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.     

Hyaluronan, a possible ligand mediating Treponema denticola binding to periodontal tissue

Binding of Treponema denticola ATCC 35405 to glycosaminoglycans, fibrinogen, type I collagen and porcine periodontal ligament epithelial cells was studied using an enzyme-linked immunosorbent assay. T. denticola bound to hyaluronan (hyaluronic acid) and its hexameric fragments, whereas little or no binding was detected to chondroitin-4-sulfate or dermatan sulfate proteoglycan. Binding of T. denticola to hyaluronan gradually increased during the 2-h incubation time. In contrast, binding to fibrinogen and type I collagen was more rapid, peaking within 5 min. T. denticola also bound to microbeads coated with hyaluronan and formed visible aggregates in solution. Pretreatment of the bacteria with hyaluronan or fibrinogen inhibited binding to hyaluronan. Gelatin, bovine serum albumin, chondroitin-4-sulfate, chondroitin-6-sulfate, heparin, dermatan sulfate, glucuronic acid, N -acetylglucosamine and N -acetyl-galactosamine did not inhibit binding. Binding was also inhibited by heating T. denticola and by pretreatment of the spirochetes with sodium periodate, phenylmethylsulfonyl fluoride, and p-chloromercurybenzoic acid. All these treatments also inhibited the chymotrypsin-like activity of T. denticola. Hyaluronan strongly inhibited binding of T. denticola to epithelial cells, whereas the other glycosaminoglycans and N -acetyl-glucosamine did not. The results show that T. denticola binds to hyaluronan, possibly by a mechanism involving the chymotrypsin-like surface protein of T. denticola.     

Steroid 5α‐reductase activity of Treponema denticola

Introduction: Previously we have shown that reference and freshly isolated Treponema denticola cultures possess 5α‐reductase (5α‐R) and 3β‐ and 17β‐hydroxysteroid dehydrogenase activity. A gene matching the 3‐oxo‐5α‐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 5α‐R activity of T. denticola ATCC 33520. Methods: 5α‐R activity of cell‐free preparations was assayed with radioactive steroid substrates. 5α‐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 5α‐R was 5.5 and the preferred co‐factor was NADPH. The order of the steroids with respect to their 5α‐R substrate specificities was (in descending order): progesterone, 4‐androstenedione, testosterone and corticosterone. There are at least two intermediates in the synthesis of 5α‐dihydrocholesterol from cholesterol. Conclusion: These results suggest that the 3‐oxo‐5α‐steroid 4‐dehydrogenase family protein gene of T. denticola codes for a functional protein that resembles mammalian 5α‐R isoenzyme 2 with regard to co‐factor requirement and pH optimum.     

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.     

Inhibition of peptidase and glycosidase activities of Porphyromonas gingivalis, Bacteroides intermedius and Treponema denticola by plant extracts

Aqueous extracts from 5 plants used widely in Kenya as chewing sticks (mswaki) for the control of oral hygiene were tested for their ability to inhibit extracellular peptidase and glycosidase enzyme activities produced by the periodontopathic bacteria Porphyromonas gingivalis (formerly Bacteroides gingivalis), Bacteroides intermedius and Treponema denticola. The plants studied were Rhus natalensis, Cupressus hisitanica, Sida cordifolia, Olea africana and Euclea divinorum. Protease activities, including glycylprolyl dipeptidase and trypsin-like activities of P. gingivalis, chymotrypsin-like and glycylprolyl dipeptidase activities of B. intermedius and the trypsin-like activity of T. denticola, were particularly affected by extracts from Rhus natalensis and Euclea divinorum. Glycosidase activities were generally less affected with the notable exceptions of the inhibition of beta-mannosidase activity of P. gingivalis by all extracts and the inhibition of neuraminidase activity of T. denticola by Rhus natalensis and Euclea divinorum. Generally, these same proteolytic and glycosidic activities were inhibited by tannic acid and to lesser extents by gallic acid and gallic acid methyl ester. An inhibitory component, present in all extracts, exhibited physical and chemical properties identical to those of tannic acid. The inhibition of these enzyme activities is likely to reduce the virulence of these periodontophathic bacteria and to reduce the rate of dental plaque formation.     

Conservation and revised annotation of the Treponema denticola prcB‐prcA‐prtP locus encoding the dentilisin (CTLP) protease complex

Interstrain differences in antigenic surface proteins may reflect immunological pressure or differences in receptor specificity of the antigen. Treponema denticola exhibits considerable interstrain variability in its major surface protein (Msp), but no studies have addressed this issue in dentilisin (CTLP), a surface protease complex that has a significant role in T. denticola–host interactions in periodontal disease. Furthermore, the genome annotation of the prcB‐prcA‐prtP operon encoding dentilisin contains apparent errors and lacks a deduced PrtP amino acid sequence. To address these issues we analysed the protease operon from diverse T. denticola strains, as well as clones of the ATCC 35405 Type strain from which the genome sequence and original GenBank prtP sequence were derived. 6xHis‐tagging of the PrtP C‐terminus in ATCC 35405 demonstrated absence of the ‘authentic frameshift’ in PrtP reported in the genome databases. We propose that T. denticola genome annotations be updated to reflect this new information. PrcB and the PrtP N‐terminal region that includes the catalytic domain were highly conserved in common laboratory strains and clinical isolates of T. denticola. Dentilisin proteolytic activity varied considerably between strains. Antibodies against PrcB, PrcA and PrtP from the type strain recognized these proteins in most T. denticola strains. PrtP varied up to 20% over the C‐terminal 270 residues between strains. The PrtP C‐terminal eight‐residues (DWFYVEYP) was present in all strains, with two strains containing an additional Y‐residue preceding the stop codon. Such conserved PrtP domains may be required for interactions with PrcA and PrcB, or for substrate interactions.     

Isolation and chemical analysis of a lipopolysaccharide from the outer membrane of the oral anaerobic spirochete Treponema pectinovorum

Isolation of a putative lipopolysaccharide from the surface of the oral treponeme, Treponema pectinovorum, revealed it to contain larger amounts of 3-deoxy-D-manno-octulosonic acid compared with other oral Treponema species. This molecule was isolated from the outer membrane of T. pectinovorum and had chemical characteristics of a putative lipopolysaccharide. The yield of lipopolysaccharide was between 0.6% and to 1.1% of the bacterial dry weight. The purified molecule was resistant to the action of proteinases and consisted of both sugars and lipids. 3-Deoxy-D-manno-octulosonic acid and hexoses accounted for 6.1-8.7% and 17.6-20.2%, respectively of the dry weight. Carbohydrate compositional analysis revealed the presence of glucose, galactose, 2-acetamido-2-deoxy-glucose, rhamnose and 6-deoxy-talose in the molar ratio of 1.00:0.96:0.19:0.88:0.98, respectively. No heptose was detected. The fatty acid analysis determined the presence of straight chain, C13:00, C14:00, C15:00 and C17:00 acids, as well as branched chain, C13:00, C14:00 and two species of C15:00, acids. Electrophoretic analysis indicated that the lipopolysaccharide was present as two major species.     

Detection of Treponema denticola in saliva obtained from patients with various periodontal conditions

The aim of the study was to determine the prevalence of Treponema denticola in saliva of periodontally diseased and healthy patients and its relationship with the periodontal status. A 16S rRNA-based polymerase chain reaction detection method was used to determine the prevalence of T. denticola in whole saliva samples from patients with chronic periodontitis (CP, n = 37), aggressive periodontitis (AgP, n = 24), and healthy subjects (n = 28). The periodontal status of each subject was assessed by criteria based on probing depth, clinical attachment loss, and extent of periodontal breakdown. Risk factors were assessed individually and adjusted for confounding using a binary logistic regression model. The results showed that the prevalence of T. denticola in CP patients was significantly higher than those in healthy and AgP subjects (P < 0.05). Odds ratio analysis revealed a positive association for CP group/T. denticola-positive and smoking/T. denticola-positive subjects. Furthermore, all clinical measurements were significantly greater (P < 0.05) for T. denticola-positive subjects compared to T. denticola-negative subjects. After binary logistic regression analysis, both T. denticola and smoking were independently and strongly associated with development of CP. It was concluded that when used in conjunction with an optimized clinical examination protocol, this assay may offer a rapid, useful, and cost-effective tool for monitoring the presence of T. denticola in noninvasive clinical samples from both healthy and diseased patients and correlating it with the amount and extent of periodontal breakdown.     

Development of an FhbB based chimeric vaccinogen that elicits antibodies that block Factor H binding and cleavage by the periopathogen Treponema denticola

Treponema denticola is a proteolytic anaerobic spirochete and key contributor to periodontal disease of microbial etiology. As periodontal disease develops and progresses, T. denticola thrives in the hostile environment of the subgingival crevice by exploiting the negative regulatory activity of the complement protein, factor H (FH). FH bound to the cell surface receptor, FhbB (FH binding protein B), is competent to serve as a cofactor for the Factor I mediated‐cleavage of the opsonin C3b. However, bound FH is ultimately cleaved by the T. denticola protease, dentilisin. As the T. denticola population expands, the rate of FH cleavage may exceed its rate of replenishment leading to local FH depletion and immune dysregulation culminating in tissue and ligament destruction and tooth loss. The goal of this study was to develop a T. denticola FhbB based‐vaccine antigen that can block FH binding and cleavage and kill cells via antibody‐mediated bactericidal activity. Tetra (FhbB‐ch4) and pentavalent fhbB (FhbB‐ch5) chimerics were engineered to have attenuated FH binding ability. The chimerics were immunogenic and elicited high‐titer bactericidal and agglutinating antibody. Anti‐Fhb‐ch4 antisera blocked FH binding and cleavage by the T. denticola protease, dentilisin, in a dose dependent manner. Precedent for the use of FH binding proteins comes from the successful development of two FDA approved vaccines for type B Neiserria meningitidis. This study is the first to extend this approach to the development of a preventive or therapeutic vaccine (or monoclonal Ab) for periodontal disease.     

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.     

Fibronectin levels of unstimulated saliva from naval recruits with and without chronic inflammatory periodontal disease

Past studies have suggested that gingival crevicular fluid is produced more readily in persons with severe periodontal diseases than in persons with healthy gingivae. In this study, salivary fibronectin was selected as an index of total gingival crevicular fluid flow. Our purpose was to determine whether a relationship could be found between salivary fibronectin level and periodontal disease status. Unstimulated saliva was collected from 20 healthy and 20 periodontally-diseased naval recruits. The periodontally-diseased subjects included 10 with localized juvenile periodontitis and 10 with moderate to severe periodontitis. Mean subject ages and salivary flow rates were similar for the 2 groups. Although 2 of the periodontally-diseased subjects showed unusually high fibronectin levels, the mean level for the remaining 18 subjects did not differ significantly from the mean of the healthy group, and no association of periodontal disease status with salivary fibronectin content was seen. Consequently, it was not evident from salivary fibronectin levels that the content of gingival crevicular fluid in unstimulated whole saliva differed significantly for persons with or without severe periodontal disease, except possibly for extreme cases of disease.     

Detection of tet(M) and tet(Q) using the polymerase chain reaction in bacteria isolated from patients with periodontal disease

Olsvik B, Olsen I, Tenover FC. Detection of tet (M) and tet (O) using the polymerase chain reaction in bacteria isolated from patients with periodontal disease.
The polymerase chain reaction was used to examine 114 tetracycline-resistant anaerobic and facultative anaerobic bacterial isolates from patients with periodontal disease for the tet (M) and tet (O) genes. A 740-base-pair fragment of the tet (M) gene was amplified from 84 of 114 isolates, and a 519-base-pair fragment of the tet (O) gene was amplified from 13 streptococcal isolates. Six of 7 tet racycline-resistant isolates of Veillonella spp. and tet racycline-resistant isolates of En-bacterium spp. ( n =3), Eubacterium saburreum ( n =1), Streptococcus intermedius ( n =5) and Gemella morbillorum ( n =2) all harbored the tet (M) gene. The tet (M) and tet (O) negative as well as selected positive isolates were tested for the tet (K) and tet (L) genes using DNA probes. All isolates of Staphylococcus spp. ( n =11) hybridized with the tet (K) probe. None of the isolates tested hybridized with the probe for tet (L). This is the first report of the tet (M) gene in the facultative bacterium G. morbillorum and in E. saburreum .