Purification and characterization of cell-associated glucosyltransferase synthesizing insoluble glucan from Streptococcus mutans serotype c

Streptococcus mutans Ingbritt (serotype c) was shown to have a significant amount of cell-associated glucosyltransferase activity which synthesizes water-insoluble glucan from sucrose. The enzyme was extracted from the washed cells with SDS, renatured with Triton X-100, adsorbed to 1,3-alpha-D-glucan gel, and then eluted with SDS. The enzyme preparation was electrophoretically homogeneous, and the specific activity was 7.3 i.u. (mg protein)-1. The enzyme had an Mr of 158,000 as determined by SDS-PAGE, and was a strongly hydrophilic protein, as judged by its amino acid composition. The enzyme gradually aggregated in the absence of SDS. The enzyme had an optimum pH of 6.5 and a Km value of 16.3 mm for sucrose. Activity was stimulated 1.7-fold by dextran T10, but was not stimulated by high concentrations of ammonium sulphate. Below a sodium phosphate buffer concentration of 50 mm, activity was reduced by 75%. This enzyme synthesized an insoluble D-glucan consisting of 76 mol% 1,3-alpha-linked glucose and 24 mol% 1,6-alpha-linked glucose.     

Substrate specificity of hydrolase activity of the primer-dependent glucosyltransferases from Streptococcus sobrinus.

Four kinds of glucosyltransferases, P1, P2, P3 and P4, were separately purified from the culture supernatant of Streptococcus sobrinus. Their dependencies on primer were analysed. There were two primer-dependent glucosyltransferases (P3 and P4). In the absence of primer 1,6-alpha-D-glucan, P3 was not able to produce glucan from sucrose. However, P3 showed sucrose hydrolase activity, whereas P4 was still able to produce glucan without primer 1,6-alpha-D-glucan. Consequently, glucosyltransferase activity of P4 was incompletely primer-dependent. Both P3 and P4 showed high substrate specificity for sucrose, failing to use melezitose, raffinose, or stachyose as the substrates.     

Production, characterization, and application of monoclonal antibodies which distinguish four glucosyltransferases from Streptococcus sobrinus

A 1,3-alpha-glucan synthase (GTF-I), a highly branched 1, 6-alpha-glucan synthase (GTF-U) and a 1,6-alpha-glucan synthase (GTF-T) were purified to near homogeneity from the culture fluid of Streptococcus sobrinus strain B13N (serotype d) and characterized. In addition, a crude preparation of a recombinant oligo-isomaltosaccharide synthase (rGTF-S) was prepared from a cell-free extract of Escherichia coli MD124 transformant. Using four homogeneous GTF preparations including previously purified rGTF-S as antigens for immunization, 11 murine hybridomas producing a monoclonal antibody (MAb) were established through the fusion of myeloma cells (P3X63-Ag8-U1) and spleen cells of immunized BALB/c mice. When the immunoreactivities of the resultant MAbs were tested, all five MAbs raised against GTF-I, all three MAbs raised against GTF-T, and two of three MAbs raised against GTF-U reacted specifically with the homologous enzyme alone, while one MAb (B86) raised against GTF-U cross-reacted strongly with all GTFs. Although no MAb monospecific for rGTF-S was obtained, precise recognition of GTF-S was possible using the nonspecific B86 antibody together with the MAbs monospecific for the three glucan synthases. Thus, a set of four typical MAbs (B17, B76, B19 and B86) were successfully used for the identification of gene products expressed in 24 previously constructed E. coli phage clones, and the findings suggested that six phage clones might express a gtfU gene encoding GTF-U which has not been hitherto isolated.     

Immunochemical study of polysaccharide antigen in Streptococcus sobrinus and Streptococcus downei with a cross-reactive monoclonal antibody

Abstract A monoclonal antibody (mAb h-448) was prepared after cell fusion of mouse myeloma cells(SP2/0-Ag-14) to the spleen cells of mice immunised with serotype h strain (MF25) of Streptococcus downei . The antibody (IgM class) reacted in enzyme immunoassay only with whole cells as well as purified polysaccharide (PS) antigen of Streptococcus sobrinus (types d and g) and Streptococcus downei (serotypy h), but not with cells or purified PS antigen from any other serotypes of the mutants group of streptococci. mAb h-448 also quantitatively precipitated in solution with the purified antigens. Competitive hapten inhibition tests demonstrated that β-methylgalactopyranoside inhibited the reaction most strongly. Although rhamnose also showed a substantial inhibitory effect, the results of this study indicate that the antigenic determinant of the PS antigen has a structure similar to the β-methylgalactopyranoside molecule.     

Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synthetase).

The gene encoding glucosyltransferase responsible for water-insoluble glucan synthesis (GTF-I) of Streptococcus sobrinus (formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S. sobrinus 6715 DNA was constructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound alpha-1,6 glucan. GTF-I activity was found in only two large peptides: one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3' end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments: that for sucrose splitting (approximately 1,100 residues), that for glucan binding (approximately 240 residues), and that of unknown function (approximately 260 residues), in order from the N terminus. The primary structure of the GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologous protein from another strain of S. sobrinus.     

Influence of cranberry phenolics on glucan synthesis by glucosyltransferases and Streptococcus mutans acidogenicity

AIMS: To investigate the influence of several phenolic compounds isolated from cranberry fruit (Vaccinium macrocarpon) on some of the virulence properties of Streptococcus mutans associated with glucan synthesis and acidogenicity. METHODS AND RESULTS: Individual phenolic acids, flavonols and proanthocyanidins were isolated by semi-preparative high-performance liquid chromatography from fresh cranberry fruit. Flavonols and proanthocyanidins (at 500 micromol l(-1)) moderately inhibited the activity of surface-adsorbed glucosyltransferases (GTFs) B and C and F-ATPases (15-35% inhibition; P < 0.05), and also disrupted acid production by S. mutans cells without affecting bacterial viability. Phenolic acids displayed minimal biological effects. Quercetin-3-arabinofuranoside, myricetin and procyanidin A2 displayed the most inhibition of S. mutans virulence traits; a combination of these compounds displayed enhanced effects. CONCLUSIONS: Specific flavonoids from cranberries exhibit statistically significant but moderate biological activity against S. mutans. The biological activity of cranberry extracts may be a result from the complex mixture of flavonoids rather than a single active compound. SIGNIFICANCE AND IMPACT OF STUDY: This is the first study to identify the bioactive constituents in cranberry against an oral bacterium using highly purified isolated compounds. The combined effects of specific flavonols and proanthocyanidins from cranberry on GTFs activity, acid production and acid tolerance of S. mutans make them attractive compounds to fully explore for their anti-biofilm and cariostatic properties.     

Isolation and purification of Flavobacterium alpha-1,3-glucanase-hydrolyzing, insoluble, sticky glucan of Streptococcus mutans.

Studies were made on the physical and chemical properties of polysaccharides synthesized by cell-free extracts of Streptococcus mutans, Streptococcus sanguis, and Streptococcus sp. and their susceptibilities to dextranases. Among the polysaccharides examined, insoluble glucans were rather resistant to available dextranase preparations, and the insoluble, sticky glucan produced by S. mutans OMZ 176, which could be important in formation of dental plaques, was the most resistant. By enrichment culture of soil specimens, using OMZ 176 glucans as the sole carbon source, an organism was isolated that produced colonies surrounded by a clear lytic zone on opaque agar plates containing the OMZ 176 glucan. The organism was identified as a strain of Flavobacterium and named the Ek-14 bacterium. EK-14 bacterium was grown in Trypticase soy broth, and an enzyme capable of hydrolyzing the OMZ 176 glucan was concentrated from the culture supernatant and purified by negative adsorption on a diethylaminoethyl-cellulose (DE-32) column and gradient elution chromatography with a carboxymethyl-cellulose (CM-32) column. The enzyme was a basic protein with an isoelectric point of pH 8.5 and molecular weight of 65,000. Its optimum pH was 6.3 and its optimal temperature was 42 C. The purified enzyme released 11% of the total glucose residues of the OMZ 176 glucan as reducing sugars and solubilized about half of the substrate glucan. The products were found to be isomaltose, nigerose, and nigerotriose, with some oligosaccharides. The purified enzyme split the alpha-1,3-glucan endolytically and was inactive toward glucans containing alpha-1,6, alpha-1,4, beta-1,3, beta-1,4, and/or beta-1,6 bonds as the main linkages.     

Characterization of the product of the gtfS gene of Streptococcus downei, a primer-independent enzyme synthesizing oligo-isomaltosaccharides

The gtfS gene, coding for a glucosyltransferase which synthesizes water-soluble glucan and previously cloned from Streptococcus downei strain MFe28 (mutans serotype h) into a bacteriophage vector, was subcloned into a plasmid vector. The gtfS gene products expressed in Escherichia coli were compared to the primer-independent, oligo-isomaltosaccharide synthase in Streptococcus sobrinus strain AHT (mutans serotype g) and shown to resemble it closely in molecular mass, isoelectric point, immunological properties, optimum pH and Km values. The glucans produced from sucrose by the gtfS gene products are alpha-1,6-linked linear oligo-isomaltosaccharides without any branching sites. A similar gtfS gene was also detected on chromosomal DNA from S. sobrinus strain AHT.     

Single-molecule imaging of interaction between dextran and glucosyltransferase from Streptococcus sobrinus

Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of Streptococcus sobrinus. Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constraints estimated for TMR-labeled GTF at pH 6.8 and 25 degrees C in the absence and presence of sucrose were 9.2 and 13.3 s(-1), respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (> or = 25 s(-1)) under comparable conditions.     

Identification of amino acid residues in Streptococcus mutans glucosyltransferases influencing the structure of the glucan product.

The glucosyltransferases (GTFs) of mutans streptococci are important virulence factors in the sucrose-dependent colonization of tooth surfaces by these organisms. To investigate the structure-function relationship of the GTFs, an approach was initiated to identify amino acid residues of the GTFs which affect the incorporation of glucose residues into the glucan polymer. Conserved amino acid residues were identified in the GTF-S and GTF-I enzymes of the mutans streptococci and were selected for site-directed mutagenesis in the corresponding enzymes from Streptococcus mutans GS5. Conversion of six amino acid residues of the GTF-I enzyme to those present at the corresponding positions in GTF-S, either singly or in multiple combinations, resulted in enzymes synthesizing increased levels of soluble glucans. The enzyme containing six alterations synthesized 73% water-soluble glucan in the absence of acceptor dextran T10, while parental enzyme GTF-I synthesized no such glucan product. Conversely, when residue 589 of the GTF-S enzyme was converted from Thr to either Asp or Glu, the resulting enzyme synthesized primarily water-insoluble glucan in the absence of the acceptor. Therefore, this approach has identified several amino acid positions which influence the nature of the glucan product synthesized by GTFs.     

Purification of a fourth glucosyltransferase from Streptococcus sobrinus.

Recently, we found a novel primer-independent, water-soluble glucan synthase as a fourth glucosyltransferase (GTF) in a culture supernatant of strain AHT-k of Streptococcus sobrinus (Y. Yamashita, N. Hanada, and T. Takehara, Biochem. Biophys. Res. Commun. 150:687-693, 1988). In the present study, four kinds of purified GTFs, including the novel GTF, were prepared. They were composed of two primer-dependent GTFs and two primer-independent GTFs. Of the primer-dependent GTFs, one was a water-insoluble glucan synthase and the other was a water-soluble glucan synthase; both of the primer-independent GTFs were water-soluble glucan synthases (GTF-Sis). Using antisera against four purified GTFs, we concluded that the immunological properties of each were completely different from those of the others. Additionally, it was shown that the novel GTF-Si, which was previously shown to have a molecular weight of 137,000, was proteolytically degraded and could be isolated at a molecular weight of 152,000 and that Streptococcus cricetus secreted an enzyme that immunologically cross-reacted with GTF-Si. While the product of the novel GTF-Si was not an effective primer for both of the primer-dependent enzymes (water-soluble and -insoluble glucan synthases), the product of the enzyme affected the molecular size of the products of the other GTF-Sis.     

Purification and properties of extracellular mutacin, a bacteriocin from Streptococcus sobrinus.

Mutacin MT6223, a cell-free bacteriocin produced by Streptococcus sobrinus MT6223, was purified by ammonium sulphate precipitation, chromatofocusing with PBE 94 and column chromatography on SP Sephadex C-25. The specific activity of the purified mutacin was increased 1950-fold with a recovery of 9.7%. The molecular mass of the purified mutacin preparation was estimated to be 6.5 kDa. The mutacin activity was stable from pH 2-7, and was resistant to treatment at 100 degrees C for 20 min. It was inactivated by papain or ficin digestion, and was partially inhibited by alpha-chymotrypsin. The mutacin was found to be active against strains of serotypes c, e and f of Streptococcus mutans and the addition of purified mutacin MT6223 to growing cells of S. mutans MT8148 resulted in a rapid inhibition of incorporation of [3H]thymidine, [3H]uracil or L-[3H]glutamic acid into DNA, RNA or protein, respectively. Specific pathogen-free Fischer rats fed diet 2000 and infected with S. mutans MT8148R showed significantly fewer caries and lower plaque scores when mutacin was administered through drinking water. The present study demonstrates that mutacin MT6223 inhibited the growth of mutans streptococci. Thus, mutacin MT6223 may be a candidate for use in dental caries prevention.     

Homology between surface protein antigen genes of Streptococcus sobrinus and Streptococcus mutans

The structural gene (pag gene) for a 210 kDa protein antigen of Streptococcus sobrinus serotype g was cloned and compared with that (pac gene) of a 190 kDa protein antigen of Streptococcus mutans serotype c. Immunodiffusion analysis revealed that the product of the pag gene immunologically cross-reacted with that of the pac gene. Southern blot and nucleotide sequence analyses revealed that a significant homology existed between the middle regions of the two structural genes.     

Purification and characterization of glucosyltransferases from Streptococcus mutans 6715

A water-soluble glucan-synthesizing glucosyltransferase (GTase-S) and a water-insoluble glucan-synthesizing glucosyltransferase (GTase-I) were purified from culture supernatant of Streptococcus mutans 6715 (serotype g) by ammonium sulphate precipitation, chromatofocusing on a Polybuffer exchanger PBE 94 column, and subsequent phenyl-Sepharose CL-4B or hydroxyapatite column chromatography. The GTase-S and GTase-I activities were purified 4019- and 4714-fold, respectively, and the molecular weights were calculated to be 160000 and 165000, respectively. GTase-S had a pH optimum of 5.0, a Km of 8.8 mM for sucrose in the presence of 20 microM-dextran T10, and an isoelectric point of pH 4.3. GTase-I had two pH optima of 5.0 and 7.0, Km values of 4.9 mM (at pH 5.0) and 7.0 mM (at pH 7.0), mM (at pH 7.0), and an isoelectric point of pH 4.9. Methylation analysis indicated that the water-soluble glucan produced by GTase-S was a highly branched 1,6-alpha-linked D-glucan with 1,3-linked glucose residues, and that the water-insoluble glucan synthesized by GTase-I was composed of 1,3-alpha-linked glucose units.     

Nucleotide sequence and molecular characterization of a dextranase gene from Streptococcus downei

DNA fragments encoding the Streptococcus downei dextranase were amplified by PCR and inverse PCR based on a comparison of the dextranase gene (dex) sequences from S. sobrinus, S. mutans, and S. salivarius, and the complete nucleotide sequence of the S. downei dex was determined. An open reading frame (ORF) of dex was 3,891 bp long. It encoded a dextranase protein (Dex) consisting of 1,297 amino acids with a molecular mass of 139,743 Da and an isoelectric point of 4.49. The deduced amino acid sequence of S. downei Dex had homology to those of S. sobrinus, S. mutans and S. salivanus Dex in the conserved region (made of about 540 amino acid residues). DNA hybridization analysis showed that a dex DNA probe of S. downei hybridized to the chromosomal DNA of S. sobrinus as well as that of S. downei, but did not to other species of mutans streptococci. The C terminus of the S. downei Dex had a membrane-anchor region which has been reported as a common structure of C termini of both the S. mutans and S. sobrinus Dex. The recombinant plasmid which harbored the dex ORF of S. downei produced a recombinant Dex enzyme in Escherichia coli cells. The analysis of the recombinant enzyme on SDS-PAGE containing blue dextran showed multiple active forms as well as dextranases of S. mutans, S. sobrinus and S. salivarius.     

Cloning and nucleotide sequence analysis of the Streptococcus sobrinus gtfU gene that produces a highly branched water-soluble glucan

Streptococcus sobrinus has four gtf genes, gtfI, gtfS, gtfT, and gtfU, on the chromosome. These genes correspond respectively to the enzymes GTF-I, GTF-S1, GTF-S2, and GTF-S3. An Escherichia coli MD66 clone that contained the S. sobrinus gtfU gene was characterized. Immunological properties showed that the protein produced by the E. coli MD66 clone was similar to S. sobrinus GTF-S1. Biological properties and a linkage analysis of the glucans by 13C NMR spectrometry revealed that the protein produced by the E. coli MD66 clone was GTF-S1.     

Identification and Characterization of an Antigen I/II Homologous Gene, pah, from Streptococcus downei

Antigen I/II of Streptococcus mutans is a cell surface protein involved in the adherence of cells to tooth surfaces. In this study, an antigen I/II homologous gene, pah, was identified and sequenced from Streptococcus downei MFe28 using degenerate polymerase chain reaction (PCR) and the gene-walking method. The pah gene encodes a cell-wall-anchoring protein, PAh, containing 1565 amino acids. At the deduced amino acid sequence level, PAh shows a strong similarity to PAg of S. sobrinus (97.6% identity). Southern hybridization analysis indicated that a single copy of the pah gene was preserved in the chromosomal DNA of S. downei. Two pah mutants, SES-1 and SES-2, were constructed and analyzed by Western blotting. Two bands corresponding to 200- and 160-kDa proteins were observed in the parent strain, whereas no band was detected in pah mutant strains. In an adhesion assay of cells, pah mutants failed to adhere to tube surfaces in contrast to the parent strain. Furthermore, saliva-induced aggregation was decreased in pah mutants compared to the parent strain. Together, PAh is associated with the adhesion of cells to abiotic surfaces and whole saliva.     

Nucleotide sequence of a glucosyltransferase gene from Streptococcus sobrinus MFe28.

The complete nucleotide sequence was determined for the Streptococcus sobrinus MFe28 gtfI gene, which encodes a glucosyltransferase that produces an insoluble glucan product. A single open reading frame encodes a mature glucosyltransferase protein of 1,559 amino acids (Mr, 172,983) and a signal peptide of 38 amino acids. In the C-terminal one-third of the protein there are six repeating units containing 35 amino acids of partial homology and two repeating units containing 48 amino acids of complete homology. The functional role of these repeating units remains to be determined, although truncated forms of glucosyltransferase containing only the first two repeating units of partial homology maintained glucosyltransferase activity and the ability to bind glucan. Regions of homology with alpha-amylase and glycogen phosphorylase were identified in the glucosyltransferase protein and may represent regions involved in functionally similar domains.     

Purification and properties of extracellular glucosyltransferases from Streptococcus mutans serotype a

Extracellular glucosyltransferases (sucrose: 1,6-alpha-D-glucan 3-alpha- and 6-alpha-glucosyltransferase) of Streptococcus mutans HS6 (serotype a) were purified from the culture supernatant by DEAE-Sepharose chromatography, ConA-Sepharose chromatography and chromatofocusing. The enzymes I and II with specific activities of 6.20 and 5.86 i.u. mg-1, respectively, exhibited slightly different isoelectric points (pI 4.5 and 4.2) and the molecular weights were estimated to be 161000 and 174000, respectively, by SDS-PAGE. The enzymes had the same optimum pH of 5.5 and the same Km values of 1.3 mM for sucrose and of 83 microM-glucose equivalent for dextran T10. By double immunodiffusion test on agar, these enzymes were immunologically identical to each other. Analysis by GLC of the glucans synthesized de novo from sucrose by the enzymes (I and II) established that they were 1,6-alpha-D-glucans with 20 and 24.5 mol% 1,3,6-branch points, respectively. Both are therefore bifunctional enzymes.