Streptococcal Glucosyltransferases Research Articles

Expression of a streptococcal glucosyltransferase as a fusion to a solute-binding protein in Lactobacillus fermentum BR11

BspA is a non-covalently anchored cystine-binding protein from Lactobacillus fermentum BR11. It has previously been used to present antigens derived from infectious organisms on the L. fermentum BR11 cell surface. In this study, the capacity of BspA to present a very large polypeptide was tested. A temperature sensitive plasmid was constructed that encodes a 175-kDa chimeric protein consisting of a fusion between BspA and an N-terminally truncated derivative of the Streptococcus salivarius ATCC 25975 glucosyltransferase GtfJ. This plasmid was introduced into the L. fermentum genome. Integrants were able to incorporate 20–40 nmol sucrose derived glucose into glucan per ml culture per optical density unit. The glucosyltransferase activity was external to the cytoplasmic membrane and bound to the cell. Unlike native BspA, the BspA–GtfJ fusion could not be removed from the cell by 5 M LiCl wash.

Lactoperoxidase Inhibits Glucosyltransferases from Streptococcus mutans in vitro

This study examines the possible effect of the antimicrobial peroxidase system on the activity of streptococcal glucosyltransferases B, C and D (GtfB, GtfC and GtfD), either in solution (GtfB and GtfC) or when adsorbed to hydroxyapatite (GtfC and GtfD) at pH 6.5. The lactoperoxidase (LP) system (LP, H₂O₂, SCN^–) had no effect on the activity of dissolved GtfC, but the activity of dissolved GtfB was enhanced. The LP system, however, strongly inhibited the activities of both GtfC and GtfD in their adsorbed form. LP enzyme, without its substrates, inhibited all three Gtf enzymes: GtfB and GtfC in concentrations between 10 and 100 µg/ml in liquid phase and adsorbed GtfC and GtfD in concentrations between 25 and 50 µg/ml. This inhibition was in part abolished in liquid phase, but not in solid phase, if the substrates of LP were added. This study shows that the lactoperoxidase system can exert inhibitory activity against streptococcal Gtfs without generating oxidizing agents.

Role of the C-terminal YG repeats of the primer-dependent streptococcal glucosyltransferase, GtfJ, in binding to dextran and mutan.

The recombinant primer-dependent glucosyltransferase GtfJ of Streptococcus salivarius possesses a C-terminal glucan-binding domain composed of eighteen 21 aa YG repeats. By engineering a series of C-terminal truncated proteins, the position at which truncation prevented further mutan synthesis was defined to a region of 43 aa, confirming that not all of the YG motifs were required for the formation of mutan by GtfJ. The role of the YG repeats in glucan binding was investigated in detail. Three proteins consisting of 3.8, 7.2 or 11.0 C-terminal YG repeats were expressed in Escherichia coli. Each of the three purified proteins bound to both the 1,6-alpha-linked glucose residues of dextran and the 1,3-alpha-linked glucose residues of mutan, indicating that a protein consisting of nothing but 3.8 YG repeats could attach to either substrate. Secondary structure predictions of the primary amino acid sequence suggested that 37% of the amino acids were capable of forming a structure such that five regions of beta-sheet were separated by regions capable of forming beta-turns and random coils. CD spectral analysis showed that the purified 3.8 YG protein possessed an unordered secondary structure with some evidence of possible beta-sheet formation and that the protein maintained this relatively unordered structure on binding to dextran.

Facilitated intranasal induction of mucosal and systemic immunity to mutans streptococcal glucosyltransferase peptide vaccines.

Synthetic peptide vaccines which are derived from functional domains of Streptococcus mutans glucosyltransferases (GTF) have been shown to induce protective immunity in Sprague-Dawley rats after subcutaneous injection in the salivary gland region. Since mucosal induction of salivary immunity would be preferable in humans, we explored methods to induce mucosal antibody in the rat to the GTF peptide vaccines HDS and HDS-GLU after intranasal administration. Several methods of facilitation of the immune response were studied: the incorporation of peptides in bioadhesive poly(D,L-lactide-coglycolide) (PLGA) microparticles, the use of monoepitopic (HDS) or diepitopic (HDS-GLU) peptide constructs, or the use of mucosal adjuvants. Salivary immunoglobulin A (IgA) responses were not detected after intranasal administration of diepitopic HDS-GLU peptide constructs in alum or after incorporation into PLGA microparticles. However, significant primary and secondary salivary IgA and serum IgG antibody responses to HDS were induced in all rats when cholera holotoxin (CT) or a detoxified mutant Escherichia coli heat-labile enterotoxin (R192G LT) were intranasally administered with HDS peptide constructs in PLGA. Coadministration of LT with HDS resulted in predominantly IgG2a responses in the serum, while coadministration with CT resulted in significant IgG1 and IgG2a responses to HDS. Serum IgG antibody, which was induced to the HDS peptide construct by coadministration with these adjuvants, also bound intact mutans streptococcal GTF in an enzyme-linked immunosorbent assay and inhibited its enzymatic activity. Thus, immune responses which are potentially protective for dental caries can be induced to peptide-based GTF vaccines after mucosal administration if combined with the CT or LT R192G mucosal adjuvant.

Effects of Apis mellifera Propolis on the Activities of Streptococcal Glucosyltransferases in Solution and Adsorbed onto Saliva–Coated Hydroxyapatite

Propolis, a resinous hive product collected by Apis mellifera bees, has been used for thousands of years in folk medicine. Ethanolic extracts of propolis (EEP) have been shown to inhibit the activity of a mixture of crude glucosyltransferase (Gtf) enzymes in solution. These enzymes synthesize glucans from sucrose, which are important for the formation of pathogenic dental plaque. In the present study, the effects of propolis from two different regions of Brazil on the activity of separate, purified Gtf enzymes in solution and on the surface of saliva–coated hydroxyapatite (sHA) beads were evaluated. The EEP from Minas Gerais (MG; Southeastern Brazil) and Rio Grande do Sul (RS; Southern Brazil) were tested for their ability to inhibit the enzymes GtfB (synthesis of insoluble glucan), GtfC (insoluble/soluble glucan) and GtfD (soluble glucan). The effects of propolis on Gtf from Streptococcus sanguis (soluble glucan synthesis) was also explored. The EEP from both regions effectively inhibited the activity of all Gtfs in solution (75–95%) and on the surface of sHA beads (45–95%) at concentrations between 0.75 and 3.0 mg of propolis/ml. However, the two samples of propolis showed different levels of inhibition on each of the enzymes tested. In general, EEP RS demonstrated a significantly higher inhibitory activity on GtfB and C activities (both solution and surface assays) than EEP MG at concentrations between 0.047 and 0.187 mg/ml (p<0.05). EEP MG, on the other hand, exhibited a greater inhibitory effect on the activities of surface GtfD (at 0.375, 0.75 and 1.5 mg/ml) and S. sanguis Gtf (at 1.5 and 3.0 mg/ml; p<0.05). These data indicate that EEP is a potent inhibitor of Gtf enzymes in solution and adsorbed on an experimental pellicle; however, its effect on Gtf activity is variable depending on the geographical origin of the propolis samples. There is a need to identify the active compounds of propolis.

Nucleotide sequencing and transcriptional analysis of two tandem genes encoding glucosyltransferase (water-soluble-glucan synthetase) in Streptococcus cricetus HS-6.

Two tandem genes encoding glucosyltransferase synthesizing water-soluble glucan (GTF-S) were cloned from the lambda gene library of Streptococcus cricetus HS-6 (serotype a) using anti-GTF-S antibody, and the nucleotide sequences were analyzed. The two genes (ORF1 and ORF2) were identified as streptococcal glucosyltransferases based on the following evidence: [1] the deduced amino acid sequences of their products have an active site for catalytic action and C-terminal repeated units for dextran binding, and [2] a homology search revealed that the ORF1 and ORF2 products are homologous to the GtfS protein (77.4%) of S. downei Mfe28 and GtfT protein (83.8%) of S. sobrinus OMZ176, respectively, which are both known to have GTF-S activity. Therefore, ORF1 and ORF2 might be designated gtfS and gtfT of S. cricetus, respectively. A Northern blotting and RNase protection assay suggested that the gtfS and gtfT of S. cricetus are transcribed as a single bicistronic mRNA as well as separate monocistronic mRNAs. Primer extension analysis indicated multiple transcriptional start points for each gene.

Coimmunization with complementary glucosyltransferase peptides results in enhanced immunogenicity and protection against dental caries.

Peptide constructs from the catalytic (CAT) and glucan-binding (GLU) regions of the mutans streptococcal glucosyltransferase enzymes (GTF) can provide immunity to dental caries infection. A strategy of coimmunization was tested to determine whether protection could be enhanced. Rats were immunized with one of the previously described peptide constructs from the CAT or GLU region of the GTF of mutans streptococci or coimmunized with a combination of these constructs (CAT-GLU). Coimmunized animals demonstrated significantly higher serum immunoglobulin G (IgG) and salivary IgA antibody levels to CAT or GTF than rats immunized with either construct alone. To assess the functional significance of coimmunization with these constructs, animals were immunized as above or with Streptococcus sobrinus GTF and then infected with S. sobrinus to explore the effects of immunization on immunological, microbiological, and disease (dental caries) parameters. Serum antibody from the communized group inhibited S. sobrinus GTF-mediated insoluble glucan synthesis in vitro above that of the individual-construct-immunized groups. Immunization with CAT or GLU constructs resulted in significantly reduced dental caries after infection with S. sobrinus compared with sham-immunized animals. Coimmunization produced greater reductions in caries than after immunization with either CAT or GLU. Also, significant elevations in lymphocyte proliferative responses to CAT, GLU, and GTF were observed after coimmunization with CAT-GLU compared with the responses after immunization with the individual constructs. The results suggested that increased numbers of memory T cells, which could proliferate to CAT, were generated by coimmunization. The experiments support the functional significance of these GTF domains in dental caries pathogenesis and present coimmunization as a simple alternative to intact GTF to enhance protective immunity against cariogenic microorganisms.

Gene encoding a dextransucrase-like protein in Leuconostoc mesenteroides NRRL B-512F.

A gene, dsrT, encoding a dextransucrase-like protein was isolated from the genomic DNA libraries of Leuconostoc mesenteroides NRRL B-512F dextransucrase-like gene. The gene was similar to the intact open reading frames of the dextransucrase gene dsrS of L. mesenteroides NRRL B-512F, dextransucrase genes of strain NRRL B-1299 and streptococcal glucosyltransferase genes, but was truncated after the catalytic domain, apparently by the deletion of five nucleotides. dsrT mRNA was produced in this strain L. mesenteroides when cells were grown in a sucrose medum, but at a level of 20% of that of dsrS mRNA. The molecular weight of the dsrT gene product was 150,000 by SDS-PAGE. The product did not synthesize dextran, but had weak sucrose cleaving activity. The insertion of five nucleotides at the putative deletion point in dsrT resulted in an enzyme with a molecular weight of 210,000 and with dextransucrase activity.

Structure and enzymatic properties of genetically truncated forms of the water-insoluble glucan-synthesizing glucosyltransferase from Streptococcus sobrinus.

Glucosyltransferase-I (GTF-I: 175 kDa) of a cariogenic bacterium, Streptococcus sobrinus 6715, mediates the conversion of water-soluble dextran (alpha-1,6-glucan) into a water-insoluble form by making numerous alpha-1,3-glucan branches along the dextran chains with sucrose as the glucosyl donor. The structures and catalytic properties were compared for two GTF-I fragments, GTF-I' (138 kDa) and GS (110 kDa). Both lack the N-terminal 84 residues of GTF-I. While GTF-I' still contains four of the six C-terminal repeats characteristic of streptococcal glucosyltransferases, GS lacks all of them. Electron microscopy of negatively stained samples indicated a double-domain structure for GTF-I', consisting of a spherical head with a smaller spherical tail, which was occasionally seen as a long extension. GS was seen just as the head portion of GTF-I'. In the absence of dextran, both fragments simply hydrolyzed sucrose with similar K(m) and k(cat) values at low concentrations (<5 mM). At higher sucrose concentrations (>10 mM), however, GTF-I' exhibited glucosyl transfer activity to form insoluble alpha-1, 3-glucans. So did GS, but less efficiently. Dextran increased the rate and efficiency of the glucosyl transfer by GTF-I'. On removal of the C-terminal repeats of GTF-I' by mild trypsin treatment, this dextran-stimulated transfer was completely lost and the dextran-independent transfer became less efficient. These results indicate that the N-terminal two-thirds of the GTF-I sequence are organized as a structurally and functionally independent domain to catalyze not only sucrose hydrolysis but also glucosyl transfer to form alpha-1,3-glucan chains, although not efficiently; the C-terminal repeat increases the efficiency of the intrinsic glucosyl transfer by the N-terminal domain as well as rendering the whole molecule primer-dependent for far more efficient insoluble glucan synthesis.

Binding properties of streptococcal glucosyltransferases for hydroxyapatite, saliva-coated hydroxyapatite, and bacterial surfaces

The binding specificities of Streptococcus glucosyltransferase (Gtf) B, C and D for hydroxyapatite (HA), saliva-coated hydroxyapatite (SHA), and bacterial surfaces were examined. For HA beads the following values were obtained: ( K=affinity; N=number of binding sites) GtfB, K=46×10 5 ml/ μmol, N=0.65×10 −6 μmol/m 2; GtfC, K=86×10 5 ml/ μmol, N=4.42×10 −6 μmol/m 2; GtfD, K=100×10 5 ml/ μmol, N=0.83×10 −6 μmol/m 2. For SHA beads, the following values were obtained: GtfB, K=14.7×10 5 ml/ μmol, N=1.03×10 −6 μmol/m 2; GtfC, K=21.3×10 5 ml/ μmol, N=3.66×10 −6 μmol/m 2; GtfD, K=1.73×10 5 ml/ μmol, N=8.88×10 −6 μmol/m 2. The binding of GtfB to SHA beads was reduced in the presence of parotid saliva, but the binding of GtfC and D was unaffected. The binding of GtfB to SHA in the presence of parotid saliva supplemented with GtfC and D was reduced when compared with its binding to SHA in the presence of parotid saliva alone. In contrast, the binding of GtfC and D to SHA was unaffected when parotid saliva was supplemented with the other Gtf enzymes. GtfB bound to several bacterial strains ( Strep. mutans GS-5, Actinomyces viscosus OMZ105E and Lactobacillus casei 4646) in an active form, while GtfC and D did not bind to bacterial surfaces. It is concluded that of the three Gtf enzymes, GtfC has the highest affinity for HA and SHA surfaces and can adsorb on to the SHA surface in the presence of the other two enzymes. GtfD also binds to SHA in the presence of the other enzymes but has a very low affinity for the surface. GtfB does not bind to SHA in the presence of the other Gtf enzymes but binds avidly to bacterial surfaces in an active form. Therefore, GtfC most probably binds to apatitic surfaces, while GtfB binds to bacterial surfaces.

Glucan Binding Regions of Dextransucrase fromLeuconostoc mesenteroidesNRRL B-512F

We isolated glucan-binding peptides of a dextransucrase from Leuconostoc mesenteroides B-512F. The dextransucrase was bound to DEAE-Sephadex A-50, Sephadex G-100, and mutan from Streptococcus mutans. Mild trypsin digestion dissociated the enzyme and glucan binding. In the presence of ammonium sulfate, several peptides were bound to glucan after trypsin digestion. Four main mutan-binding peptides were obtained by this method, and those amino acid sequences were analyzed. One of them was identical with the dextran-binding peptide that contains lysine, which was previously isolated by differential chemical modification with o-phthalaldehyde. We also found mutan-binding peptides in sucrose- and dextran-binding regions and a lysine-rich region. Also, there was a peptide similar in sequence to glucan-binding A-repeat of streptococcal glucosyltransferases.

Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene, and secrete its active gene product

A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H. K. Kuramitsu, Plasmid, 1995, 34, 85–95). This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb BglII fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3′-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3′-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.

Immunogenicity and protective immunity induced by synthetic peptides associated with a catalytic subdomain of mutans group streptococcal glucosyltransferase.

We examined the immunogenicity and induction of protective immunity of two 19-mer sequences (GGY and AND) which overlapped a highly conserved region which has recently been implicated in the enzymatic activity of glucosyltransferases (GTFs) of the mutans group streptococci. These peptides were synthesized as eight-branched constructs on a lysine core. Serum immunoglobulin G (IgG) antibody, induced by subcutaneous (s.c. [salivary gland vicinity]) injection with these peptide constructs, reacted with the inciting antigen, with mutans streptococcal GTFs, and with a 21-mer peptide (CAT) containing an aspartate previously shown to covalently bind sucrose. Several of these antisera also inhibited the ability of Streptococcus sobrinus GTF to synthesize insoluble glucan. Significant levels of salivary IgA antibody were also induced by GGY and AND peptide constructs after s.c. injection. The effect of immunization with the GGY and AND peptide constructs on the cariogenicity of Streptococcus mutans was studied in three experiments by immunization of weanling Sprague-Dawley rats, twice at 7- to 14-day intervals with peptides, S. sobrinus GTF, or phosphate-buffered saline. All rats were then orally infected with S. mutans SJ. After 63-day infection periods, the GGY and AND-injected groups had significant dental caries reductions compared with sham-injected groups in most experiments. These studies support the existence of an additional catalytic subdomain within the sequence defined by the GGY and AND peptides. Furthermore, the epitopes defined in these sequences have significant immunogenicity, can induce immune responses which interfere with GTF-mediated glucan synthesis in vitro, and can protect rats from experimental dental caries.

Cloning and sequencing of a gene coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only α(1–6) and α(1–3) linkages

The coding region for a Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene ( dsrA) was isolated and sequenced. Using a pair of primers designed on the basis of two highly conserved amino-acid (aa) sequences in L. mesenteroides NRRL B-512F dextransucrase and streptococcal glucosyltransferases (GTFs), a fragment of dsrA was amplified by the polymerase chain reaction (PCR). This PCR product was used as an hybridization probe to isolate a 1.8-kb fragment identified as the central region of dsrA. Cleavage by Sac I of this fragment allowed two probes to be obtained to isolate the 5′ and the 3′ ends of dsrA. The nucleotide sequence of the dsrA gene was determined and found to consist of an open reading frame (ORF) of 4870 base pairs (bp) coding for a 1290-aa protein with an M r of 145 590. The aa sequence exhibited a high similarity with other GTFs. The two domains previously described in GTFs are conserved in DSRA: an N-terminal conserved domain and a C-terminal domain composed of a series of repeats. Surprisingly, the expected signal peptide was not detected. The entire gene was reconstructed and the activity of DSRA was investigated. The dextran produced appeared to be composed of 85% α(1–6) and 15% α(1–3) linkages and the oligosaccharides synthesized in the presence of maltose were mainly composed of α(1–6) linkages. This enzyme is a novel dextransucrase from L. mesenteroides NRRL B-1299 producing no α(1–2) linkages and is the first glucosyltransferase having no signal peptide described.

Interactions of streptococcal glucosyltransferases with α-amylase and starch on the surface of saliva-coated hydroxyapatite

The salivary pellicle consists of various proteins and glycoproteins which may interact with one another. Experiments were performed to elucidate the interactions of streptococcal glucosyltransferase (Gtf) enzymes with human salivary α-amylase in solution and on the surface of saliva-coated hydroxyapatite (SHA) beads. The Gtf enzymes -B, -C and -C, when immobilized on to SHA beads, reduced the activity of adsorbed amylase; GtfD showed the highest inhibition of salivary amylase activity. The presence of glucan produced by immobilized GtfD did not further reduce amylase activity. The amount of amylase adsorbed on to hydroxyapatite beads was reduced when salivary amylase was added simultaneously with any of the Gtf enzymes, suggesting that amylase and Gtfs may compete with each other for binding sites on hydroxyapatite. Starch hydrolysates produced by SHA-surface-bound salivary amylase were tested for their effect on glucan production from sucrose by Gtf enzymes in solution and on SHA beads; glucan production by SHA-immobilized GtfB was stimulated in the presence of starch hydrolysates. Glucan synthesized by SHA-immobilized GtfB in the presence of starch hydrolysates was less susceptible to hydrolysis by the fungal enzyme mutanase than was glucan made by SHA-immobilized GtfB in the absence of starch hydrolysates. Glucan production by GtfB associated with streptococci immobilized on to SHA was also enhanced in the presence of starch hydrolysates. The adhesion of oral micro-organisms to SHA coated with glucan made in the presence and absence of starch hydrolysates was investigated, and some bacteria displayed higher adhesion activities for the glucan made in the presence of the hydrolysates. Therefore, the interaction of amylase and Gtf enzymes on a SHA surface may modulate the formation of glucan and the adherence of oral micro-organisms.

Immunization of rats with synthetic peptide constructs from the glucan-binding or catalytic region of mutans streptococcal glucosyltransferase protects against dental caries.

Previously, we have described peptide constructs from two regions of glucosyltransferase (GTF) of mutans streptococci. A putative catalytic site in the amino-terminal half of the molecule and a repeated glucan-binding site in the carboxyl-terminal half of GTF were the regions upon which sequences were based. The present study explored the effects of immunization with these peptide constructs (called CAT or GLU) and with streptococcal GTFs from Streptococcus sobrinus and S. mutans on immunological, microbiological, and disease parameters. Groups of immunized Sprague-Dawley rats were infected with either 10(8) S. sobrinus 6715 or 10(8) S. mutans SJ32 organisms. Serum immunoglobulin G antibody levels, determined by enzyme-linked immunosorbent assay, to the respective peptide constructs and to the appropriate streptococcal GTF were significantly increased (after immunization) prior to infection and at the end of the experiment. Also, serum antibody from CAT-, GLU-, and S. sobrinus GTF-immunized rats inhibited S. sobrinus GTF-mediated insoluble glucan synthesis (all) and S. mutans GTF-mediated soluble glucan synthesis (all except anti-GLU) from sucrose. Immunization with the CAT or GLU peptide construct resulted in significantly reduced smooth surface and sulcal caries after infection with S. sobrinus. Sulcal dental caries after infection with S. mutans SJ32 were also significantly reduced in CAT- and GLU-immunized rats. Thus, immunization with peptides whose sequences are based on putative functional domains of mutans streptococcal GTF are protective toward a cariogenic S. sobrinus or S. mutans infection.

The effect of milk and kappa casein on streptococcal glucosyltransferase.

Many dietary components such as carbohydrates, lipids and proteins may be incorporated into the salivary pellicle and thus may affect glucosyltransferase (GTF) activity on pellicle surfaces. The effect of milk on streptococcal GTF activity was determined. Milk, when coated onto buffer-coated hydroxyapatite or saliva-coated hydroxyapatite (sHA), reduced the subsequent adsorption of GTF onto the surfaces. Milk also reduced the expression of enzymatic activity of GTF adsorbed onto sHA. kappa-Casein, when present on the surface of sHA, reduced the adsorption of GTF activity onto sHA, resulting in reduced glucan formation. alpha-Casein had no effect on the adsorption of GTF onto sHA or on subsequent glucan formation. Both milk and kappa-casein reduced activity of the enzyme in solution. The presence of milk and kappa-casein fractions on the surface of sHA and in solution with GTF can clearly modulate glucan formation in vitro.

Aggregated form of dextransucrases from Leuconostoc mesenteroides NRRL B-512F and its constitutive mutant.

Purified dextransucrases [EC 2.4.1.5], DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE-Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively. DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively. Although the sugar contents of the purified enzymes were different, their molecular masses by SDS-PAGE were all 170 kDa. DSW-D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V0) on Sepharose 6B, while the DSM-G was eluted at 1.2 x V0 volume. On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively. The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other.

Immunological characteristics of a synthetic peptide associated with a catalytic domain of mutans streptococcal glucosyltransferase.

The immunogenicity of a multiple antigenic peptide construct consisting of four copies of the synthetic 21-mer peptide DANFDSIRVDAVDNVDADLLQ was measured. The composition of this peptide was derived from a sequence in the N-terminal region of mutans streptococcal glucosyltransferases (GTFs) containing an aspartic acid implicated in catalysis. The peptide (CAT) construct was synthesized as a tetramer on a lysine backbone and subcutaneously injected into Sprague-Dawley rats for polyclonal antibody formation or intraperitoneally injected into BALB/c mice, and then spleen cell fused with Sp2/0Ag14 murine myeloma cells for monoclonal antibody formation. The resulting rat antisera and mouse monoclonal antibodies reacted with CAT and with native GTF isozymes from Streptococcus sobrinus and Streptococcus mutans (in enzyme-linked immunosorbent assay and Western blot [immunoblot] analyses). Functional inhibition of the water-insoluble glucan synthetic activity of S. sobrinus GTF-I was demonstrated with an immunoglobulin M anti-CAT monoclonal antibody (> 80% inhibited) and with rat sera (approximately 17% inhibited). The monoclonal antibody preparation also modestly inhibited the water-soluble glucan synthetic activity of an S. mutans GTF mixture. These results suggest that the CAT peptide contains B-cell epitopes that are similar to those of intact mutans streptococcal GTFs and has the potential to elicit antibody that can inhibit GTF function. Thus, sequences within this peptide construct may have value for inclusion in a synthetic dental caries vaccine.

Definition of a Fundamental Repeating Unit in Streptococcal Glucosyltransferase Glucan-binding Regions and Related Sequences

The C-termini of the glucosyltransferases (Gtfs) of oral streptococci are responsible for glucan binding. These glucan-binding domains (GBDs) are composed of a series of repeated sequences that have been classified into four different classes (A-D) by virtue of sequence similarity and which, by inference, have been suggested to be of functional importance. In contrast, we propose that repeat sequences evolve in response to selection for an increase in the number of copies of a particular domain through multiple duplication events occurring at different times. According to this hypothesis, repeats should possess various degrees of similarity, especially if only key residues are of functional importance. Analysis of the GBDs of the Gtfs indicated that a common fundamental repeat, designated the "YG" repeat, could be discerned within the "A", "B", "C", and "D" repeats. Similar elements were also conserved in the ligand-binding repeats of the Clostridium difficile toxins and the lysins and the PspA protein of Streptococcus pneumoniae, suggesting that similar selective pressures had also been imposed on these sequences. Analysis of the "YG" repeats present in the GtfJ and GtfK of Streptococcus salivarius indicated that some of the "YG" repeats in the GBDs of these proteins had arisen as a result of duplication events involving a series of three sequential "YG" repeats.