Purification of saliva agglutinin of<Emphasis Type="Italic"> Streptococcus intermedius</Emphasis> and its association with bacterial aggregation and adherence

Streptococcus intermedius strain 1208-1 cells were aggregated in the presence of saliva. The saliva agglutinin was purified by centrifugation, filtration, and gel filtration. SDS-PAGE analyses indicated that the purified agglutinin consisted of two high-molecular-mass proteins. Aggregation was dependent on calcium over pH 5.5, with 1 mM being the most effective concentration. Boiling inactivated purified agglutinin. S. intermedius strain 3 and Streptococcus mutans strain 1 were aggregated in the purified agglutinin. After adsorption with strain 1208-1 cells, the saliva sample did not exhibit any aggregation activity, and the agglutinin bands were no longer visible by SDS-PAGE. Adherence analyses demonstrated that the purified agglutinin immobilized on the surfaces of polystyrene wells, actinomyces cells, and apatite beads accounted for the binding of streptococcus cells. Agglutinin also effectively inhibited adherence to apatite beads coated with native saliva.     

Effective Immunity to Dental Caries: Protection of Gnotobiotic Rats by Local,Immunization with a Ribosomal Preparation from Streptococcus mutans

A ribosomal preparation from Streptococcus mutans 6715 was characterized for its ability to induce an immune response in gnotobiotic rats which was protective against S. mutans-induced dental caries. Animals injected in the salivary gland region with the S. mutans ribosomal vaccine developed significantly higher (P < 0.01) salivary IgA and serum IgG antibody activities against whole S. mutans cells and ribosomal preparations than nonimmunized rats. Vaccinated animals had significantly lower (67%; P < 0.01) levels of S. mutans adherent to their molar surfaces than the control rats after infection with the homologous, cariogenic S. mutans. The immunized animals had significantly fewer (P < 0.01) carious lesions on their buccal, sulcal, and proximal molar surfaces than the nonimmunized rats following challenge with the virulent organism. Animals injected with the ribosomal preparation developed salivary IgA and serum IgG antibodies with specificities to various cell surface-associated antigens such as lipoteichoic acid and glucosyltransferase, suggesting that the observed protection may be due to antibodies against cell surface contaminants of the ribosomal vaccine. These results are the first demonstration that a ribosomal preparation from S. mutans protected rats from caries formation after challenge with the homologous, virulent S. mutans.     

Cloning and characterization of an alpha-enolase of the oral pathogen Streptococcus mutans that binds human plasminogen

Streptococcus mutans is the etiologic agent of dental caries and is a causative agent of infective endocarditis. While the mechanisms by which S. mutans cells colonize heart tissue is not clear, it is thought that bacterial binding to extracellular matrix and blood conponents is crucial in the development of endocarditis. Previously, we have demonstrated that S. mutans cells have the capacity to bind and activate plasminogen to plasmin. Here we report the first cloning and characterization of an α-enolase of S. mutans that binds plasminogen. The functional identity of the purified recombinant α-enolase protein was confirmed by its ability to catalyze the conversion of 2-phosphoglycerate to phosphoenolpyruvate. The protein exhibited a K_m of 9.5 mM and a V_max of 31.0 mM/min/mg. The α-enolase protein was localized in the cytoplasmic, cell wall and extracellular fractions of S. mutans. Binding studies using an immunoblot analysis revealed that human plasminogen binds to the enolase enzyme of S. mutans. These findings identify S. mutans α-enolase as a binding molecule used by this oral pathogen to interact with the blood component, plasminogen. Further studies of this interaction may be critical to understand the pathogenesis of endocarditis caused by S. mutans.     

Dental Caries Induction in Experimental Animals by Clinical Strains of Streptococcus mutans Isolated from Japanese Children

Oral implantation and the cariogenic activity of clinical strains of Streptococcus mutans which had been isolated from Japanese children and labeled with streptomycin-resistance were examined in specific pathogen-free Sprague-Dawley rats. All the seven strains tested were easily implanted and persisted during the experimental period. Extensive carious lesions were produced in rats inoculated with clinical strains of S. mutans belonging to serotypes c, d, e, and f, and maintained on caries-inducing diet #2000. Noninfected rats did not develop dental caries when fed diet #2000. Type d S. mutans preferentially induced smooth surface caries in the rats. Strains of other serotypes primarily developed caries of pit and fissure origin. Caries also developed in rats inoculated with reference S. mutans strains BH-TR and FAIR (type b) that had been maintained in the laboratories for many years. However, the cariogenicity of the laboratory strains was found to have decreased markedly. All three S. sanguis strains could be implanted, but only one strain induced definite fissure caries. Two S. salivarius strains could not be implanted well in the rats and therefore they were not cariogenic. Four different species of lactobacilli also failed to induce dental caries in rats subjected to similar caries test regimen on diet #2000. S. mutans strain MT6R (type c) also induce caries in golden hamsters and ICR mice, but of variable degrees.     

Putative down-stream signaling molecule of GTPase in Porphyromonas gingivalis

Porphyromonas gingivalis is a strict anaerobic bacterium mainly responsible for periodontal disease in oral cavity. Putative GTPase gene (pgp) of this bacterium was cloned and its recombinant protein (rPGP) was produced in Escherichia coli. Based on the amino acid sequence of SGP that is a GTP-binding protein of Streptococcus mutans, putative GTPase amino acid sequence was deduced in the data base of genome sequences of Porphyromonas gingivalis. A 900-bp PCR fragment was amplified with P. gingivalis genomic DNA as a template and cloned into E. coli JM109. Then pgp was transferred into pQE-30 expression vector to make pQE-PGP for production of rPGP. This protein was produced and purified by Ni-NTA affinity column chromatography. Anti-PGP antibody was also produced in Sprague Dawley rats. Using Westernblot analysis with this antibody, it was confirmed that the rPGP produced in E. coli was identical to that of donor strain. Furthermore, by Southernblot analysis it was revealed that the pgp was originated from P. gingivalis. By immunoprecipitation with anti-PGP antibody and N-terminal amino acid sequence analysis it was found that PGP was able to bind to acetate kinase, which was reported to be a secondary signaling molecule in anaerobic microorganisms. Therefore, these results imply that P. gingivalis produces putative GTPase and this protein might play a potential role in signaling pathway in oral biofilm formation.     

Distribution of Streptococcus troglodytae and Streptococcus dentirousetti in chimpanzee oral cavities

The aim of this study was to analyze the distribution and phenotypic properties of the indigenous streptococci in chimpanzee (Pan troglodytes) oral cavities. Eleven chimpanzees (aged from 9 to 44 years, mean ± SD, 26.9 ± 12.6 years) in the Primate Research Institute of Kyoto University were enrolled in this research and brushing bacterial samples collected from them. Streptococci were isolated from the oral cavities of all chimpanzees. The isolates (n = 46) were identified as thirteen species by 16S rRNA genes analysis. The predominant species was Streptococcus sanguinis of mitis streptococci from five chimpanzees (45%). Mutans streptococci were isolated from six chimpanzees (55%). The predominant species in the mutans streptococci were Streptococcus troglodytae from four chimpanzees (36%), this species having been proposed as a novel species by us, and Streptococcus dentirousetti from three chimpanzees (27%). Streptococcus mutans was isolated from one chimpanzee (9%). However, Streptococcus sobrinus, Streptococcus macacae and Streptococcus downei, which are indigenous to human and monkey (Macaca fasciclaris) oral habitats, were not isolated. Of the mutans streptococci, S. troglodytae, S. dentirousetti, and S. mutans possessed strong adherence activity to glass surface.     

Effects of Panose on Glucan Synthesis and Cellular Adherence by Streptococcus mutans

The effects of panose on glucan synthesis and sucrose-dependent cellular adherence by Streptococcus mutans were investigated. Panose effectively inhibited glucan synthesis from sucrose by glucosyltransferases from S. mutans strain 6715, but increasing amounts of panose increased the release of fructose from sucrose by the enzymes. On the other hand, production of a series of oligosaccharides of increasing size by the enzymes was markedly enhanced in the presence of panose. These results indicate that panose activates the enzymes and that the inhibition of glucan synthesis by panose is due to the transfer of the glucosyl group of sucrose to panose. Sucrose-dependent adherence of cells of various S. mutans strains to a glass surface was also inhibited by panose.     

Mutacin H-29B is identical to mutacin II (J-T8)

Background  

Streptococcus mutans produces bacteriocins named mutacins. Studies of mutacins have always been hampered by the difficulties in obtaining active liquid preparations of these substances. Some of them were found to be lantibiotics, defined as bacterial ribosomally synthesised lanthionine-containing peptides with antimicrobial activity. The goal of this study was to produce and characterize a new mutacin from S. mutans strain 29B, as it shows a promising activity spectrum against current human pathogens.     

Therapeutic effect of llama derived VHH fragments against Streptococcus mutans on the development of dental caries

Streptococcus mutans is the main cause of dental caries. We evaluated the therapeutic effect of variable regions of a llama heavy chain antibody fragments directed against S. mutans named S36-VHH (S for Streptococcus) alone or fused with glucose oxidase (GOx) from Aspergillus niger. Western blot analysis and ELISA revealed binding of the S36-VHH to the streptococcal antigen I/II adhesin molecule of S. mutans serotype C. In a rat-desalivated caries model, daily administration of S36-VHH significantly reduced the development of smooth surface caries. No additional therapeutic effect of GOx was observed. Our results suggest that llama VHH antibodies may be a potential benefit as prophylaxis against dental caries.     

Purification of dextran-binding protein from cariogenic Streptococcus mutans

An extracellular protein produced by $\text{Streptococcus mutans}$ was purified to electrophoretic homogeneity by affinity chromatography on Sephadex G50 followed by gel filtration. The protein is devoid of both dextransucrase and dextranase activity but binds dextran and therefore probably is implicated in the adherence of $\text{S. mutans}$ cells to the host tooth surface. The presence of the dextran-binding protein may be a determinant of the pathogenicity of such cariogenic micro-organisms.     

Investigating Acid Production by Streptococcus mutans with a Surface-Displayed pH-Sensitive Green Fluorescent Protein

Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins) to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.     

Characterization of monoclonal antibodies raised toStreptococcus mutans

Monoclonal antibodies (MAb) to whole cells ofStreptococcus mutants P-4 (serotype e) were generated. Of the four MAb used in this investigation, three were found to cross-react with mouse heart tissue (MAb-HT) in the solid-phase ELISA, while the remaining MAb was found to be specific forS. mutants only (MAb-SM). Serotypic characterization has shown that MAb-SM as well as MAb-HT recognize strains ofS. mutans representing all of the eight currently known serological groups. In addition, MAb-HT showed reactivity withStreptococcus pyogenes, a Lancefield group-A streptococcus frequently implicated in rheumatic fever. Western blot analysis has shown that MAb-SM recognizes a 14,000-dalton component present in a partially purified membrane fraction recovered following glass-bead breakage ofS. mutans P-4 cells. Two of the MAb-HT were found to react with a 35,000-dalton component from this same preparation.     

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.     

Interaction of Salivary alpha-Amylase and Amylase-Binding-Protein A (AbpA) of <Emphasis Type="Italic">Streptococcus gordonii</Emphasis> with Glucosyltransferase of <Emphasis Type="Italic">S. gordonii</Emphasis> and <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Background  

Glucosyltransferases (Gtfs), enzymes that produce extracellular glucans from dietary sucrose, contribute to dental plaque formation byStreptococcus gordoniiandStreptococcus mutans. The alpha-amylase-binding protein A (AbpA) ofS. gordonii, an early colonizing bacterium in dental plaque, interacts with salivary amylase and may influence dental plaque formation by this organism. We examined the interaction of amylase and recombinant AbpA (rAbpA), together with Gtfs ofS. gordoniiandS. mutans.     

A Highly Arginolytic Streptococcus Species That Potently Antagonizes Streptococcus mutans

The ability of certain oral biofilm bacteria to moderate pH through arginine metabolism by the arginine deiminase system (ADS) is a deterrent to the development of dental caries. Here, we characterize a novel Streptococcus strain, designated strain A12, isolated from supragingival dental plaque of a caries-free individual. A12 not only expressed the ADS pathway at high levels under a variety of conditions but also effectively inhibited growth and two intercellular signaling pathways of the dental caries pathogen Streptococcus mutans. A12 produced copious amounts of H₂O₂ via the pyruvate oxidase enzyme that were sufficient to arrest the growth of S. mutans. A12 also produced a protease similar to challisin (Sgc) of Streptococcus gordonii that was able to block the competence-stimulating peptide (CSP)–ComDE signaling system, which is essential for bacteriocin production by S. mutans. Wild-type A12, but not an sgc mutant derivative, could protect the sensitive indicator strain Streptococcus sanguinis SK150 from killing by the bacteriocins of S. mutans. A12, but not S. gordonii, could also block the XIP (comX-inducing peptide) signaling pathway, which is the proximal regulator of genetic competence in S. mutans, but Sgc was not required for this activity. The complete genome sequence of A12 was determined, and phylogenomic analyses compared A12 to streptococcal reference genomes. A12 was most similar to Streptococcus australis and Streptococcus parasanguinis but sufficiently different that it may represent a new species. A12-like organisms may play crucial roles in the promotion of stable, health-associated oral biofilm communities by moderating plaque pH and interfering with the growth and virulence of caries pathogens.     

Cloning and Nucleotide Sequencing of l-Lactate Dehydrogenase Gene from Streptococcus thermophilus M-192

The gene encoding l-lactate dehydrogenase (LDH) was cloned from an industrial dairy strain of Streptococcus thermophilus M-192 using a synthetic oligonucleotide probe based on the N-terminal amino acid sequence of the purified enzyme, and its nucleotide sequence was determined. The enzyme was deduced to have 328 amino acid residues with a molecular weight of 35,428 and found to have high sequence similarity to LDHs from other lactic acid bacteria (89.0% to Streptococcus mutans, 76.3% to Lactococcus lactis subsp. lactis, 67% to Lactobacillus casei, and 60% to Lactobacillus plantarum). The gene contained a promoter-like sequence similar to the Escherichia coli promoter consensus, and expression of the S. thermophilus LDH gene was observed in E. coli cells.     

Inhibition of Rat Dental Caries by Dextranase from a Strain of Spicaria violacea

Dextranase AD17 obtained from a culture liquor of a strain of Spicaria violacea was assessed for its ability to inhibit the development of dental caries in conventional Sprague-Dawley rats which had been infected with one of the Streptococcus mutans strains, MT6R (serotype c), OMZ 176R (d), or MT-703R (e). These experiments showed that caries was significantly inhibited when rats were given cariogenic diet # 2000 and drinking water containing AD17 at a concentration of 10 units/g, as compared to control rats not given dextranase. The inhibitory effects of AD17 were more prominent in smooth surface caries than in total caries. AD17 had a tendency to retard both the establishment of inoculated S. mutans and plaque deposition on tooth surfaces. However, S. mutans could be implanted in the rat oral cavity after repeated inoculation of the bacteria, even in the presence of AD17. These results suggest that the anticaries activity of AD17 is due to not only inhibition of adherence of S. mutans cells on tooth surfaces but also to physicochemical changes of dental plaque formed under the enzymatic action of AD17. Preliminary histopathological examination showed that AD17 had no significant toxicity in rats.     

Antibacterial activity of disodium succinoyl glycyrrhetinate,a derivative of glycyrrhetinic acid against Streptococcus mutans

Streptococcus mutans is a cariogenic bacterium that localizes in the oral cavity. Glycyrrhetinic acid (GRA) is a major component of licorice extract. GRA and several derivatives, including disodium succinoyl glycyrrhetinate (GR‐SU), are known to have anti‐inflammatory effects in humans. In this study, the antimicrobial effect of GRA and its derivatives against the S. mutans UA159 strain were investigated. Minimum inhibitory concentrations (MICs) of GRA and GR‐SU showed antibacterial activity against the S. mutans strain, whereas other tested derivatives did not. Because GR‐SU is more soluble than GRA, GR‐SU was used for further experiments. The antibacterial activity of GR‐SU against 100 S. mutans strains was evaluated and it was found that all strains are susceptible to GR‐SU, with MIC values below 256 µg/mL. A cell viability assay showed that GR‐SU has a bacteriostatic effect on S. mutans cells. As to growth kinetics, sub‐MICs of GR‐SU inhibited growth. The effect of GR‐SU on S. mutans virulence was then investigated. GR‐SU at sub‐MICs suppresses biofilm formation. Additionally, GR‐SU greatly suppresses the pH drop caused by the addition of glucose and glucose‐induced expression of the genes responsible for acid production (ldh and pykF) and tolerance (aguD and atpD). Additionally, expression of enolase, which is responsible for the carbohydrate phosphotransferase system, was not increased in the presence of GR‐SU, indicating that GR‐SU suppresses incorporation of sugars into S. mutans. In conclusion, GR‐SU has antibacterial activity against S. mutans and also decreases S. mutans virulence.     

Purification and Properties of a Lytic Enzyme from Streptomyces griseus H-402

A lytic enzyme which was capable of lysing cells of Streptococcus mutans was purified from the culture filtrate of Streplomyces griseus H–402 by Amberlite CG–50 treatment, CM-cellulose and hydroxylapatite column chromatographies, and Sephadex G–150 gelfiltration. The lytic enzyme was obtained in a crystalline form which was homogeneous in polyacrylamide gel electrophoresis. The molecular weight was estimated to be 2×10⁴ by the thin-layer gel-filtration method on Sephadex G–75, and 2.3 × 10⁴ by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was found to be a N-acetylmuramidase whose activity was lost by N-bromosuccinimide as an inhibitor.     

Caries in the infundibulum of the second upper premolar tooth in the horse

Background  

Swedish equine dental practices have empirically found that the prevalence of infundibular caries as a primary disorder in the first permanent premolar teeth (P2) of the horse upper jaw has increased during the last 10 years. A previously unknown bacterial species, Streptococcus devriesei (CCUG 47155^T), which is related to Streptococcus mutans, has recently been isolated from these carious lesions. To understand the aetiology of caries in horses, it is essential to elucidate the relationship between S. devriesei and P2 infundibular caries.