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.

Immunochemical features of a macromolecule of Treponema denticola.

In this study the extraction and the immunochemical features of a lipopolysaccharide-like (LPSL) macromolecule of T. denticola strains 35405, 35404, 33521 and 11 were investigated. The yield of LPSL molecule ranged between 0.5-0.9% of the cell dry weight, it possessed Limulus amebocyte lysate clotting activity, and it contained glucosamine, phosphate, heptose, glucose, small amounts of KDO, myristic and beta hydroxy myristic acid. Sera obtained from healthy individuals (ADA type I) periodontitis, from 3-8 month old infants, or the mouse monoclonal antibody, diluted 1:2, against T. pallidum did not react with the LPSL antigens of T. denticola strains 35405, 35404, 33521, and 11. Sera from patients with ADA type III-IV periodontitis were reactive with two 8-14 kDa bands even at serum dilutions of 1:2000. Sera from patients with ADA type II periodontitis showed good antibody response to the 8-14 kDa band at a dilution of 1:50, but were weekly reactive, or nonreactive at serum dilutions of 1:200. This study indicates that extraction of a lipopolysaccharide-like macromolecule is feasible from the assay spirochetes, and this macromolecule may be used as an antigen for the diagnosis of ADA types II-IV 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)

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 micrograms 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 micrograms/ml, based on 3-deoxy-2-manno-octulosonate analysis. Heat treatment of outer membrane (60 degrees 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 micrograms 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.

Phosphorylated nutrient uptake by Treponema denticola and other potential periodontopathogens.

Nutrient uptake may contribute to the survival of certain potential periodontopathogens in the competitive environment of the gingival sulcus. This study was conducted to assess the uptake of commercially available, key radiolabelled phosphorylated compounds by Treponema denticola. There was a linear relationship in the uptake of ATP and G-1-P with cell concentrations of 0.1-2.5 mg of cell dry weight. This uptake reached a steady state within 15 min, and at temperatures of 25-37 degrees C the uptake ranged between 13-21%. At 50 degrees C, or above 50 degrees C the assay cells took up 0-1% of the assay nutrients. The maximum uptake of ATP was 47.5, 38 and 47.6 pmoles for serovars a, b and c. G-1-P yielded maximum uptake values of 306, 304, and 306 pmoles respectively. Significant uptake of ATP, or G-1-P occurred at pH 4.8 for serovars a, or c, while serovar b showed a broad pH range for the uptake of G-1-P. Stannous fluoride at a concentration of 0.17 mM enhanced the uptake of ATP while chlorhexidine digluconate inhibited the uptake of ATP. At concentrations usually employed (0.05-0.5 mM) the metabolic poisons 2,4-dinitrophenol, sodium azide, sodium arsenite and the ATPase inhibitor N,N1-dicyclohexylcarbodiimide did not alter significantly the uptake of ATP. The uptake of ATP, by Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum and T. denticola at pH 4.8 was 13.4, 1.5, 6.9, and 18-20.4%, respectively. At pH 6.5 the uptake for the microorganisms indicated above was 22, 1.3, 6.6 and 6.2%, respectively. G-1-P showed the same trend in its uptake by A. actinomycetemcomitans, P. gingivalis, F. nucleatum and T. denticola. This study provides for the first time specific examples of phosphorylated nutrient uptake by potential periodontopathogens, and that the uptake of phosphorylated nutrients can be suppressed by oral hygienic compounds.

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.

A study of the acid phosphatase of Treponema denticola.

This study describes some of the properties of the acid phosphatase of the potential periodontopathogen Treponema denticola. The highest enzyme activity was found in 87 h old cells. Two optimum pHs for enzyme activity were detected, one at pH 4.8 and another at pH 6.2. Divalent cations did not influence the acid phosphatase of T. denticola. The anion F- added in the form of NaF and at a level greater than 20 micrograms/ml F- diminished the activity of the acid phosphatase of intact cells of T. denticola. The addition of 10 micrograms/ml F- as SnF2 induced a statistically significant reduction of acid phosphatase activity. The apparent Km for the acid phosphatase was 7.3 mM with p-nitrophenyl phosphate as substrate. Fluoride appeared to be a noncompetitive inhibitor of the enzyme with an apparent Ki of 0.3 mM. Acid phosphatase may be released partially in osmotic shock fluids. Also, 7-diazonium-1, 3-naphthalene disulfonate, which is incapable of penetrating the bacterial permeability barrier and is known to inactivate enzymes found in the bacterial periplasmic place, suppressed the activity of the acid phosphatase in intact cells of T. denticola.

The action of sodium deoxycholate on Escherichia coli.

Sodium deoxycholate is used in a number of bacteriological media for the isolation and classification of gram-negative bacteria from food and the environment. Initial experiments to study the effect of deoxycholate on the growth parameters of Escherichia coli showed an increase in the lag time constant and generation time and a decrease in the growth rate constant and total cell yield of this microorganism. Cell fractionation studies indicated that sodium deoxycholate at levels used in bacteriological media interferes with the incorporation of [U-14C]glucose into the cold-trichloroacetic acid-soluble, ethanol-soluble, and trypsin-soluble cellular fractions of E. coli. Finally, sodium deoxycholate interfered with the flagellation and motility of Proteus mirabilis and E. coli. It would appear then that further improvement of the deoxycholate medium may be in order.

Effect of fluoride on Treponema denticola.

The effect of fluoride on the growth of Treponema denticola was studied. Fluoride, at a concentration of 20 micrograms/ml, was found to suppress the growth of all three strains tested. Growth was completely inhibited by 40 micrograms of fluoride per ml.

Cytoplasmic proteins of Streptococcus mutans (serotype c) and their interaction with fluoride.

The protein profile of the cytoplasmic proteins of Streptococcus mutans GS-5 was determined by two-dimensional gel electrophoresis. Use of this recently developed, high-resolution analytical tool showed in excess of 140 cytoplasmic proteins. The profile consisted of mostly acidic components with pI values between 3.70 and 5.30 and relative molecular weights mainly in the 13,000 to 90,000 range. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were resolved into 40 to 45 components. The binding of fluoride by the proteins reached a maximum value in 15 min, and it was linear with exogenous F- doses of up to 60 to 80 ppm per mg of protein (60 to 80 micrograms/g). The proteins bound 22 to 138 times more F- from assay mixtures containing 1 mM CaCl2 than from assay mixtures containing such ions as HgCl2, ZnCl2, CuCl2, MgCl2, MnCl2, or SnCl2. When NaF, SnF2, NH4F, CsF, (CH3)4NF, and Na2PO3F were used as sources of F- (adjusted to 10 ppm of F- in all cases), the proteins bound 2.1, 1.8, 1.6, 1.4, and 0.3 ppm of F- per mg of protein, respectively. Initial fractionation of the plasma proteins by preparative column isoelectric focusing indicated that proteins with pI values of 4.1 to 4.5 as well as those with pI values of 5.0 to 5.3 bound twice as much F- as did the proteins outside these pI values.

Binding of 18F by cell membranes and cell walls of Streptococcus mutans.

The binding of 18F to isolated cell membranes and cell walls of Streptococcus mutans GS-5 or other bacteria was assayed. The attachment of 18F to these cell envelopes proceeded slowly and reached equilibrium within 60 min. 18F binding was stimulated by Ca2+ (1 mM). The binding of 18F to cellular components was dependent upon the pH, as well as the amount of 18F and dose of the binder employed. The binding of 18F by cell walls prepared from fluoride-sensitive and fluoride-resistant cells of S. salivarius and S. mutans did not differ significantly. The pretreatment of cell walls or cell membranes for 60 min at 30 degrees C with 1 mg of RNase, DNase, or trypsin per ml did not influence the binding of 18F by the walls and membranes of S. mutans GS-5. However, prior exposure of cell membranes to sodium dodecyl sulfate caused a significant reduction in the number of 18F atoms bound by the membranes. In saturated assay systems, cell membranes of S. mutans GS-5 bound 10(15) to 10(16) atoms of 18F per mg (dry weight), whereas cell walls from S. mutans GS-5, FA-1, and HS-6 or Actinomyces viscosus T14V and T14AV bound 10(12) to 10(13) atoms of 18F per mg (dry weight). 18F in this quantity (10(12) to 10(13) atoms) cannot be detected with the fluoride electrode. The data provide, for the first time, a demonstration of 18F binding by cell membranes and walls of oral flora.

The action of selected agents on the accumulation of 18F by Streptococcus mutans.

The action of certain substances known to induce cellular alterations, or encounted in the oral cavity, on the accumulation of 18F by Streptococcus mutans GS-5 has been investigated. A 62-67% inhibition in the number of 18F atoms bound per mg dry weight of cells could be induced by a 15 min pretreatment with 2.7 X 10(-4) M cetyltrimethylammoniumbromide, 1 X 10(-1) M acetic anhydride, or 7 X 10(-2) M HCl. Plate counts indicated that alteration of the cellular composition rather than viability was responsible for this diminution in 18F accumulation. Prior exposure for 15 min of this organism to 1 M HCHO or 0.1 M NaOH did not alter 18F accumulation. Of the common salts encountered in the oral cavity, CaCl2 enhanced 18F binding. Pretreatment of the assay cells for 15-160 min with 0.1-10 mg/ml of trypsin, pronase, protease, alpha-glucosidase, dextranase, or lactoferrin had no significant effect on the accumulation of 18F. However, pre-exposure of cells for 60 min to 1-10 mg/ml of either amylase or lipase induced a 40-67% inhibition in the binding of 18F, while lysozyme enhanced the binding of 18F by the cells. It would appear then that the binding of 18F by S. mutans may be altered by certain substances encountered in the oral cavity.

Solubilization and electrophoretic analysis of Staphylococcus aureus membrane proteins.

The protein composition of homogeneous Staphylococcus aureus 6538P cytoplasmic membranes was examined under denaturing electrophoretic conditions. A comparative analysis on the effectiveness of a variety of membrane solubilizing agents revealed the membrane protein extracts to be qualitatively similar as determined electrophoretically but different in the quantity of protein released; Zwittergent-314, sodium dodecyl sulfate, Triton X-100, Nonidet P-40, and sodium deoxycholate all proved to be effective solubilizing agents under the conditions examined. Fifty-five to sixty protein components were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis from homogeneous late-exponential phase membranes. The profile was unaffected when phenylmethylsulfonyl fluoride was included during membrane isolation and solubilization. Analysis of the solubilized membrane proteins by two-dimensional gel electrophoresis demonstrated in excess of 100 membrane protein components in a pH gradient between 3.5 to 7.7. The profile consisted of a heterogeneous mixture of mostly acidic components with isoelectric points between pH 4 and 5 and relative molecular weights between 158,000 and 35,000. Periodic acid-Schiff staining following sodium dodecyl sulfate gel electrophoresis revealed six to ten reactive bands with two of these bands also exhibiting a reaction with concanavalin A.

Analysis of Staphylococcus aureus cytoplasmic membrane proteins by isoelectric focusing.

Cytoplasmic membranes were isolated from late-exponential phase Staphylococcus aureus 6539 P and the membrane proteins examined under non-denaturing conditions by thin-layer isoelectric focusing (TLIEF) in a pH 3.5-9.5 gradient. Isolated membrane preparations retained protein integrity as judged by the demonstration of membrane bound adenosine triphosphatase (ATPase) activity in addition to four other solubilized membrane enzyme markers. Membranes were effectively solubilized with 2.5% Triton X-100 (final concentration). Examination of Triton X-100 solubilized membrane preparations established the presence of 22 membrane proteins with isoelectric points between 3.7 and 6.0. The focused proteins displayed the following enzymatic activities and isoelectric points by zymogram methods: ATPase (EC 3.6.1.3), 4.20; malate dehydrogenase (EC 1.1.1.37), 3.90; lactate dehydrogenase (EC 1.1.1.27), 3.85; two membrane proteins exhibited multiple bands upon enzymatic staining NADH dehydrogenase (EC 1.6.99.3), 4.25, 4.35; succinate dehydrogenase (EC 1.3.99.1), 4.85, 5.10, 5.35.

Staphylococcus aureus adenosine triphosphatase: inhibitor sensitivity and release from membrane.

Homogeneous preparations of cytoplasmic membrane isolated from Staphylococcus aureus 6538P exhibited membrane-associated adenosine triphosphatase (ATPase) activity. Membrane ATPase activity was activated by divalent cations (4.0 mM: Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+), and ATP was hydrolyzed more readily than other nucleoside triphosphates and phosphorylated substrates. The pH optimum for the membrane ATPase was 6.5. The ATPase could not be released from the membrane by differential osmotic treatments, but detergent treatment effectively solubilized active enzyme. The nonionic detergent Triton X-100 (1%) released a protein with ATPase activity, after substrate-dependent staining in polyacrylamide gels, that differed slightly in electrophoretic migration when compared to the active enzyme solubilized with sodium dodecyl sulfate (0.1%). Membrane-associated ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (0.001 to 1 mM) and NaF (50% inhibition at 5 mM NaF). Azide and trypsin inhibited activity, whereas ouabain had a slight inhibitory effect. Diethylstilbestrol showed appreciable activation of the membrane ATPase over the range employed (0.001 to 1 mM).