Streptococcal Glucosyltransferases Research Articles

Role of glucosyltransferase R in biofilm interactions between Streptococcus oralis and Candida albicans

Streptococcal glucosyltransferases (Gtf) synthesize α-glucan exopolymers which contribute to biofilm matrix. Streptococcus oralis interacts with the opportunistic pathogen Candida albicans to form hypervirulent biofilms. S. oralis 34 has a single gtf gene (gtfR). However, the role of gtfR in single and mixed species biofilms with C. albicans has never been examined. A gtfR deletion mutant, purified GtfR, and recombinant GtfR glucan-binding domain were tested in single and mixed biofilms on different substrata in vitro. A mouse oral infection model was also used. We found that in single species biofilms growing with sucrose on abiotic surfaces S. oralis gtfR increased biofilm matrix, but not bacterial biomass. In biofilms with C. albicans, S. oralis encoding gtfR showed increased bacterial biomass on all surfaces. C. albicans had a positive effect on α-glucan synthesis, and α-glucans increased C. albicans accretion on abiotic surfaces. In single and mixed infection of mice receiving sucrose S. oralis gtfR enhanced mucosal burdens. However, sucrose had a negative impact on C. albicans burdens and reduced S. oralis burdens in co-infected mice. Our data provide new insights on the GtfR-mediated interactions between the two organisms and the influence of biofilm substratum and the mucosal environment on these interactions.

Activated human valvular interstitial cells sustain interleukin-17 production to recruit neutrophils in infective endocarditis.

The mechanisms that underlie valvular inflammation in streptococcus-induced infective endocarditis (IE) remain unclear. We previously demonstrated that streptococcal glucosyltransferases (GTFs) can activate human heart valvular interstitial cells (VIC) to secrete interleukin-6 (IL-6), a cytokine involved in T helper 17 (Th17) cell differentiation. Here, we tested the hypothesis that activated VIC can enhance neutrophil infiltration through sustained IL-17 production, leading to valvular damage. To monitor cytokine and chemokine production, leukocyte recruitment, and the induction or expansion of CD4(+) CD45RA(-) CD25(-) CCR6(+) Th17 cells, primary human VIC were cultured in vitro and activated by GTFs. Serum cytokine levels were measured using an enzyme-linked immunosorbent assay (ELISA), and neutrophils and Th17 cells were detected by immunohistochemistry in infected valves from patients with IE. The expression of IL-21, IL-23, IL-17, and retinoic acid receptor-related orphan receptor C (Rorc) was upregulated in GTF-activated VIC, which may enhance the proliferation of memory Th17 cells in an IL-6-dependent manner. Many chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), were upregulated in GTF-activated VIC, which might recruit neutrophils and CD4(+) T cells. Moreover, CXCL1 production in VIC was induced in a dose-dependent manner by IL-17 to enhance neutrophil chemotaxis. CXCL1-expressing VIC and infiltrating neutrophils could be detected in infected valves, and serum concentrations of IL-17, IL-21, and IL-23 were increased in patients with IE compared to healthy donors. Furthermore, elevated serum IL-21 levels have been significantly associated with severe valvular damage, including rupture of chordae tendineae, in IE patients. Our findings suggest that VIC are activated by bacterial modulins to recruit neutrophils and that such activities might be further enhanced by the production of Th17-associated cytokines. Together, these factors can amplify the release of neutrophilic contents in situ, which might lead to severe valvular damage.

Sequence and phylogenetic analyses of the water‐soluble glucan synthesizing‐glucosyltransferase genes of Streptococcus dentirousetti

Two tandemly aligned glucosyltransferase (GTF) genes whose gene products are responsible for water-soluble glucan synthesis were isolated from Streptococcus dentirousetti NUM1303 and sequenced. One of the GTF genes of S. dentirousetti consisted of a 4110 bp open reading frame (ORF) that encoded for a 1369 amino acid protein and was revealed to be a S. sobrinus gtfS homolog. The percent similarity of amino acid sequences of the GTF-S from S. dentirousetti compared to those from S. sobrinus was 99%. In addition, a putative gtfT was found in tandem in the downstream region of the S. dentirousetti gtfS. The gtfT of S. dentirousetti consisted of a 4527 bp ORF encoding for 1508 amino acids. The similarity of amino acid sequences of the GTF-T from S. dentirousetti and S. sobrinus was 94%. Phylogenetic analysis based on amino acid sequences from other related streptococcal GTFs suggested that both GTF-S and GTF-T of S. dentirousetti are closely related to S. sobrinus.

Evolution of Cariogenic Character in Streptococcus mutans: Horizontal Transmission of Glycosyl Hydrolase Family 70 Genes

Acquisition of the ability to produce polysaccharides from sucrose, i.e. the gtf gene encoding glucosyltransferase (GTF), is the key evolutionary event enabling dental biofilm formation by streptococci. To clarify the ancestry of streptococcal GTFs, time of its occurrence, and order of specific events, we investigated the distribution of GTFs among bacteria by phylogenetic analysis of the glycosyl hydrolase family 70 enzymes. We found that streptococcal GTFs were derived from other lactic acid bacteria such as Lactobacillus and Leuconostoc, and propose the following evolutionary model: horizontal gene transfer via transposons occurred when streptococci encountered lactic acid bacteria contained in fermented food. Intra-genomic gene duplication occurred by a secondary selection pressure such as consumption of refined sugar. Our findings concerning this evolution in Streptococcus mutans provide an important background for studies of the relationship between the historical spread of dental caries and anthropological factors.

Exopolysaccharides produced by Streptococcus mutans glucosyltransferases modulate the establishment of microcolonies within multispecies biofilms.

Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces.

Sequence and Phylogenetic Analyses of the Glucosyltransferase Gene of Mutans Streptococci Isolated from the Fruit Bat Oral Cavity

The nucleotide sequence of the glucosyltransferase (GTF) gene was determined for the mutans streptococci isolated from the fruit bat oral cavity, Streptococcus dentirousetti NUM1303T, which encodes a GTF enzyme that synthesizes a water-insoluble glucan. The gtf of S. dentirousetti consisted of a 4,398-bp open reading frame that encoded for a 1,466 amino acid protein and was revealed to belong to the gtfI group. The deduced 38-amino acid sequence of the N-terminal was thought to be a signal peptide for the secretion of GTF-I enzyme, which has high similarity to known GTFs from other streptococci, and three major functional domains of GTFs : an N-terminal variable region, a conserved catalytic site for the hydrolysis of sucrose, and C-terminal repeating units for glucan binding. The percent homology of the amino acid sequence of the GTF-I from S. dentirousetti and Streptococcus orisuis is 99%, however, this score shows 78% when compared to Streptococcus sobrinus. Phylogenetic analysis suggested that this gtfI was closely related to Streptococcus orisuis based on the amino acid sequences from the other related streptococcal GTFs.

A traditional Sudanese fermented camel's milk product, Gariss, as a habitat of Streptococcus infantarius subsp. infantarius

Samples of the traditional Sudanese fermented camel's milk product Gariss representing 9 different regions in Sudan were microbiologically characterized using an integrated approach including phenotypic and genotypic methods. Lactic acid bacteria [log(CFU/g) = 7.76–8.66] and yeasts [log(CFU/g) = 6.05–7.79] were detected in high numbers. A total of 180 LAB isolates were identified of which 123 isolates were found to belong to the Streptococcus bovis group. Thirteen representative isolates were characterized by PCR amplification and sequencing of the housekeeping genes rpoB and sodA and the streptococcal glucosyltransferase gene ( gtf). All thirteen isolates were identified as Streptococcus infantarius subsp. infantarius, a potential human pathogen. The gene encoding the virulence determinant gtf was detected in 10 of the 13 tested strains. The same isolates were able to survive exposure to 0.3% (w/v) oxgall for 4 h and pH=2.7 for 1--4 h. Also Lactobacillus fermentum were detected in high numbers, whereas Enterococcus faecium and Lactobacillus helveticus were detected more occasionally. The yeast microflora in all Gariss samples consisted of Kluyveromyces marxianus and Issatchenkia orientalis with the former being predominant in 7 out of 9 samples.

Mutans Streptococcal Infection Induces Salivary Antibody to Virulence Proteins and Associated Functional Domains

The interplay between mucosal immune responses to natural exposure to mutans streptococci and the incorporation and accumulation of these cariogenic microorganisms in oral biofilms is unclear. An initial approach to explore this question would be to assess the native secretory immunity emerging as a consequence of Streptococcus mutans infection. To this end, we analyzed salivary immunoglobulin A (IgA) antibody to mutans streptococcal glucosyltransferase (Gtf) and glucan binding protein B (GbpB) and to domains associated with enzyme function and major histocompatibility complex (MHC) class II binding in two experiments. Salivas were collected from approximately 45-day-old Sprague-Dawley rats, which were then infected with S. mutans SJ32. Infection was verified and allowed to continue for 2 to 2.5 months. Salivas were again collected following the infection period. Pre- and postinfection salivas were then analyzed for IgA antibody activity using peptide- or protein-coated microsphere Luminex technology. S. mutans infection induced significant levels of salivary IgA antibody to Gtf (P < 0.002) and GbpB (P < 0.001) in both experiments, although the levels were usually far lower than the levels achieved when mucosal immunization is used. Significantly (P < 0.035 to P < 0.001) elevated levels of postinfection salivary IgA antibody to 6/10 Gtf peptides associated with either enzyme function or MHC binding were detected. The postinfection levels of antibody to two GbpB peptides in the N-terminal region of the six GbpB peptides assayed were also elevated (P < 0.031 and P < 0.001). Interestingly, the patterns of the rodent response to GbpB peptides were similar to the patterns seen in salivas from young children during their initial exposure to S. mutans. Thus, the presence of a detectable postinfection salivary IgA response to mutans streptococcal virulence-associated components, coupled with the correspondence between rat and human mucosal immune responsiveness to naturally presented Gtf and GbpB epitopes, suggests that the rat may be a useful model for defining mucosal responses that could be expected in humans. Under controlled infection conditions, such a model could prove to be helpful for unraveling relationships between the host response and oral biofilm development.

C-Terminal Repeats ofClostridium difficileToxin A Induce Production of Chemokine and Adhesion Molecules in Endothelial Cells and Promote Migration of Leukocytes

The C-terminal repeating sequences of Clostridium difficile toxin A (designated ARU) are homologous to the carbohydrate-binding domain of streptococcal glucosyltransferases (GTFs) that were recently identified as potent modulins. To test the hypothesis that ARU might exert a similar biological activity on endothelial cells, recombinant ARU (rARU), which was noncytotoxic to cell cultures, was analyzed using human umbilical vein endothelial cells. The rARU could bind directly to endothelial cells in a serum- and calcium-dependent manner and induce the production of interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein 1 in a dose-dependent manner. An oligosaccharide binding assay indicated that rARU, but not GTFC, binds preferentially to Lewis antigens and 3'HSO3-containing oligosaccharides. Binding of rARU to human endothelial or intestinal cells correlated directly with the expression of Lewis Y antigen. Bound rARU directly activated mitogen-activated protein kinases and the NF-kappaB signaling pathway in endothelial cells to release biologically active chemokines and adhesion molecules that promoted migration in a transwell assay and the adherence of polymorphonuclear and mononuclear cells to the endothelial cells. These results suggest that ARU may bind to multiple carbohydrate motifs to exert its biological activity on human endothelial cells.

Assessment of Human Immune Response to Mutans Streptococcal Glucosyltransferase Peptides Selected by MHC Class II Binding Probability

Dental decay is a major public health challenge, causing substantial social and economic burdens. In animals, vaccination against mutans streptococci, the causative organism, interferes with dental caries. The mutans streptococcal glucosyltransferase (GTF) has been effectively used as a protein antigen in experimental dental caries vaccines. Compared to whole proteins, peptide subunits can focus immune responses on protective epitopes, and not on potentially harmful cross reactive antigens. In the past we selected peptide subunits of GTF for vaccine discovery based on putative functional significance and conservation of GTF primary structure. To focus on the immunogenicity of peptides, we estimated the probability of MHC class II binding. Twenty 20-mer linear GTF peptides were synthesized on this basis and their immunoreactivity explored. Significant human peripheral blood mononuclear cell (PBMC; n = 12) proliferation was observed in response to amino acids (AA) 502–521 (peptide 7), located in the catalytic domain of GTF. Human serum (n = 36) antibody reactivity was observed to AA 438–457 (peptide 5), AA 502–521 (peptide 7), and AA 1376–1395 (peptide 16). Whole saliva mutans streptococcal levels were used as markers of mutans infection, and dental examinations to determine existing and historic caries (DMFS score) were performed. DMFS scores correlated with mutans streptococcal counts, but not with immune responses. We have identified peptides with projected avid MHC-binding activity that reacted with human PBMC and serum antibody, implying that these peptides are immunogenic and may be of significance in a subunit dental caries vaccine.

Periodontal Bacterial DNA Suppresses the Immune Response to Mutans Streptococcal Glucosyltransferase

Certain CpG motifs found in bacterial DNA enhance immune responses through Toll-like receptor 9 (TLR-9) and may also demonstrate adjuvant properties. Our objective was to determine if DNA from bacteria associated with periodontal disease could affect the immune response to other bacterial antigens in the oral cavity. Streptococcus sobrinus glucosyltransferase (GTF), an enzyme involved in dental caries pathogenesis, was used as a test antigen. Rowett rats were injected with aluminum hydroxide (alum) with buffer, alum-GTF, or alum-GTF together with either Escherichia coli DNA, Fusobacterium nucleatum DNA, or Porphyromonas gingivalis DNA. Contrary to expectation, animals receiving alum-GTF plus bacterial DNA (P. gingivalis in particular) demonstrated significantly reduced serum immunoglobulin G (IgG) antibody, salivary IgA antibody, and T-cell proliferation to GTF compared to animals immunized with alum-GTF alone. A diminished antibody response was also observed after administration of alum-GTF with the P. gingivalis DNA either together or separately, indicating that physical complexing of antigen and DNA was not responsible for the reduction in antibody. Since TLR triggering by DNA induces synthesis of prospective suppressive factors (e.g., suppressor of cytokine signaling [SOCS]), the effects of P. gingivalis DNA and GTF exposure on rat splenocyte production of SOCS family molecules and inflammatory cytokines were investigated in vitro. P. gingivalis DNA significantly up-regulated SOCS1 and SOCS5 expression and down-regulated interleukin-10 expression by cultured splenocytes. These results suggested that DNA from periodontal disease-associated bacteria did not enhance, but in fact suppressed, the immune response to a protein antigen from cariogenic streptococci, potentially through suppressive SOCS components triggered by innate mechanisms.

Immunogenic and Protective Potential of Mutans Streptococcal Glucosyltransferase Peptide Constructs Selected by Major Histocompatibility Complex Class II Allele Binding

Mutans streptococcal glucosyltransferases (GTF) have been demonstrated to be effective components of dental caries vaccines. We had previously selected peptide subunits of GTF for vaccine development based on putative functional significance and conservation of GTF primary structure among enzyme isoforms. In this study, 20 20-mer linear GTF peptides were synthesized, 17 identified on the basis of the highest potential major histocompatibility complex (MHC) class II-binding activity using computer-generated algorithms (Epimatrix and ProPred) and 3 with previously demonstrated functional significance. The immunoreactivities of these peptides were explored with rodent systems. Sera from GTF-immunized rats, assessed for binding to linear peptides by enzyme-linked immunosorbent assay, demonstrated immunoglobulin G antibody reactivity with peptides 6 and 11 and a T-cell proliferation response to peptides 6, 9, 11, and 16. Multiple antigenic peptide (MAP) constructs were synthesized from promising linear sequences. Rats that were immunized with MAP 7, 11, or 16, respectively, responded well to the immunizing MAP. Most importantly, a robust immune response (antibody and T-cell proliferation) was observed to native GTF following MAP 11 (amino acids 847 to 866; VVINNDKFVSWGITDFEM) immunization. This response inhibited GTF enzyme function. Two dental caries pathogenesis experiments were performed wherein rats were immunized with MAP constructs 11, 16, and/or 11 plus 16, followed by infection with cariogenic Streptococcus sobrinus. In both experiments cariogenic bacterial recoveries were reduced relative to total streptococci in the MAP 11- and MAP 11 plus 16-immunized groups, and the extent of dental caries was also significantly reduced in these groups. Thus, we have identified a peptide with projected avid MHC-binding activity that elicited immunoreactivity with native GTF and demonstrated protection against dental caries infection after immunization, implying that this peptide may be important in a subunit dental caries vaccine.

Glucosyltransferases of Viridans Group Streptococci Modulate Interleukin-6 and Adhesion Molecule Expression in Endothelial Cells and Augment Monocytic Cell Adherence

Recruitment of monocytes plays important roles during vegetation formation and endocardial inflammation in the pathogenesis of infective endocarditis (IE). Bacterial antigens or modulins can activate endothelial cells through the expression of cytokines or adhesion molecules and modulate the recruitment of leukocytes. We hypothesized that glucosyltransferases (GTFs), modulins of viridans group streptococci, may act directly to up-regulate the expression of adhesion molecules and also interleukin-6 (IL-6) to augment monocyte attachment to endothelial cells. Using primary cultured human umbilical vein endothelial cells (HUVECs) as an in vitro model, we demonstrated that GTFs (in the cell-bound or free form) could specifically modulate the expression of IL-6, and also adhesion molecules, in a dose- and time-dependent manner. Results of inhibition assays suggested that enhanced expression of adhesion molecules was dependent on the activation of nuclear factor kappaB (NF-kappaB) and extracellular signal-regulated kinase and that p38 mitogen-activated protein kinase pathways also contributed to the release of IL-6. Streptococcus-infected HUVECs or treatment with purified IL-6 plus soluble IL-6 receptor alpha enhanced the expression of ICAM-1 and the adherence of the monocytic cell line U937. These results suggest that streptococcal GTFs might play an important role in recruiting monocytic cells during inflammation in IE through induction of adhesion molecules and IL-6, a cytokine involved in transition from neutrophil to monocyte recruitment.

Immunological and Protective Effects of Diepitopic Subunit Dental Caries Vaccines

As a prelude to development of broader-spectrum vaccines for dental caries, we explored the immune potential of constructs combining epitopes from mutans streptococcal glucosyltransferases (GTF) and glucan binding protein B (GbpB). Two diepitopic peptide constructs were synthesized in a multiple antigenic peptide (MAP) format. Both constructs contained SYI, a 20-mer GbpB peptide that included a sequence having major histocompatibility complex class II binding characteristics. One diepitopic construct (SYI-CAT) also contained a 22-mer sequence from the catalytic domain of GTF. Another diepitopic construct (SYI-GLU) contained a 22-mer sequence from the glucan binding domain of GTF. To assess the ability of each construct to induce antibody reactive with GbpB and GTF native proteins, rats were injected subcutaneously with SYI-CAT, SYI-GLU, or the constituent monoepitopic constructs. Only the SYI-CAT construct induced significant levels of serum immunoglobulin G (IgG) and IgA antibody to both pathogenesis-associated proteins. Also, immunization with SYI-CAT significantly (P < 0.001) enhanced the antibody response to the CAT peptide. Experiments then compared experimental dental caries after immunization with SYI-CAT, SYI, or CAT MAP constructs, followed by infection with Streptococcus mutans strain SJr. Dental caries were lower in each peptide-immunized group than in the sham-injected group. The level of protection after SYI-CAT immunization was similar to that after immunization with constituent MAP constructs. In another experiment, rats were infected with Streptococcus sobrinus strain 6715 under an identical protocol. Significant protection was observed on buccal surfaces in both SYI-CAT and CAT construct-immunized, but not in the SYI construct-immunized, groups. Thus, addition of the GbpB-derived SYI peptide to the GTF-derived CAT peptide construct not only enhanced the immunological response to CAT and GTF epitopes, but also extended the protective effect of the construct to include both S. mutans and S. sobrinus.

Conserved Repeat Motifs and Glucan Binding by Glucansucrases of Oral Streptococci and Leuconostoc mesenteroides

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.

Synergistic Effects of Streptococcal Glucosyltransferases on Adhesive Biofilm Formation

Glucosyltransferases (GTF)-I and GTF-SI of Streptococcus mutans synthesize water-insoluble and both water-soluble and -insoluble glucans, respectively, and play essential roles in the sucrose-dependent adhesion of the organism to tooth surfaces. To examine the interactions of different GTFs on artificial biofilm formed by S. mutans and other oral streptococci, we generated GTF-I- and GTF-SI-hyperproducing isogenic mutant strains. Transformant B42-21, which hyperexpressed GTF-SI, exhibited firm adhesion in the presence of sucrose, whereas transformant B42-10, which hyperexpressed GTF-I, failed to exhibit firm adhesion. Furthermore, co-culture of transformant B42-21 with water-soluble glucan-synthesizing Streptococcus sanguinis yielded firm adhesion, while the addition of dextran T10 to B42-21 growing culture had no effect on adhesion. These findings suggest that GTF-SI has a strong effect on sucrose-dependent adhesion and is essential for biofilm formation on smooth surfaces, in cooperation with water-soluble glucans synthesized de novo by oral streptococci that inherently lack cell adhesion ability.

Effects of Magnolol and Honokiol on the activities of streptococcal glucosyltransferases both in solution and adsorbed on an experimental pellicle.

To investigate the inhibitory effects of Magnolol and Honokiol on the activity of streptococcal glucosyltransferases (Gtfs). The effect of Magnolol and Honokiol that inhibits the activities of streptococcal GtfB, GtfC, GtfD and GtfS was explored with standard assays. The results showed that both samples can efficiently inhibit the activity of all Gtfs in solution (66.4-96.3%) and adsorbed on the surface of saliva-coated hydroxyapatite (sHA) beads (65.5-92.7%) at concentrations between 1.25 and 5.0 mg ml(-1). Furthermore, Magnolol had a stronger inhibition of four kinds of Gtfs than Honokiol both in solution and adsorbed on the surface of sHA beads at concentrations between 0.04 and 0.63 mg ml(-1) (P < 0.05). Magnolol had significant effects on the activities of streptococcal Gtfs. Magnolol as a natural herb can be developed into a new oral hygiene product to prevent plaque formation.

The influence of mutanase and dextranase on the production and structure of glucans synthesized by streptococcal glucosyltransferases

Glucanohydrolases, especially mutanase [α-(1 → 3) glucanase; EC 3.2.1.59] and dextranase [α-(1 → 6) glucanase; EC 3.2.1.11], which are present in the biofilm known as dental plaque, may affect the synthesis and structure of glucans formed by glucosyltransferases (GTFs) from sucrose within dental plaque. We examined the production and the structure of glucans synthesized by GTFs B (synthesis of α-(1 → 3)-linked glucans) or C [synthesis of α-(1 → 6)- and α-(1 → 3)-linked glucans] in the presence of mutanase and dextranase, alone or in combination, in solution phase and on saliva-coated hydroxyapatite beads (surface phase). The ability of Streptococcus sobrinus 6715 to adhere to the glucan, which was formed in the presence of the glucanohydrolases was also explored. The presence of mutanase and/or dextranase during the synthesis of glucans by GTF B and C altered the proportions of soluble to insoluble glucan. The presence of either dextranase or mutanase alone had a modest effect on total amount of glucan formed, especially in the surface phase; the glucanohydrolases in combination reduced the total amount of glucan. The amount of (1 → 6)-linked glucan was reduced in presence of dextranase. In contrast, mutanase enhanced the formation of soluble glucan, and reduced the percentage of 3-linked glucose of GTF B and C glucans whereas dextranase was mostly without effect. Glucan formed in the presence of dextranase provided fewer binding sites for S. sobrinus; mutanase was devoid of any effect. We also noted that the GTFs bind to dextranase and mutanase. Glucanohydrolases, even in the presence of GTFs, influence glucan synthesis, linkage remodeling, and branching, which may have an impact on the formation, maturation, physical properties, and bacterial binding sites of the polysaccharide matrix in dental plaque. Our data have relevance for the formation of polysaccharide matrix of other biofilms.

Remote glucosyltransferase-microparticle vaccine delivery induces protective immunity in the oral cavity.

Intranasally administered dental caries vaccines show significant promise for human application. Alternate mucosal routes may be required, however, to induce caries-protective salivary IgA antibody in children with respiratory diseases. Since rectal mucosa contains inductive lymphoid tissue, we hypothesized that the rectal route could be used to induce salivary immunity to mutans streptococcal glucosyltransferase (GTF), resulting in protective immunity to experimental dental caries. We first explored the ability of glucosyltransferase, incorporated into polylactide-co-glycolide (PLGA) microparticles (MP), and administered rectally together with mucosal adjuvant, to induce a salivary IgA antibody response. Groups of Sprague-Dawley rats (6/group) were immunized rectally on days 0, 7, 14 and 21 with a) GTF-MP alone, b) GTF-MP with cholera toxin, c) GTF-MP with detoxified mutant Escherichia coli toxin (dLT), or d) sham immunized with PLGA and cholera toxin. An additional group was immunized intranasally with GTF-MP alone. Saliva and nasal washes of all intranasally immunized rats contained IgA antibody to glucosyltransferase on day 28. Salivary IgA antibody was also detected in 7/12 rats rectally immunized with GTF-MP and cholera toxin or dLT, although responses were lower than those obtained by intranasal immunization. Most fecal extracts from rectally delivered GTF-MP plus cholera toxin or dLT rats contained IgA antibody to GTF-MP. Low levels of fecal IgA antibody were detected in 3/6 intranasally immunized rats and 2/6 rats rectally immunized with GTF-MP alone. We then examined the extent to which salivary IgA antibody induced by the rectal route could be protective. At 25, 31 and 38 days of age, two groups of female Sprague-Dawley rats (13/group) were rectally immunized with GTF-MP and cholera toxin or with empty microparticles and cholera toxin (sham group). A third group was intranasally immunized with GTF-MP alone. After demonstrating salivary IgA responses to GTF in most GTF-immunized rats, all animals were infected with streptomycin-resistant Streptococcus sobrinus and placed on diet 2000. After 79 days of infection, total caries on molar surfaces were lower in both rectally (7.9 +/- 1.0) and intranasally (7.1 +/- 0.9; P < 0.0.03) immunized groups compared with the sham-immunized group (11.9 +/- 1.6). Smooth surface caries were significantly lower (P < 0.05) in both rectally and intranasally immunized groups. These results support the interconnectedness of the mucosal immune system and indicate that rectal immunization with GTF-MP, together with adjuvant, or intranasal immunization with GTF-MP alone, can induce protective levels of salivary antibody in rats.

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.