-glutamyltransferase (GGT), cysteinylglycinase (CGase) and cystalysin, are required for the catabolism of glutathione to produce hydrogen sulfide (H₂S) in Treponema denticola . In this study, we examined glutathione catabolism in Aggregatibacter actinomycetemcomitans .
Methods:  The GGT and CGase of A. actinomycetemcomitans were determined by biological methods and GGT was characterized using a molecular biological approach.
Results:  A. actinomycetemcomitans showed GGT and CGase activity, but could not produce H₂S from glutathione. The addition of recombinant T. denticola cystalysin, an l -cysteine desulfhydrase, to whole cells of A. actinomycetemcomitans resulted in the production of H₂S from glutathione. Subsequently, we cloned A. actinomycetemcomitans GGT gene ( ggt ) and overexpressed the 63 kDa GGT protein. The recombinant A. actinomycetemcomitans GGT was purified and identified. The K _cat/ K _m of the recombinant GGT from N -γ- l -glutamyl-4-nitroaniline as substrate was 31/μ m /min. The activity of GGT was optimum at pH 6.9–7.1 and enhanced by thiol-containing compounds.
Conclusion:  The results demonstrated that A. actinomycetemcomitans had GGT and CGase activities and that the GGT was characterized. The possible role of A. actinomycetemcomitans in glutathione metabolism and H₂S production from oral bacteria was discussed.     

Serine protease activity is essential for thrombin-induced protein synthesis in cultured human dental pulp cells: modulation roles of prostaglandin E2

Chang MC, Lan WH, Chan CP, Lin CP, Hsieh CC, Jeng JH: Serine protease activity is essential for thrombin-induced protein synthesis in cultured human dental pulp cells: modulation roles of prostaglandin E₂. J Oral Pathol Med 1998; 27: 23–9. © Munksgaard, 1998.
Irritations and injuries to the dental pulp usually lead to different degrees of pulpal inflammation. To investigate the roles of thrombin and prostaglandins in the healing and inflammatory processes of dental pulp as well as their effects on pulpal protein synthesis, human dental pulp cell cultures were established and their protein production was measured with or without the presence of exogenous thrombin and prostaglandins. At concentrations of 1–25 U/ml, a-thrombin increased the protein synthesis to 1.4–2.3 fold over the vehicle control. On the contrary, 0.1 (μg/ml of prostaglandin E] (PGE₁ suppressed protein synthesis by 60%. Prostaglandin E₂ (PGE₂) also inhibited protein synthesis with an IC50 of 0.4 ug/ml. The stimulatory effects of thrombin (10 U/ml) can be inhibited by antithrombin III (2 U/ml) (a natural thrombin inhibitor) with heparin (2 U/ml), PPACK (D-Phe-Pro-ArgCH₂Cl) (20–50 ug/ml) (a serine protease inhibitor), and PGE₂ (0.5–1.0 μg/ml). Moreover, TRAP (20–40 μg/ml), a thrombin receptor agonist peptide, also exerted a stimulatory effect (1.21–1.37 fold). In conclusion, thrombin-induced protein synthesis by pulp cells is dependent on proteolytic activity, but not on binding to receptors. Both PGE₁ and PGE₂ exert suppressive effects on protein synthesis, indicating that interactions between thrombin and prostaglandins are important in regulating the inflammation, repair and regeneration of pulp tissue following injury.     

Common and specific antigens of several treponemes detected by polyclonal antisera against major cellular proteins

Thirteen polypeptide antigens with molecular weights ranging from 34 kDa to 83 kDa were selected and their antigenic behaviors and distribution were examined in 12 strains of microorganisms including Treponema, Borrelia, Leptospira and Leptonema. Immunoblot analysis demonstrated that 45 kDa and 83 kDa polypeptides of Treponema socranskii subsp. buccale ATCC 35534, 53 kDa antigen of Treponema denticola ATCC 33520 and 44 kDa polypeptide of the strain G7201 were strain-specific. The 34, 62, 66 and 84 kDa polypeptide antigens were detected in all 8 treponemal strains examined. T. denticola ATCC 33520 and ATCC 35404 possessed 38 kDa, 48 kDa, 52 kDa and 72 kDa common polypeptide antigens. All 12 strains possessed the 84 kDa polypeptide antigen. Immunoelectron microscopy demonstrated that the 34 kDa and 38 kDa polypeptide antigens were located on the axial flagella and that other polypeptide antigens were located on the outer envelopes or wall-membrane complexes.     

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.     

Combined inhibitory effect of fluoride and hypothiocyanite on the viability and glucose metabolism of Streptococcus mutans, se retype c

The separate and combined effects of peroxidase-generated hypothiocyanite (HOSCN/OSCN⁻) and F⁻ ions on glucose uptake and growth of Streptococcus mutans ATCC 25175 were investigated. S. mutans cells were grown to late exponential or stationary growth phase, harvested, washed and suspended in 2.0 ml of sterilized human whole saliva supplemented with 10 mM D-glucose. This saliva-bacteria mixture was supplemented with 5–150 μM H₂O₂ at pH 5.0 or 6.5. At pH 5.0, up to 103±21 uM HOSCN/OSCN⁻ was generated. After 20 h of incubation at 37°C, the saliva-bacteria suspension exposed to HOSCN/OSCN⁻ were plated on mitis salivarius agar plates and incubated anaerobically for 2 days. Identical experiments were made with F⁻ ions (0.5, 1.0 and 5.0 mM). Both HOSCN/OSCN⁻ and F⁻ caused a significant dose-dependent growth inhibition at pH 5.0, whereas no inhibition was observed at pH 6.5. When F- and HOSCN/OSCN⁻ were added simultaneously at pH 5.0, an additive effect of growth inhibition was observed. In glucose incorporation experiments the bacteria-saliva mixture was exposed to 1 μM HOSCN/OSCN⁻, 0.5 mM F⁻ or both. F⁻, HOSCN/OSCN⁻ or their combination in sterilized whole saliva at pH 5.0 caused 14.2, 67.8 and 74.2% inhibition, respectively. These observations indicate that F⁻ and HOSCN/OSCN⁻ ions have an additive inhibitory effect on S. mutans and therefore their combination is likely to be more antibacterial than either agent alone.     

The influence of chlorhexidine on superoxide generation by induced human neutrophils

Chlorhexidine directly stimulates the generation of superoxide anion (O₂) by neutrophils at nontoxic concentrations of the drug (less than 0.01%). The kinetics of drug-induced O₂ generation simulated that of receptor-mediated FMLP induction of O₂, but not that of PMA-induced synthesis. Nontoxic concentrations of chlorhexidine inhibited FMLP-induced generation of O₂ if the neutrophils were pretreated with chlorhexidine or if chlorhexidine and FMLP were added simultaneously. Cells activated first by FMLP were unaffected by chlorhexidine. These data support the concept that chlorhexidine interferes with the binding of FMLP to its receptor. Chlorhexidine inhibited both the velocity and the duration of FMLP-induced O₂ synthesis. In contrast, chlorhexidine promoted PMA-induced O₂ generation irrespective of the sequence of addition of chlorhexidine and PMA. Chlorhexidine decreased the lag interval between PMA induction and O₂ synthesis, and increased the velocity of synthesis, which leads us to propose that chlorhexidine increases PMA penetrance of neutrophils. Since neither nonionic detergents nor polycationic polymers separately minic the effect of chlorhexidine treatment, it appears that more than surfactant or charge effects are involved. The interrelation between the positive charge and the hydrophobic domain of chlorhexidine plays a role in the drug-neutrophil interaction which directly stimulates O₂ generation, and which modulates neutrophil responsiveness to FMLP and to PMA.     

Effect of outer membrane of Treponema denticola on bone resorption

The effect of the outer membrane (outer sheath) of Treponema denticola on bone resorption was studied. Bone resorption was measured by the release of previously incorporated ⁴⁵Ca from the shafts of the radii and ulnae of 19-day fetal rats. A treated-over-control ratio (T/C ratio) significantly greater than 1 indicated the stimulation of bone resorption by the test substance. The addition of outer membrane of T. denticola increased the release of ⁴⁵Ca from the assay bones. The minimum concentrations required to yield significant ⁴⁵Ca release from the assay bones were 15, 22 and 75 μg protein/ml for serovars a, b and c, respectively. These protein values corresponded to estimated lipopolysaccharide contents of 0.6, 0.8 and 2.8 μg/ml, based on 3-deoxy-2- manno -octulosonate analysis. Heat treatment of outer membrane (60° for 30 min) did not change the effect on ⁴⁵Ca release. Parathyroid hormone or prostaglandin E₂ known to act synergistically with lipopolysaccharides in bone resorption, was also added to the assay system. Neither prostaglandin E₂ at 10^-7 M nor parathyroid hormone at 40 ng/ml, by itself, increased ⁴⁵Ca release. However, in the presence of 10 μg protein/ml of outer membrane of serovar b at 120 h, the T/C ratio was increased to 1.31±0.07 and 1.58±0.118, respectively. These results suggest that a lipopolysaccharide-like material is present in the outer membrane of T. denticola that may be responsible for bone resorption in the in vitro system.     

Salivary effects on polymorphonuclear leukocyte functions

Respiratory burst, enzymatic degranulation and bacterial killing were investigated on peripheral blood polymorphonuclear leukocytes (polymorphonuclear leukocytes) incubated with a pool of salivary fluids elicited from healthy donors. Low saliva concentrations primed polymorphonuclear leukocytes for enhancement of O₂ consumption, O₂- and β-glucuronidase release and Staphylococcus aureus killing. Whole saliva, on the contrary, depressed all tested phagocytic activities.     

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 .     

The action of sodium fluoride on suspected periodontopathogens

Certain oal bacteria have been implicated in the initiation of chronic gingivitis and the progression of periodontitis. The present study was conducted to assess the influence of sodium fluoride on the in vitro growth and on various enzymes of Treponema denticola, Actinomyces actinomycetemcomitans, Fusobacterium nucleatum and 3 Bacteroides species. Complete inhibition of growth for 5 strains of T. denticola , 3 strains of A. actinomycetemcomitans , 3 black-pigmented strains of Bacteroides and 3 strains of F. nuclatum was observed with 40, 80, 160, and 320 μg/ml of F- respectively. The API system, a convenient and rapid method designed to detect 19 enzymes, was employed primarily to determine the relative sensitivity of demonstrable enzymes to F^- for the assay microorganisms. F^-concentrations of 10–60 μg/ml inhibited the acid phosphatase of T. denticola or the acid and alkaline phosphatase of A. actinomycetemcomitans . However, F^- at a concentration of 10–60 μg/ml did not influence any of the detectable enzymes of the other assay bacteria. Periplasmic phosphatases hydrolyze impermeable phosphate esters to compounds that could then be transported and utilized by the cells.     

Ultrasonic dispersion of pure cultures of plaque bacteria and plaque

Abstract – This study compared th somic sensitivity of 12 Gram-negative and two Gram-positive bacteria commonly encountered in plaque associated with periontal diseases. Pure bacterial cultures were groun to standard turbidity, diluted in 1/4 strength jprereduced anaerobically sterilized Ringer's solution, and aliquots dispersed for 0–180s, using and MSE sonic oscillator at 6 μm under 80% N² 10% H² and 10% CO². CO². Viable recoveries were determined on anaerobically cultured trypticase soy 5% blood agar plates. Breakage of T. denticola was assessed by electron microscopy. Gram-positive organisms tolerated sonication better than Gram-negative. A. viscosus was more resitant than Strep. sanguis. Gram-negative bacteria could be divided into groups according to their sensitivity. Eikenella corrodens was most resistant, followed by F. nucleatum. B. asaccharolyticus, Capnocytophaga gingivalis, A. actinomycetemcomitans, a strain (2097) of Group IV Racteroides, and B. melaninogenicus ss. intermedius ressted sonication better than "corroding" Bacteroides and oral Campylobacter. T. denticola, Selenomonas sputigena and Wolinella were most sensitive with viable counts which declined after sonication for 5–10s. Recoveries from plaque taken from five patients with periodontal diseases increased with sonication time, reaching higher values for supragingival than for subgingival samples.     

Effect of in vitro aging on Campylobacter rectus lipopolysaccharide-stimulated PGE2 release from human gingival fibroblasts

OBJECTIVE: We examined the influence of in vitro aging on Campylobacter rectus (C. rectus) lipopolysaccharide (LPS)-stimulated prostaglandin (PG) E₂ release from human gingival fibroblasts (HGFs).
MATERIALS AND METHODS: LPS was prepared from C. rectus ATCC33238. HGFs were established from healthy gingival tissue removed from three patients (donors A, B and C), aged 10–12 years. Aging of the cells in culture was determined with increasing population doubling. The cells were cultured until confluence, then stimulated with LPS (1.0 μg ml^-1), and the levels of PGE₂ in the medium were measured after 24 h by radioimmunoassay.
RESULTS: The LPS-stimulated PGE₂ production in each old cell (passage 17–20) was significantly increased to about 1.6–2.6 times than that in the corresponding young cells (passage 5–6). The gene expression of cyclooxygenase-2 mRNA in the old cells was higher than that in the young cells in response to LPS. In the absence of LPS, PGE, production levels in both the young and old cells were very low, and also at the same level. However, there was a higher level of LPS-stimulated PGE₂ production in the young cells from donor C compared to that in the old cells from donor B. The LPS-stimulated PGE₂ production in each young cell from donors A and C was almost equal to that in each old cell from donors B and A, respectively.
CONCLUSIONS: The results suggested that aging in HGFs may be one of the factors that take part in the stimulation of C. rectus LPS-stimulated PGE₂ production in old cells.     

PLAQUE PH IN CARIES-ACTIVE AND INACTIVE SUBJECTS MODIFIED BY SUCROSE AND FLUORIDE, WITH AND WITHOUT BICARBONATE-PHOSPHATE

abstract – Plaques were grown for 3 d by 16 caries-active and 17 caries-inactive subjects. During the growth, the subjects consumed tablets containing sucrose (C), sucrose with a 3% supplement of NaHCO₃+ KH₂PO₄ combination (BP, mole ratio 9 82/1), sucrose with the NaHCO₃+ KH₂PO₄+10 parts/10⁶ fluoride as NaF (BPF) or 10 parts/10⁶ F as NaF alone in the sucrose (F). Ten tablets of the respective qualities were consumed within 30 min in the experimental sessions. Plaque samples were taken before, during and after ingestion of the tablets and suspended in a small volume of distilled water. The pH dropped immediately after the start of ingestion of the sucrose, remained practically at the decreased level during ingestion, and after ingestion dropped even more in the caries-active plaques. Throughout the experiment, these plaques showed a lower average pH than the caries-inactive. The BPF additive elevated significantly the pH of the caries-active plaques from the respective sucrose values and the BP values, both during and after ingestion. The BP additive elevated the plaque pH only during ingestion and the F additive had no significant influence.     

Accelerated corrosion analysis of dental amalgams

Abstract – The corrosion of powdered conventional and high Cu dental amalgam was studied in vitro under fixed conditions (100% oxygen, pH 4, and constant weak mechanical action). Results were retrieved from X-ray diffraction of samples of amalgam and solid corrosion products Formed, in combination with recording of the HC1 consumed to maintain the fixed pH. In the conventional amalgam no corrosion of γ₁ occurred until all γ₂ had corroded, whereas in the high Cu amalgam corrosion of γ₁ occurred from the beginning, concurrent with corrosion of ε and ń. Corrosion products found were AgCl, Hg₂Cl₂, CuCl₂-3Cu(OH)₂, and SnO₂. The results may be interpreted as follows: in the conventional amalgam the matrix phase γ₁ is anodically protected against corrosion as long as any γ₂ remians; in the high Gu amalgam the least noble phases ε and ń do not protect γ₁ in a similar way.     

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

Protease activities in cell sonicates of denned 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 ala-nine- and lysine-specific activity, but its nature was unclear.     

Effect on plaque formation and acidogenicity of stored aqueous solutions of stannous fluoride

Abstract – Stored aqueous solutions of SnF₂ showed a reduced effect on plaque acidogenicity after 24 weeks, this being paralleled by reduced concentrations of Sn²⁺ and lowered pH. The plaque inhibiting effect was, however, maintained unchanged, indicating a different mechanism for this activity. Stored solutions caused a yellow dental stain probably consisting of SnS₂.