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.

The utility of the BANA test for monitoring anaerobic infections due to spirochetes (Treponema denticola) in periodontal disease.

Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus each possesses an enzyme(s) that hydrolyzes the synthetic substrate benzoyl-DL-arginine-naphthylamide (BANA). The presence of these organisms in a subgingival plaque sample can be determined by the ability of the plaque to hydrolyze BANA. In the present study, we describe the usefulness of the BANA test at various stages of a clinical trial of the efficacy of metronidazole in the treatment of periodontal disease. A BANA-positive test was significantly associated with high levels and proportions of spirochetes in the plaque, so that it provided information comparable with that which could be obtained by a microscopic examination of the plaque. Patients with such anaerobic spirochetal infections were randomly assigned to a group receiving either metronidazole or placebo (250 mg, three times a day) for one week and whose teeth were scaled and root-planed. The advantages of the decision that metronidazole be used were apparent from the comparison with the results obtained in the patients who received only the scaling and root planing. The initially BANA-positive teeth in the patients treated with metronidazole, scaling, and root planing gained attachment and exhibited a significant reduction in the need for periodontal surgery, when compared with the BANA-positive teeth in the patients who received only placebo, scaling, and root planing. After the conclusion of this therapy, those teeth with persistent BANA-positive plaques had significantly higher proportions and levels of spirochetes than did the teeth with BANA-negative plaques.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and immunochemical characterisation of strains of Treponema hyodysenteriae.

The protein composition of 18 clinical isolates of Treponema hyodysenteriae from pigs with swine dysentery in Australia were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Coomassie Blue stained SDS-PAGE-profiles of whole cell and outer membrane (OM) proteins demonstrated the same gel pattern among the T. hyodysenteriae isolates, particularly the OM proteins in the molecular mass (Mr) range of 30 kDa to 40 kDa. The T. hyodysenteriae isolates were categorised into two distinct groups (A and B) based on the strain-variability in the 37 kDa OM protein. Immunoblotting of whole cell proteins after SDS-PAGE using serum from rabbits and pigs immunised with known T. hyodysenteriae serotypes revealed a number of common immunoreactive bands in all isolates. LPS typing of the T. hyodysenteriae isolates by immunoblotting with the rabbit antiserum revealed one additional serotype emphasising the LPS heterogeneity among the strains isolated from geographic locations in Australia, Great Britain and the U.S.A. Immunoblotting of the OM preparations revealed several common immunoreactive polypeptides corresponding to Mr values of 34 kDa to 30 kDa among the T. hyodysenteriae and T. innocens isolates but a distinct 39 kDa found only in the T. hyodysenteriae isolates. Trypsin proteolysis of intact. T. hyodysenteriae cells caused selective loss of these and other major abundant proteins identifying the location of the 39 kDa, 36 kDa, 34 kDa and 30 kDa proteins on the cell surface and suggesting a possible role of these proteins in the pathogenesis of swine dysentery.

Pathogenesis of Treponema hyodysenteriae: induction of interleukin-1 and tumor necrosis factor by a treponemal butanol/water extract (endotoxin).

Biological activities of lipopolysaccharide-like (LPS-like, phenol/water extract) and endotoxin-like (butanol/water extract) preparations from Trepomena hyodysenteriae were examined. The treponemal phenol/water and butanol/water extracts were less toxic than E. coli LPS for murine peritoneal exudate cells (PECs). The treponemal phenol/water extract did not stimulate the production of interleukin-1 (IL-1) or tumor necrosis factor (TNF) from murine PECs. The treponemal butanol/water extract did induce production of IL-1 and TNF but at doses 5- to 50-fold higher than E. coli LPS. Natural killer cell activity was augmented by the treponemal butanol/water extract but not by the phenol/water extract. Suppression of a splenic anti-SRBC plaque forming cell response was observed when the LPS-like and endotoxin-like preparations from T. hyodysenteriae were administered 24 h prior to injection of the SRBC. These findings indicate that the butanol/water extracted material from T. hyodysenteriae is more biologically active than the phenol/water extracted material and that the treponemal endotoxin may contribute to the inflammatory response of swine dysentery by inducing IL-1 and TNF production.

Characterization of the attachment of Treponema hyodysenteriae to Henle intestinal epithelial cells in vitro.

Properties of the attachment of Treponema hyodysenteriae to Henle intestinal epithelial (HIE 407) cells were examined. The frequency of attachment depended on the motility and viability of the spirochetes. Rabbit hyperimmune and swine convalescent antisera inhibited attachment. Treatment of HIE cells with neuraminidase had no effect on attachment; however, treatment of spirochetes with the enzyme decreased adherence significantly (P = 0.01). Attachment was inhibited by N-acetylneuraminic acid, D-glucuronic acid, and fetuin. Adherence was increased following coincubation with N-acetylglucosamine or yeast mannan. Surface antigens of T hyodysenteriae, isolated by chemical extraction, competitively inhibited adherence. Concentrated T hyodysenteriae culture supernatant fractions inhibited adherence, but concentrated phosphate buffered-saline washings of the spirochete and concentrated uninoculated media did not inhibit adherence. Sialic acid was detected in unwashed T hyodysenteriae and spent culture supernatant fractions in higher concentrations than from washed spirochetes and uninoculated media. It was concluded that the binding adhesins on T hyodysenteriae for cultured HIE cells may contain sialic acid residues.

Analysis of the axial filaments of Treponema hyodysenteriae by SDS-PAGE and immunoblotting.

Purified axial filaments from eight serotypes of Treponema hyodysenteriae and two non-pathogenic intestinal spirochaetes were characterized by SDS-PAGE and Western blotting. Axial filaments of all ten strains had similar SDS-PAGE profiles; five major axial filament polypeptides were identified, with molecular masses of 43.8, 38, 34.8, 32.8 and 29.4 kDa. Hyperimmune gnotobiotic pig serum raised against purified axial filaments of strain P18A (serotype 4) cross-reacted with all other serotypes and with the non-pathogens, and convalescent serum taken from a pig with persistent swine dysentery also showed a strong response to the axial filament polypeptides. Hyperimmune gnotobiotic pig serum raised against axial filaments failed to agglutinate viable organisms and did not inhibit growth in vitro. Hence, the axial filaments of T. hyodysenteriae have been identified as major immunodominant antigens, although the role that antibodies to these antigens play in protection has yet to be established.

Optimizing the solid-phase immunofiltration assay. A rapid alternative to immunoassays.

The technical variables of the solid-phase immunofiltration assay (SPIA) for the detection of antibodies bound to antigens on a solid-phase filter have been investigated. The binding to solid-phase filters of 125I-labelled axial filament proteins derived from Treponema phagedenis and the optimal conditions for blocking non-specific protein binding were analysed. Axial filament was applied to nitrocellulose, Hybond Nylon and Zeta Probe. After extensive rinsing, the highest amount (68%) of axial filament was observed bound to Zeta Probe. However, blocking non-specific protein binding by pre-wetting the filter with rinsing buffer containing 0.5% Tween 20, prevented the binding of protein to the filter only when nitrocellulose was used as solid phase. Tween 20 (0.5%) in the rinsing and incubation solutions was found to be necessary for the reduction of non-specific binding of contaminants in turbid sera. However, the use of such solutions resulted in a substantial leakage of antigen (47%) during rinsing procedures. Binding of antigen-specific antibody was analysed using 125I-labelled protein A. The maximal possible binding of the antibody occurred within 5 min when the antibody solution was filtered. For optimal binding of 125I-labelled protein A an incubation time of 1 h was needed. It is suggested that solid-phase immunofiltration may provide a rapid alternative for radioimmunoassays or enzyme immunoassays for the detection of specific antibodies.

Lipooligosaccharides from Treponema hyodysenteriae and Treponema innocens.

Lipooligosaccharides from Treponema hyodysenteriae serotypes 1 through 7, attenuated T. hyodysenteriae serotypes 1 and 2, and five strains of T. innocens were extracted with hot phenol water. The extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and analyzed by lipopolysaccharide selective silver staining and Western blot (immunoblot) immunodetection. Silver staining revealed the presence of two bands that ranged between 18,000 and 24,000 daltons and that were serotype specific for T. hyodysenteriae. Attenuation of pathogenic strains resulted in the loss of the higher-molecular-weight band. Four of five T. innocens strains also lacked this particular band. T. innocens 421 had six bands between 17,000 and 26,900 daltons. Western blots with hyperimmune rabbit sera and convalescent-phase swine sera revealed antigenic variation among serotypes of T. hyodysenteriae and attenuated serotypes of T. hyodysenteriae. Convalescent-phase swine sera failed to recognize lipopolysaccharides from T. innocens. Differences in results obtained by lipopolysaccharide selective silver staining versus immunoblotting of the lipopolysaccharide preparations probably indicate that these two methods identify separate characteristics of the same molecule.

Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis.

The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species.

Ultrastructural and electrophoretic analysis of Treponema hyodysenteriae axial filaments.

Axial filaments of Treponema hyodysenteriae were purified and examined by transmission electron microscopy. The size and fine structure of the filaments were similar to those of Treponema zuelzerae and Treponema phagedenis biotype reiterii. Filaments were dissociated by heat and 2-mercaptoethanol and examined by polyacrylamide-gel electrophoresis. Six protein bands were evident, with approximate molecular weights of 39,000, 29,000, 27,000, 22,000, 21,000, and 18,500 daltons. All the proteins reacted with T hyodysenteriae and T innocens rabbit antisera, using the immunoblot technique.

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.

Antigenic variability of Borrelia burgdorferi.

Borrelia burgdorferi strains (six isolates from North America and 28 isolates from Europe) were analyzed by physicochemical and immunological methods. By SDS-PAGE, all Borrelia burgdorferi strains tested had two major proteins with constant molecular weights of 60 and 41 kDa and one, two, or three variable low molecular weight proteins (OspA = 30-32 kDa, OspB = 34-36 kDa, pC = 21-22 kDa). All combinations--except OspB alone or OspB/pC--were observed. Borrelia burgdorferi strains were different from relapsing fever borreliae by strong reactivity with OspA- and/or pC-specific polyclonal antibodies, whereas relapsing fever borreliae were only weakly reactive. Among 25 Borrelia burgdorferi isolates, seven different serotypes of Borrelia burgdorferi were defined according to their reactivity in the Western blot with three monoclonal OspA-specific antibodies (H5332, H3TS, and LA5), four OspA- or OspB-specific polyclonal antibodies, and 12 polyclonal antibodies against whole borreliae. Antigenic differences between European CSF and skin isolates were observed, all skin isolates (n = 11) belonging to serotype 2 in contrast to only two out of seven CSF isolates. CSF isolates were antigenically heterogenous (serotypes 1, 2, 3, 4, and 5). Serotypes 6 and 7 were represented by two tick isolates, and the other European tick isolates are not yet fully characterized. Antigenic differences between European and North American strains may play a role in differences in the clinical picture of Lyme borreliosis.

Cholesterol metabolism by Treponema hyodysenteriae.

The sterol content of cellular lipids of Treponema hyodysenteriae, the agent of swine dysentery, was determined. When cultured in lipid-depleted brain heart infusion broth containing vesicles made from [4-14C]cholesterol and phosphatidylcholine, T. hyodysenteriae cells incorporated radioactive label. Most (95%) of this radioactivity was associated with bacterial membrane preparations. Lipids were extracted from radiolabeled cells and fractionated by silicic acid column chromatography. Components of the neutral lipid fraction were separated by reversed-phase high-performance liquid chromatography and were detected by monitoring both radioactivity and UV absorption (210 nm) of the column effluent. Cholesterol represented only about 5% of the total radioactivity in the bacterial neutral lipids. The remaining radioactivity was associated with a compound that did not absorb light at 210 nm. This lipid was purified and, on the basis of results from thin-layer chromatography and mass spectrometry, was identified as cholesterol (5 alpha-cholestan-3 beta-ol), a sterol lacking the unsaturated bond of cholesterol. Cholestanol was also present in cell-free culture broth, but only after growth of the spirochete. These results are evidence that cholesterol is used by T. hyodysenteriae for membrane synthesis. Cholesterol is converted to cholestanol in T. hyodysenteriae cultures and cholestanol is a major component (approximately 9% by weight) of T. hyodysenteriae cell lipids.

Molecular characterization of proteins from porcine spirochetes.

Sonicated preparations of Treponema hyodysenteriae and Treponema innocens were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Treponemal proteins were electrophoresed on a 10% polyacrylamide slab gel in a discontinuous Tris-glycine system and either stained with Coomassie blue dye or transferred electrophoretically at 20 mA for 16 h and 30 mA for 3 h to nitrocellulose paper. Staining of the gels revealed at least 42 distinct T. hyodysenteriae and T. innocens proteins, with molecular sizes ranging from greater than 100 to 14 kilodaltons (kDa). Each species contained 12 to 16 major protein bands; five of the proteins were common to both species. Fourteen major antigens were identified in T. hyodysenteriae isolate B204 by using serum specimens from pigs in the acute stage of swine dysentery. Twelve additional antigens were detected in isolate B204 when convalescent-phase serum specimens were reacted to the blot. A wide band at 16 kDa was identified with convalescent-phase serum specimens in T. hyodysenteriae but not in T. innocens. This 16-kDa antigen was also identified in T. hyodysenteriae with colonic secretions from convalescent pigs.

Antiserum to the 33,000-dalton periplasmic-flagellum protein of "Treponema phagedenis" reacts with other treponemes and Spirochaeta aurantia.

"Treponema phagedenis" periplasmic flagella (PF) have two major protein bands at molecular weights of 33,000 and 39,800 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (R. J. Limberger and N. W. Charon, J. Bacteriol. 166:105-112, 1986). By use of Western blotting and a polyclonal antiserum directed toward the 33,000-molecular-weight PF protein, cell lysates of 12 species of spirochetes were surveyed for reactivity. Eight species of Treponema as well as Spirochaeta aurantia were positive. The results suggest that epitopes residing on the 33,000-molecular-weight PF protein of "T. phagedenis" are evolutionarily well conserved among the spirochetes.

Microscopic agglutination and polyacrylamide gel electrophoresis analyses of oral anaerobic spirochetes.

Microscopic agglutination (MA) analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to determine strain and species similarities and dissimilarities among three species of oral anaerobic spirochetes, Treponema denticola, Treponema pectinovorum, and Treponema vincentii. The MA analysis revealed a diversity of serologic reactivity or sharing of common antigens within each species. However, there was no cross-reactivity or sharing of common antigens among the three species. Distinct SDS-PAGE whole-cell electrophoretograms for each species were obtained. The banding patterns for 16 T. denticola strains revealed 30 distinct proteins, while the banding patterns for 5 strains of T. pectinovorum and 2 strains of T. vincentii revealed 26 and 35 distinct proteins, respectively. Analysis of the electrophoretograms showed that their respective banding patterns could be used to distinguish the three species from one another. In addition, strain differences within each species could be detected. There was a correlation between MA analysis and SDS-PAGE analysis. It is thus suggested that both MA and SDS-PAGE analysis be included in classification schemes for the identification of oral spirochetes.

Treponema phagedenis has at least two proteins residing together on its periplasmic flagella.

Treponema phagedenis is an anaerobic, motile spirochete with several periplasmic flagella (PFs) at each cell end. This study provides the first genetic evidence that multiple protein species are associated with the PFs. In addition, these proteins were found to reside together on a given PF. Nonmotile mutants which lacked the PFs were isolated, and spontaneous revertants to motility regained the PFs. These results suggest that the PFs are involved in the motility of T. phagedenis. Isolated PFs had two major protein bands with molecular weights of 33,000 and 39,800, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots with monoclonal and polyclonal antibodies indicated that both proteins were absent in the PF mutants but present in the revertants. Immunoelectron microscopy revealed that the 39,800-molecular-weight protein was distributed along the entire PF. Immunoprecipitation analysis suggested that the 39,800- and 33,000-molecular-weight proteins were closely associated in situ.

Isolation, and structural and chemical characterization of outer sheath carrying a polygonal array from Treponema phagedenis biotype Reiter.

An outer sheath was isolated from Treponema phagedenis biotype Reiter by our previously developed method (Masuda, K., and Kawata, T. 1982. J. Bacteriol. 150: 1405-1413). The isolated outer sheath was observed as a triple-layered, closed vesicle carrying a polygonal array by electron microscopy. The outer sheath was mainly composed of protein (41.0%), phospholipid (38.7%), and carbohydrate (11.0%). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated outer sheath in the presence of 2-mercaptoethanol (EtSH) gave one major protein band with an apparent molecular weight of about 69,000 and several minor protein bands. On the other hand, in the absence of EtSH, the major protein band disappeared but two new protein bands at positions of molecular weights of about 65,000 and 72,000 appeared. The SDS-PAGE profiles of the minor protein bands did not change with or without EtSH. Sodium deoxycholate (DOC)-solubilized materials from the isolated outer sheath were reassembled into thin membranous sheets carrying a roughly polygonal array upon removal of DOC by dialysis against Tris-HCl buffer in the absence of Mg2+.