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.

Measurement of proteases in human subgingival dental plaque by fluorescence polarization.

Fluorescence polarization (FP) was examined as a rapid quantitative method to assay the proteases in subgingival plaque. Protease activity was measured by a decrease in FP at 0.5-min intervals over 5 min, using BODIPY-alpha-casein, a protein substrate. To quantitate activity, the least absolute deviation (LAD) slope for each assay was determined. Protease activity increased with the quantity of plaque (r=0.416, P<0.001). Of the 208 subgingival plaque samples, 87 contained detectable protease activity, with a mean of about 4 microg trypsin equivalents above a general background of 1 microg per site. The mean plaque protease activity of 89 paired samples from 15 individuals had decreased by 1.1 microg trypsin equivalents per site when measured at 8 months after tooth scaling and root planing (P<0.01). Most isolates of Porphyromonas gingivalis, Treponema denticola, Prevotella nigrescens, and Prevotella intermedia implicated in the pathogenesis of adult periodontitis exhibited high activity in the FP assay. The assay is rapid, quantitative and requires only one-tenth of the plaque sampled using a single pass with a Gracey curette at a single tooth site.

Mitogenic activity of the outer membrane of Treponema denticola.

The outer membrane (OM) was isolated by detergent extraction from Treponema denticola ATCC 35405, ATCC 33521 and ATCC 35404, representing serovars a, b and c, respectively, as well as from two fresh isolates of T. denticola. Strict precautions were undertaken against the introduction of contaminant lipopolysaccharide when the OM was isolated. The OM was active in mitogenic stimulation of C3H/HeOuJ mouse spleen cultures, but to a somewhat lesser extent than purified lipopolysaccharide (LPS) from Escherichia coli 055:B5. Polymyxin B only partially inhibited the response. Unheated OM abrogated mitogenic activity of E. coli LPS, but heated preparations enhanced the mitogenic activity of E. coli LPS, suggesting the presence of a heat-labile cytolytic factor associated with T. denticola OM in addition to a putative lipopolysaccharide and/or heat-stable lipoprotein.

BODIPY-alpha-casein, a pH-independent protein substrate for protease assays using fluorescence polarization.

BODIPY-alpha-casein is a new fluorescent protein substrate designed for fluorescence polarization studies to measure proteolytic activity at any pH over the range from pH 2 to 11. Kinetic protease assays in real-time were performed in 1 to 5 min using an FPM-1 fluorescence polarization instrument. A purified enzyme or bacterial culture was mixed with the BODIPY-alpha-casein in a buffer of an appropriate pH and the decrease in fluorescence polarization was automatically recorded at 0.5-min intervals. The initial decrease in fluorescence polarization with time was dependent on protease concentration. In 3-min assays at 37 degrees C, the sensitivity of detection was 8 mU for pepsin at pH 2.0, 1 mU for papain at pH 6.0, 0.6 mU for proteinase K at pH 7.4, and 2 mU for Streptomyces griseus alkaline protease at pH 11. Only 1-10 microliters of a growing culture was necessary to assay the protease activity of Porphyromonas gingivalis or Treponema denticola, oral bacteria that possess certain proteases on their surfaces. These assays have clinical applications, since certain pathogens use proteolytic activity as a virulence mechanism and differ from their nonpathogenic counterparts in this characteristic. Fluorescence polarization assays are simple, rapid, and reproducible.

Detection of pathogen-related oral spirochetes, Treponema denticola, and Treponema socranskii in dental plaque from dogs.

Spirochetes have been observed in dental plaque from dogs, but specific spirochetes have not been identified. In particular, it is not known whether treponemes associated with periodontal diseases in humans also occur in dogs, and whether, like in humans, detection of specific treponemes correlates with periodontal status of dogs. Forty-two dogs were grouped according to the worst periodontal condition in the mouth, as determined by overt signs of inflammation and pocket probing depths. A representative specimen of dental plaque was obtained by pooling subgingival plaque collected from three uniform reference sites, irrespective of periodontal status at selected sites. The presence of pathogen-related oral spirochetes. Treponema denticola, and T. socranskii was determined using specific monoclonal antibodies in an immunocytochemical microscopic assay. All three treponemes were detected in all groups, but a significantly greater proportion of dogs with pocket probing depths > or = 5 mm had detectable treponemes, compared to dogs that were in periodontal health.

Trypsin-like activity levels of Treponema denticola and Porphyromonas gingivalis in adults with periodontitis.

Treponema denticola (Td) and Porphyromonas gingivalis (Pg) are associated with human moderate and severe adult periodontal diseases. This study quantifies these two anaerobes and their trypsin-like (TL) activities in subgingival plaque collected from both clinically healthy and periodontally diseased sites of human periodontitis patients. Antigen levels of the microorganisms were determined by monoclonal antibodies and TL activities were measured by the fluorescent substrate Z-gly-gly-arg-AFC in a disc format. Significant positive correlations were observed between the antigen levels and the TL activities when the data were subjected to statistical analyses both on a site-specific and on a patient basis. Anaerobe synergism was found between Td and Pg in a continental US population, and positive correlations were found between anaerobe levels (individually and total) and clinical indicators of adult periodontitis.

Fatty acid profiles of the outer membrane of ATCC strains 35405, 35404 and 33521 of Treponema denticola.

The fatty acid composition of the outer membrane (outer sheath) of Treponema denticola is not known. This study examined the fatty acid profiles of the outer membranes of T. denticola ATCC strains 35405, 35404, 33521. Homogeneous outer membranes were prepared from the three strains of T. denticola. The fatty acids were extracted and converted to methyl esters, and their mass spectra were determined with a sensitive Hewlett-Packard 5880A-5970 gas chromatography-mass spectrometry system. Fatty acids were identified by comparing the unknown fatty acid mass spectra to computer stored known mass spectra standards. Dodecanoic, 2 hydroxy dodecanoic, tridecanoic, tetradecanoic, pentadecanoic, hexadecanoic, 2-hydroxy hexadecanoic and octadecanoic acid were found in the assay spirochetes yielding correlation indices (r) of 0.8-1.00. Isotetradecanoic acid was found in the outer membranes of strains 33521 and 35405 (r = 0.913-0.967). Anteiso pentadecanoic and heptadecanoic acids were found in the outer membrane of strains 33521 and 35404 (r = 0.941-0.996), while cis 9, 12 octadecadienoic was found only in the outer sheath of strain 35405 (r = 0.922-0.958). The average concentration of dodecanoic, tridecanoic, tetradecanoic, pentadecanoic, hexadecanoic, heptadecanoic and octadecanoic acid in the outer membranes of strains 35405, 35404 and 33521 were as follows. Strain 35404: 138, 178, 845, 296, 751, not detected, and 699 nanogram per mg dry weight of the outer membrane. Strain 35404: 96, 125, 670, 306, 597, 38 and 249 nanograms per mg dry weight of the outer membrane. Strain 33521: 323, 135, 1650, 125, 9080, 235 and 618 nanograms per mg dry weight of the outer membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of in vitro growth of Treponema denticola by extracellular growth factors produced by Porphyromonas gingivalis.

Cell-free culture filtrates of Porphyromonas gingivalis grown in Wilkins-Chalgren broth stimulated the growth of six strains of Treponema denticola in 1186 broth when compared with the effect of uninoculated WC. The pH of the 1186 broth was not altered by the addition of either culture filtrate or WC, and all media were fully reduced prior to inoculation with T. denticola. Growth was also stimulated by factors precipitated from the culture filtrate with 90% (NH4)2SO4, 50% cold ethanol, or 50% cold acetone, and by factors retained after dialysis of the culture filtrate through a membrane with a molecular weight cut-off of 50 kDa. Growth factor activity was eliminated by heating of the culture filtrate at 55 degrees C for 4 h. An ether extract of the culture filtrate containing acetic, butyric, isobutyric, isovaleric, propionic, and phenylacetic acids did not stimulate growth. Since subgingival plaque from periodontal pockets colonized with T. denticola also contains P. gingivalis, certain extracellular proteins with molecular weights greater than 50 kDa produced by P. gingivalis may act as growth factors for T. denticola in the microenvironment of the periodontal pocket.

Treponema denticola and Porphyromonas gingivalis as prognostic markers following periodontal treatment.

Subgingival plaque samples were collected from individuals with advanced periodontitis before and 3 to 11 weeks after scaling and root planing periodontal treatment. The plaque levels of Treponema denticola and Porphyromonas gingivalis antigens were measured before and after treatment by a quantitative immunoassay procedure using monoclonal antibodies specific for these oral bacteria. A decrease in mean levels of T. denticola (P less than .05) and P. gingivalis antigens (P less than .09) were observed following periodontal therapy. Improved health, as measured by a decrease in probing depth, was associated with a decrease in T. denticola antigen (P less than .05). These results suggest that the T. denticola levels of successfully treated sites decreased, while non-responding sites had levels of this microbial marker which were equal to or greater than the pre-treatment levels. These results provide additional evidence that T. denticola is associated with human adult severe periodontal disease, and can serve as a prognostic marker for disease recurrence.

Bacterial synergy of Treponema denticola and Porphyromonas gingivalis in a multinational population.

Treponema denticola and Porphyromonas gingivalis have been associated with human adult severe periodontitis. In this study, we quantified these putative pathogens in subgingival plaque samples collected from 74 Fijians, 74 Colombians and 73 U.S. Americans stationed at the Multinational Force and Observers encampment in the Sinai Desert, Egypt. A contingency table of T. denticola and P. gingivalis frequency revealed a highly significant synergistic relationship. We discovered that the occurrence of T. denticola apparently requires the presence of P. gingivalis. This represents the first observation of a synergistic relationship between these putative oral pathogens associated with adult severe periodontal disease.

Relative proportions of pathogen-related oral spirochetes (PROS) and Treponema denticola in supragingival and subgingival plaque from patients with periodontitis.

The purpose of this study was to use monoclonal antibodies to enumerate spirochetes in dental plaque, including the newly recognized pathogen-related oral spirochete (PROS) and specific serovars of Treponema denticola. Plaque was collected from control subjects with no apparent periodontal disease and from sites of moderate to severe chronic periodontitis in patients with inflammatory periodontal disease. Individual monoclonal antibodies were used to determine whether spirochetes were present and then a double-staining protocol was employed to count total spirochetes and specific treponemes in individual microscopic fields. Results indicate that spirochetes are more common at diseased sites and in subgingival plaque than at healthy sites or in supragingival plaque. Together PROS and T. denticola comprised the majority of all spirochetes in all samples and PROS and T. denticola serovars "B" and D were most numerous in plaque from patients with periodontitis. PROS were the majority of all spirochetes in supragingival plaque (76.2% +/- 23.8%) and subgingival plaque (60.9% +/- 19.1%) from periodontitis patients, significantly larger than the percentage of T. denticola serovar "B" (P less than .001 for both supragingival and subgingival plaque) and serovar D (P less than .01 for supragingival and P less than .001 for subgingival plaque). These observations indicate that PROS are the predominant spirochete in plaque from sites of patients with periodontitis, but other analytical approaches are necessary to determine if PROS or T. denticola are pathogenic.

Pathogen-related spirochetes identified within gingival tissue from patients with acute necrotizing ulcerative gingivitis.

The purpose of this investigation was to determine whether monoclonal antibodies against pathogen-restricted antigens of Treponema pallidum subsp. pallidum could be used as probes for spirochetes in diseased gingival tissue from subjects with acute necrotizing ulcerative gingivitis. A biotin-streptavidin system was used to identify spirochetes bound by monoclonal antibodies in cryostat sections of tissue. Twelve of 16 tissue samples from diseased sites, but none of 8 tissue specimens from healthy sites, reacted with pathogen-restricted antibodies. Organisms were found in intact epithelium and connective tissues adjacent to ulcers. Staining intensity was often high in perivascular locations and around vesicular spaces. Monoclonal antibodies to Bacteroides gingivalis and Treponema denticola were each reactive with diseased gingival tissues, but staining was usually restricted to ulcerated areas. These studies extend recent observations that showed that subjects with acute necrotizing ulcerative gingivitis had both pathogen-related spirochetes in dental plaque and serum immunoglobulin G to pathogen-restricted antigens on T. pallidum subspecies, suggesting that pathogen-related spirochetes may be associated with the pathogenesis of certain periodontal diseases.

Biochemical properties of the outer membrane of Treponema denticola.

The outer membranes (OMs) from serovars a, b, and c of Treponema denticola, originally isolated from periodontal patients, were prepared. Dialysis of the OMs against 20 mM MgCl2 yielded the aggregable (A) and the nonaggregable (NA) moieties of the OMs. The absence of muramic acid, adenosine triphosphatase, hexokinase, and nucleic acid as well as electron microscopy indicated that the OM preparations were homogeneous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the A and NA moieties of the OMs showed approximately 25 Coomassie brilliant blue R-250 stain-positive bands or 47 silver-stained polypeptides. The relative molecular masses ranged between 14 and 97 kDa. The electrophoretic polypeptide profiles of the A and NA moieties shared many similarities among serovars a, b, and c. However, they exhibited variation in the overall pattern, intensity, or location of the polypeptide stained zones. This was especially true for serovar b. Two-dimensional electrophoretic studies showed an excess of 100 silver-stained spots with isoelectric points of 4.6 to 7.0 and relative molecular masses in the 14- to 97-kDa range. The OMs contained simple proteins, glycoproteins, and lipoproteins. The NA moieties of the OMs contained 4 to 6, 10 to 12, and 4 to 6 glycopeptides as well as two, seven, and two lipoprotein bands for serovars a, b, and c, respectively. The A moieties of the OMs showed 7 to 9, 11 to 13 and 5 to 6 glycopeptides as well as four, five, and three lipoprotein bands for serovars a, b, and c, respectively. Lipopolysaccharide was detected in the OMs of the three serovars following removal of proteins with proteinase K, pronase and silver staining of sodium dodecyl sulfate-polyacrylamide gels, or removal of lipopolysaccharide from the OMs by hot phenol extraction. The 66- and 53-kDa bands were present in serovars b and c, while a band with a relative molecular mass of 45 kDa was present only in serovar c. Endotoxin-like activity was also shown in the OMs of the three serovars by the Limulus amebocyte clotting assay and the chick embryo lethality test. This is the first report on selected biochemical properties of the OM macromolecules of three known serovars of T. denticola.

Use of monoclonal antibodies to enumerate spirochetes and identify Treponema denticola in dental plaque of children, adolescents and young adults.

Identification of spirochetes in dental plaque is difficult. Not all spirochetes can be cultured and microscopic analysis based on darkfield or phase optics cannot determine the genus and species of individual bacterial cells. The purpose of this study was to use monoclonal antibodies in an immunoenzyme technique to stain spirochetes in dental plaque. Separate mAb were used to estimate total spirochetes and relative numbers of 2 distinct types of Treponema denticola. Plaque samples were collected from 40 subjects grouped by age. Results showed that older subjects are more likely to have spirochetes, to have more spirochetes and to have more diverse populations or spirochetes than younger subjects. Our studies suggest that T. denticola may be the first treponeme to colonize the primary dentition, that T. denticola appears to comprise a major proportion of all spirochetes at all ages and that two distinct serotypes of T. denticola are found to coexist in plaque from most children.

Characterization of monoclonal antibodies to two Treponema denticola serotypes by the indirect fluorescent-antibody assay.

Members of the genus Treponema have been implicated as possible aetiologic agents of periodontal disease. Previously developed murine monoclonal antibodies (MAbs) to two Treponema denticola strains were characterized by an indirect fluorescent-antibody (IFA) technique. Fifteen T. denticola strains and 32 other bacteria commonly isolated from periodontal pockets were used in the screening process. Two monoclonal antibodies to T. denticola ATCC 33521, serotype B, reacted with five T. denticola isolates, and one monoclonal antibody to T. denticola ATCC 33520, serotype c, reacted with two T. denticola isolates. There was no cross-reactivity between MAbs to the two serotypes. Preliminary tests of the MAbs on human periodontal samples show that they are useful in detecting these bacteria in clinical samples.

Quantitative immunoassay of Treponema denticola serovar C in adult periodontitis.

Murine monoclonal antibodies specific for Treponema denticola serovar C were produced and characterized in this study. An immunoassay was then developed by using these monoclonal antibodies, and the T. denticola serovar C antigen content of subgingival plaque was quantitated for samples taken from patients with periodontitis and healthy volunteers. The human subgingival plaque samples were grouped by severity of disease and pocket depth measurements at the collection site. The T. denticola serovar C content per milligram of subgingival plaque from deep pockets (greater than 6 mm) of patients with severe periodontitis was found to be twice that of samples collected from deep pockets (4 to 6 mm) of patients with moderate periodontitis or samples collected from healthy subjects (pocket depth, less than 4 mm).

Quantitative relationship of Treponema denticola to severity of periodontal disease.

The Treponema denticola content of plaque was quantitatively estimated for samples taken from periodontitis patients as well as periodontally healthy subjects among two separate human populations. The populations studied included military volunteers and civilians at a university dental clinic. The plaque samples from each population were grouped according to pocket depth measurements at the collection site. A biotin-avidin enzyme-linked immunosorbent assay procedure was developed with a monoclonal antibody specific for a serovariety of T. denticola. T. denticola was present at significantly elevated levels in plaque samples collected from deep-pocket sites of patients with severe periodontitis relative to the healthy controls and a group with moderate disease. The ratio of T. denticola content per milligram of plaque in the deep pocket groups to that of the other two groups was about 2:1 for both populations. This is the first quantitative evidence of a positive relationship between a specific spirochete species and severe periodontitis.

Monoclonal antibodies that recognize a specific surface antigen of Treponema denticola.

Spirochetes have been implicated as potential etiologic agents of periodontitis in humans. Murine monoclonal antibodies (MAbs) specific for a serogroup of Treponema denticola, an oral spirochete, were developed and characterized in this study. Antibodies secreted by clone IAA11 were judged to be the most useful, since they were able to detect 8 of 15 T. denticola strains. This MAb consisted of an immunoglobulin G3 heavy chain and a kappa light chain. MAb IAA11 was found to react with an epitope target located on the outer sheath of the cell wall. This MAb should be of diagnostic and scientific value in the study of T. denticola populations in human periodontitis.

Diagnostic value of a coagglutination procedure using monoclonal antibodies to Bacteroides gingivalis.

A specific monoclonal antibody against Bacteroides gingivalis was bound to particles coated with protein A and evaluated for use in a coagglutination test. B. gingivalis was the only organism tested which gave a specific positive reaction with the CoA reagent. Subgingival plaque samples were collected from 217 patients diagnosed as having periodontitis. Organisms that gave biochemical reactions which indicated they were B. gingivalis were isolated from eleven of the 217 gingival pockets. These eleven strains were the only organisms which gave a positive reaction using the CoA test.