Complete structure of the adhesin receptor polysaccharide of Streptococcus oralis ATCC 55229 (Streptococcus sanguis H1).

This report describes the determination of the complete primary structure of the adhesin receptor polysaccharide of Streptococcus oralis ATCC 55229 (previously characterized as Streptococcus sanguis H1), a Gram-positive bacteria implicated in dental plaque formation. The polysaccharide was isolated from S. oralis ATCC 55229 cells after deproteination, enzymatic hydrolysis, and ion exchange chromatography. It was shown to consist of rhamnose, galactose, glucose, glycerol, and phosphate, in molar ratios of 2:3:1:1:1. Sequence and linkage assignments of the glycosyl residues were obtained by methylation analysis followed by gas-liquid chromatography and electron-impact mass spectrometry. 31P NMR spectroscopy revealed that phosphate was present in a diester, connecting glycerol to one of the galactosyl residues. High-performance liquid chromatography of a partial acid hydrolysate of the polysaccharide confirmed this finding by showing galactose 6-phosphate and glycerol 1-phosphate. The structural determination was completed by the combination of two-dimensional homonuclear Hartmann-Hahn and NOE experiments and heteronuclear [1H,13C] and [1H,31P] multiple-quantum coherence experiments. Thus, the adhesin receptor polysaccharide of S. oralis ATCC 55229 was found to be a polymer composed of hexasaccharide repeating units that contain glycerol linked through a phosphodiester to C6 of the alpha-galactopyranosyl residue and are joined end-to-end through galactofuranosyl-beta(1-->3)-rhamnopyranosyl linkages: [formula: see text] This structure is novel among bacterial cell surface polysaccharides in general and specifically among those implicated in dental plaque formation.     

Isolation of a coaggregation-inhibiting cell wall polysaccharide from Streptococcus sanguis H1.

Coaggregation between Streptococcus sanguis H1 and Capnocytophaga ochracea ATCC 33596 cells is mediated by a carbohydrate receptor on the former and an adhesin on the latter. Two methods were used to release the carbohydrate receptor from the gram-positive streptococcus, autoclaving and mutanolysin treatment. The polysaccharide released from the streptococcal cell wall by either treatment was purified by ion-exchange chromatography; this polysaccharide inhibited coaggregation when preincubated with the gram-negative capnocytophaga partner. After hydrolysis of the polysaccharide by hydrofluoric acid (HF), the major oligosaccharide of the polysaccharide was purified by high-performance liquid chromatography. By analysis of the HF hydrolysis of the polysaccharide and the purified oligosaccharide, this major oligosaccharide appeared to be the repeating unit of the polysaccharide, with minor components resulting from internal hydrolysis of the major oligosaccharide. Gas chromatography results showed that the oligomer was a hexasaccharide, consisting of rhamnose, galactose, and glucose, in the ratio of 2:3:1, respectively. By weight, the purified hexasaccharide was a fourfold-more-potent inhibitor of coaggregation than the native polysaccharide. Resistance to hydrolysis by sulfuric acid alone and susceptibility to hydrolysis by HF suggested that oligosaccharide chains of the polysaccharide are linked by phosphodiester bonds. Studies with a coaggregation-defective mutant of S. sanguis H1 revealed that the cell walls of the mutant contained neither the polysaccharide nor the hexasaccharide repeating unit. The purification of both a polysaccharide and its constituent hexasaccharide repeating unit, which both inhibited coaggregation, and the absence of this polysaccharide or hexasaccharide on a coaggregation-defective mutant strongly suggest that the hexasaccharide derived from the polysaccharide functions as the receptor for the adhesin from C. ochracea ATCC 33596.     

The cell wall polysaccharide of Streptococcus gordonii 38: structure and immunochemical comparison with the receptor polysaccharides of Streptococcus oralis 34 and Streptococcus mitis J22

As part of our ongoing investigations involving lectinmediatedadhesion among oral bacteria, the receptor polysaccharide fromStreptococcus gordonii 38 was isolated and characterized. Carbohydrateanalysis of the hydrolysed S.gordonii 38 polysaccharide by high-performanceanionexchange chromatography with pulsed amperometric detection(HPAEC-PAD) showed galactose (Gal) (2 mol), N-acetylgalactosamine(GalNAc) (1 mol), rhamnose (Rha) (2 mol), glucose (Glc) (1 mol)and galactosamine-6-phosphate (1 mol). Mild acid hydrolysisof the polysaccharide yielded a heptasaccharide repeating unit.The structure of the heptasaccharide repeating unit was determinedby high-resolution NMR spectroscopy which includes various homonuclear(DOF—COSY, TQF-COSY, NOESY and HOHAHA) and heteronuclearexperiments (HMQC), including linkage assignments by ¹H-¹³Clong-range correlation (HMBC). Complete ¹H and ₁₃C NMR assignmentsfor the intact polysaccharide yielded the covalent structureof a heptasaccharide repeating unit:     

Lipoteichoic Acids from Streptococcus sanguis

Two lipoteichoic acids, membrane (MLTA) and wall (WLTA), have been purified from Streptococcus sanguis by Sepharose and Ecteola-cellulose column chromatographies and concanavalin A-conjugated Sepharose affinity column chromatography. The teichoic acids were homogenous as judged by disc gel electrophoresis, column chromatography, and double diffusion tests. Both MLTA and WLTA consisted of glycerol, phosphate, glucose, and fatty acids in the ratios of 0.95:1:0.71:0.046 and 0.99:1:0.79:0.023, respectively. alpha-Glycerol-phosphate was obtained by the partial acid hydrolysis of the lipoteichoic acids suggesting that their backbone structure consists of the glycerol moieties linked by 1, 3-phosphodiester bonds. Both WLTA and MLTA form aggregates, perhaps due to micelle formation, in concentrated solution. The aggregate form of MLTA dissociates to a much greater extent than that of WLTA under similar conditions.     

A polysaccharide from Streptococcus sanguis 34 that inhibits coaggregation of S. sanguis 34 with Actinomyces viscosus T14V.

Coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34 depends on interaction of a lectin on A. viscosus T14V with a cell surface carbohydrate on S. sanguis 34. This carbohydrate was isolated, and its chemical makeup was established. The carbohydrate remained attached to S. sanguis 34 cells through extraction with Triton X-100 and treatment with pronase. It was cleaved from the cell residue by autoclaving and purified by differential centrifugation and column chromatography on DEAE-Sephacel and Sephadex G-75. The polysaccharide contained phosphate which was neither inorganic nor monoester. Treatment with NaOH-NaBH4, followed by Escherichia coli alkaline phosphatase, or with 48% HF at 4 degrees C, followed by NaBH4, yielded inorganic phosphate and oligosaccharide alditols. Therefore, the polysaccharide is composed of oligosaccharide units joined together by phosphodiester bridges. The structure and stereochemistry of the main oligosaccharide alditol was established previously (F. C. McIntire, C. A. Bush, S.-S. Wu, S.-C. Li, Y.-T. Li, M. McNeil, S. Tjoa, and P. V. Fennessey, Carbohydr. Res. 166:133-143). Permethylation analysis, 1H and 31P nuclear magnetic resonance studies on the whole polysaccharide revealed the position of the phosphodiester linkages. The polysaccharide is mainly a polymer of (6) GalNAc(alpha 1-3)Rha(beta 1-4)Glc(beta 1-6)Galf(beta 1-6)GalNAc(beta 1- 3)Gal(alpha 1)-OPO3. It reacted as a single antigen with antiserum to S. sanguis 34 cells and was a potent inhibitor of coaggregation between A. viscosus T14V and S. sanguis 34. Quantitative inhibition of precipitation assays with oligosaccharides, O-allyl N-acetylgalactosaminides, and simple sugars indicated that specific antibodies were directed to the GalNAc end of the hexasaccharide unit. In contrast, coaggregation was inhibited much more effectively by saccharides containing betaGalNAc. Thus, the specificity of the A. viscosus T14V lectin is strikingly different from that of antibodies directed against the S. sanguis 34 polysaccharide.     

血链球菌细菌素抑菌活性研究

检测分离纯化的血链球菌细菌素对牙周可疑致病菌的抑制作用。通过羟基磷灰石(HA)柱层析、SephadexG-150凝胶柱层析、中空纤维柱超滤脱盐、浓缩纯化提取血链球菌细菌素,以具核梭杆菌为指示菌,洞平板法检测细菌素的抑菌活性。经HA柱层析得到4个相互分离的组分,经洞平板法检测,第Ⅱ峰的蛋白具有抑菌活性,冻干后得纯化后的细菌素,终产率为0.082%;1mg/mL的细菌素溶液可形成17mm的抑菌环,最小抑菌浓度为62.5μg/mL。血链球菌细菌素对牙周可疑致病菌具有较强的拮抗作用。     

Glycerolphosphate-containing cell wall polysaccharides from Streptococcus sanguis

Six glycerolphosphate-containing tetraheteroglycans, a, b-1, b-2, b-3, b-4, and b-5, have been purified from the formamide extracts of Streptococcus sanguis by alcohol and acetone precipitations, Sephadex G-75, and diethylaminoethyl-cellulose column chromatography. The polysaccharides were judged as at least 95% pure by analytical disc gel electrophoresis and immune double diffusion against rabbit antiserum. They were shown to be cell wall polysaccharides, since they formed a single band of identity in immune double diffusion with partially purified polysaccharide extracted from a purified cell wall preparation of S. sanguis. The polysaccharides were composed of l-rhamnose, d-glucose, and N-acetyl d-glucosamine in a similar molar ratio, but varied in their glycerol and phosphate contents. They exhibited four different mobilities in polyacrylamide disc gel electrophoresis at pH 8.9. When they were treated with formamide at 170 C for 20 min, the faster moving polysaccharide(s) yielded polysaccharides with mobilities corresponding to the other slower moving polysaccharides. These results indicate that the polysaccharides originated from the same cell wall polysaccharide and were produced as a result of breakage in the phosphodiester bonds during the formamide extraction procedure. A preliminary structural study shows that the terminal reducing sugar is l-rhamnose and that the glycerol moiety is probably linked to the polysaccharide through a phosphodiester bond.     

Protoplast and cytoplasmic membrane preparations from Streptococcus sanguis and Streptococcus mutans

Protoplasts were prepared from Streptococcus sanguis and some S. mutans serotypes by use of lysozyme (EC 3.2.1.17) under particular conditions: cells had to be grown in DL-threonine (20 mM) and harvested in early exponential phase. The efficiency of protoplast formation was enhanced by two additional steps: plasmolysis (in 12% PEG), prior to addition of lysozyme, and a swirling phase, after the enzymic action. This procedure allowed us to obtain clean protoplasts, with only 0.5% contamination by bacterial cell walls. Up to 90% protoplast lysis was obtained in 0.5 M-NaCl. Cytoplasmic membrane purification was achieved by centrifugation on a glycerol cushion.     

Complete structure of the cell surface polysaccharide of Streptococcus oralis C104: a 600-MHz NMR study

Specific lectin-carbohydrate interactions between certain oral streptococci and actinomyces contribute to the microbial colonization of teeth. The receptor molecules of Streptococcus oralis, 34, ATCC 10557, and Streptococcus mitis J22 for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii are antigenically distinct polysaccharides, each formed by a different phosphodiester-linked oligosaccharide repeating unit. These streptococci all coaggregated strongly with both A. viscosus and A. naesludii strains, whereas S. oralis C104 interacted preferentially with certain strains of the latter species. Receptor polysaccharide was isolated from S. oralis C104 cells and was shown to contain galactose, N-acetylgalactosamine, ribitol, and phosphate with molar ratios of 4:1:1:1. The 1H NMR spectrum of the polysaccharide shows that it contains a repeating structure. The individual sugars in the repeating unit were identified by 1H coupling constants observed in E-COSY and DQF-COSY spectra. NMR methods included complete resonance assignments (1H and 13C) by various homonuclear and heteronuclear correlation experiments that utilize scalar couplings. Sequence and linkage assignments were obtained from the heteronuclear multiple-bond correlation (HMBC) spectrum. This analysis shows that the receptor polysaccharide of S. oralis C104 is a ribitol teichoic acid polymer composed of a linear hexasaccharide repeating unit containing two residues each of galactopyranose and galactofuranose and a residue each of GalNAc and ribitol joined end to end by phosphodiester linkages with the following structure. [----6)Galf(beta 1----3)Galp(beta 1----6)Galf(beta 1----6)GalpNAc(beta 1----3) Galp(alpha 1----1)ribitol(5----PO4-]n     

Isolation and analysis of sacculi from Streptococcus sanguis.

Sacculi were prepared from Streptococcus sanguis 34 by exhaustive extraction of bacteria with hot 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol. Lyophilized residue was dissociated by brief sonication to single bodies closely resembling streptococci in phase-contrast microscopic density, staining properties, and morphology. Electron micrographs revealed bodies that contained variable amounts of cellular contents and were bounded by intact cell walls. Chemical analyses of sacculi demonstrated the presence of peptidoglycan, carbohydrate, protein, and phosphate. The hexose content of sacculi varied 10-fold depending upon the composition of the growth medium. When sacculi were subjected to treatment with 5 M LiCl, 8 M urea, 40% phenol (25 degrees C), or dimethyl sulfoxide most of the nitrogen and carbohydrate present was recovered in the insoluble fraction. These data suggest that sacculi contain the cell wall fraction of the extracted bacteria and that most of the carbohydrates and proteins of sacculi are firmly bound to the insoluble fraction, which contains the peptidoglycan matrix.     

Inactivation of D-glucosyltransferases from oral Streptococcus mutans and Streptococcus sanguis by photochemical oxidation

Cell-free D-glucosyltransferase of D-glucose-grown Streptococcus mutans AHT was completely inactivated in the presence of 0.002% of Methylene Blue at 25 degrees and pH 7.0 after illumination with a 150-W incandescent lamp. The rate of inactivation was decreased at pH values less than 7.0. Histidine was the only amino acid residue modified to a significant extent, and the rates of oxidation of histidine residues and loss of enzyme activity closely agreed. Production of both water-insoluble and -soluble D-glucan fractions from sucrose by the oxidized D-glucosyltransferase preparations was significantly inhibited. Photooxidation with 0.002% of Rose Bengal at pH 7.0 or higher also induced complete inactivation of the D-glucosyltransferase. These results strongly suggest that the imidazole portion of histidine may function as part of the active sites of both D-glucosyltransferase isozymes of S. mutans AHT, which are responsible for the synthesis of (1 goes to 3)- and (1 goes to 6)-alpha-D-glucosidic linkages. The D-glucosyltransferases from S. mutans 6715 and AHT-mutant M1, and Streptococcus sanguis ATCC 10558 were also almost completely inactivated by Methylene Blue-sensitized photooxidation.     

Streptococcus sanguis modulates type II collagen-induced arthritis in DBA/1J mice

Native type II collagen is tolerogenic when given orally or i.p. to DBA/1J mice and induces autoimmune arthritis when given s.c. in CFA. The tolerogenic epitope is contained in cyanogen bromide fragment 11 (CB11) and is structurally mimicked by PGEQGPK within the platelet aggregation-associated protein (PAAP) on Streptococcus sanguis. To learn whether S. sanguis modulates transmucosally the Ag-specific development of autoimmune arthritis, DBA/1J pups were given live S. sanguis, CB11, or type II collagen intragastrically. Feeding S. sanguis at 6 days postpartum delayed the onset of arthritis, and reduced the rate, final severity, and percentage of affected limbs. Next, PAAP(+) S. sanguis and type II collagen were tested for T cell cross-reactivity. T cells primed with the tolerogenic epitope of type II collagen proliferated more when incubated with PAAP(+) S. sanguis than with PAAP(-) Streptococcus gordonii or type II collagen, suggesting an Ag-specific transmucosal tolerogenic effect. In neonatal mice, therefore, bacterial surface Ags that mimic self can transmucosally stimulate Ag-specific inhibitory T cells. In adult mice immunized with type II collagen, these Ag-specific inhibitory T cells manifest later as attenuated arthritis. The PAAP(+) S. sanguis appear to activate adult memory, rather than naive, type II collagen-specific T cells, suggesting that systemic challenge with commensal self-mimicking microorganisms may perpetuate existing autoimmunity, but not initiate autorecognition.     

Complete structure of the cell surface polysaccharide of Streptococcus oralis ATCC 10557: a receptor for lectin-mediated interbacterial adherence

Lectin-carbohydrate binding is known to play an important role in a number of different cell-cell interactions including those between certain species of oral streptococci and actinomyces that colonize teeth. The cell wall polysaccharides of Streptococcus oralis ATCC 10557, S. oralis 34, and Streptococcus mitis J22, although not identical antigenically, each function as a receptor molecule for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii. Carbohydrate analysis of the receptor polysaccharide isolated from S. oralis ATCC 10557 shows galactose (3 mol), glucose (1 mol), GalNAc (1 mol), and rhamnose (1 mol). 1H NMR spectra of the polysaccharide show that is is partially O-acetylated. Analysis of the 1H NMR spectrum of the de-O-acetylated polysaccharide shows that it is composed of repeating subunits containing six monosaccharides and that the subunits are joined by a phosphodiester linkage. The 1H and 13C NMR spectra were completely assigned by two-dimensional homonuclear correlation methods and by 1H-detected heteronuclear multiple-quantum correlation (1H[13C]HMQC). The linkage of the component monosaccharides in the polymer, deduced from two-dimensional 1H-detected heteronuclear multiple-bond correlation spectra (1H[13C]HMBC), shows that the repeating unit of the de-O-acetylated polymer is a linear hexasaccharide with no branch points. The complete 1H and 13C assignment of the native polysaccharide was carried out by the same techniques augmented by a 13C-coupled hybrid HMQC-COSY method, which is shown to be especially useful for carbohydrates in which strong coupling and overlapping peaks in the 1H spectrum pose difficulties. The fully assigned spectra of the native polymer show that each of two different positions is acetylated in one-third of the repeating subunits and that the acetylation is randomly distributed along the polymer. The exact positions of acetylation were assigned by a carbonyl-selective HMBC method that unambiguously defines the positions of O-acetylation. The complete structure of the native polysaccharide in S. oralis ATCC 10557 is [formula: see text] Comparison of this structure with those previously determined for the polysaccharides of strains 34 and J22 suggests that the similar lectin receptor activities of these molecules may depend on internal galactofuranose linked (beta 1----6)- to Gal(beta 1----3)GalNAc(alpha) or GalNAc(beta 1----3)Gal(alpha).     

Transformation of Streptococcus sanguis Challis by plasmid deoxyribonucleic acid from Streptococcus faecalis.

Plasmid deoxyribonucleic acid (DNA) from Streptococcus faecalis, strain DS5, was transferred to the Challis strain of Streptococcus sanguis by transformation. Two antibiotic resistance markers carried by the beta plasmid from strain DS5, erythromycin and lincomycin, were transferred to S. sanguis at a maximum frequency of 1.8 x 10-5/colony-forming unit. Approximately 70% of the covalently closed circular DNA isolated from transformant cultures by dye buoyant density gradients was shown to be hybridizable to beta plasmid DNA. Two major differences were observed between the beta plasmid from S. faecalis and the plasmid isolated from transformed S. sanguis: (i) the beta plasmid from strain DS5 sedimented in velocity gradients at 43S, whereas the covalently closed circular DNA from transformed Challis sedimented at 41S, suggesting a 1.5-Mdal deletion from the beta plasmid occurred; (ii) although the 43S beta plasmid remained in the supercoiled configuration for several weeks after isolation, the 41S plasmid was rapidly converted to a linear double-stranded molecule. Attempts to transform S. sanguis with the alpha plasmid from S. faecalis, strain DS5, were unsuccessful.     

Photolabeling of dextransucrase from Streptococcus sanguis with p-azidophenyl alpha-D-glucopyranoside.

Dextransucrase from Streptococcus sanguis ATCC 10558 was photolabeled using p-azidophenyl alpha-D-glucopyranoside with an apparent rate constant of inactivation of 1.40 min-1. The dissociation constant for this compound, which acts as an acceptor molecule in the enzymatic reaction, is 90 microM. Apparently two acceptor binding sites exist on dextransucrase as shown by (i.) photolabeling the enzyme with p-azidophenyl-alpha-D-[5,6-3H]glucopyranoside and (ii.) fluorescence titration experiments.     

Biosynthesis of oligosaccharide-lipid in Streptococcus sanguis

An oligosaccharide-lipid containing N-acetyl d-glucosamine (GlcNAc), l-rhamnose, and d-glucose was synthesized when the particulate enzyme from Streptococcus sanguis was incubated with UDP-GlcNAc, TDP-rhamnose, and UDP-glucose. The incorporation of d-glucose into the lipid was dependent on the preincorporation of l-rhamnose, which in turn was dependent on that of GlcNAc. This indicates that the order of sugar incorporation is GlcNAc, l-rhamnose, and d-glucose. The synthesis of GlcNAc-lipid was stimulated twofold by ATP and was inhibited strongly by UDP and slightly by UMP, CDP, and TDP, but not by all other nucleoside diphosphates and nucleoside monophosphates tested. A [gamma-(32)P]ATP labeling experiment indicated that some acceptor lipid was present in nonphosphorylated form. The acid and alkaline stabilities of the GlcNAc-lipid were similar to those of glycosyl undecaprenylphosphate, and the thin-layer chromatographic mobility of the lipid was slightly faster than that of the mannosylphosphorylundecaprenol. The molar ratio of phosphate to GlcNAc in purified GlcNAc-lipid was found to be 0.96:1. These results suggested that the GlcNAc was attached to the lipid moiety, presumably undecaprenol, by phosphodiester bonds. The incorporation of l-rhamnose into the lipid was inhibited by UDP and UMP, respectively, in a manner similar to the incorporation of GlcNAc. This suggested that the oligosaccharide was also linked to the lipid moiety by phosphodiester bonds.     

Genetic and biochemical characterization of the F-ATPase operon from Streptococcus sanguis 10904

Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species Streptococcus sanguis and Streptococcus mutans. Differences in the pH optima of the respective ATPases appears to be the main reason that S. mutans is more tolerant of low pH values than S. sanguis and hence pathogenic. We have recently reported the genetic arrangement for the S. mutans operon. For purposes of comparative structural biology we have also investigated the F-ATPase from S. sanguis. Here, we report the genetic characterization and expression in Escherichia coli of the S. sanguis ATPase operon. Sequence analysis showed a gene order of atpEBFHAGDC and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both S. sanguis and S. mutans; however, it is not induced to the same extent in the nonpathogenic S. sanguis. Expression studies with an atpD deletion strain of E. coli showed that S. sanguis-E. coli hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.