Purification and properties of endo-1, 3-alpha-D-glucanase from Pseudomonas.

An endo-1, 3-alpha-D-glucanase (EC 3.2.1.59) was purified from cell-free culture supernatants of Pseudomonas NRRL-B-12324. The enzyme was purified 8.7-fold to a specific activity of 78.1 U/mg of protein. The enzyme was inducible and had an isoelectric point of 4.6 and a Km of 80.0 mM in terms of anhydroglucose units. Two distinct peaks of activity were resolved by gel filtration with two different supporting media, whereas only one peak of activity was resolved by isoelectricfocusing. The two peaks were assigned molecular weight values of 67,400 and 279,000. The pH optimum was near 5.0 and the temperature optimum was near 56 degrees C. Additional gel filtration data indicated that the enzyme functions as an endohydrolase.

Investigation of possible solvents for extracellular polysaccharides from Streptococcus mutans and Streptococcus sanguis.

The solubilities of extracellular polysaccharide fractions produced by 14 oral streptococcal strains were compared in water, aqueous lithium and guanidine salt solutions, dimethyl sulfoxide (DMSO), and a 9:1 DMSO-water mixture. The best results for solubilizing the fractions were obtained with the DMSO-water mixture procedure, which used the water and DMSO in sequence. By this method all fo the fractions were solubilized.

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.

Production and characterization of monoclonal antibodies to Bacteroides gingivalis.

The rapid detection of Bacteroides gingivalis by immunological methods using monoclonal antibodies could greatly improve the diagnosis and prognosis of severe periodontal disease in adults. In this study, three distinct hybridomas were produced which secrete monoclonal antibodies to soluble and whole-cell antigen preparations of B. gingivalis. The BGII, V F9/2D hybridoma produced a murine antibody that can detect all of the B. gingivalis isolates studied while never cross-reacting with the other oral microbial antigens tested.

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.

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.

The effects of basic and acidic synthetic polypeptides on the adherence of the oral bacteria, Streptococcus mutans and Streptococcus sanguis, to hydroxyapatite.

Two basic and two acidic synthetic polypeptides that bind strongly to hydroxyapatite at neutral pH were tested to determine their influence on adsorption of two Streptococcus mutans and two Streptococcus sanguis strains to hydroxyapatite. The adsorption of the strains was significantly enhanced or reduced by the basic and acidic agents, respectively. Study of acidic polypeptides provided evidence of competition between the polypeptides and the bacterial cells for hydroxyapatite adsorption sites.

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.

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.

Effects of dextranases on attachment of Streptococcus mutans to hydroxyapatite.

A Fusarium dextranase and a Penicillium dextranase were compared for their relative ability to quantitatively reduce the adsorption of (3)H-labeled Steptococcus mutans cells onto hydroxyapatite. Fusarium dextranase-treated hydroxyapatite disks caused a statistically significant decrease in the hydroxyapatite adsorption of both the OMZ 176 and NCTC 10449 strains of S. mutans relative to untreated control disks. The extent of initial bacterial adsorption was not promoted by sucrose-dependent glucan synthesis. Since the Fusarium dextranase has a much greater affinity for hydroxyapatite than the Penicillium dextranase, it could represent an enzyme with improved decay-preventive therapeutic properties. This was concluded because the Fusarium dextranase may interfere with both the initial attachment and later glucan-dependent accumulation of dental plaque microorganisms.

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.

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.

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).

Characterization of an extracellular dextranase from Fusarium moniliforme.

An extracellular dextranase (EC 3.2.1.11) was purified approximately 75-fold from cell-free culture filtrates of Fusarium moniliforme. The purified dextranase was of the endo type, and isomaltose was identified as the primary end product of dextran hydrolysis. The molecular weight of the dextranase was determined to be 39,000 by gel permeation chromatography. The enzyme was most active at pH 5.5, and the temperature optimum was near 55 C. Activity was not inhibited by either ethylenediaminetetraacetic acid or iodoacetate. The Km for dextran with an average molecular weight of 10,000 was estimated to be 1.1 X 10(-4) M. The electrophoretic mobility of the dextranase was distinctly different from that of a Penicillium-derived commercial dextranase. The F. moniliforme dextranase was also found to differ from the commercial preparation by its greater relative activity against glucans isolated from Streptococcus mutans.

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)