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.     

Studies on collagen-tannic acid-collagenase ternary system: Inhibition of collagenase against collagenolytic degradation of extracellular matrix component of collagen

We report the detailed studies on the inhibitory effect of tannic acid (TA) on Clostridium histolyticum collagenase (ChC) activity against degradation of extracellular matrix component of collagen. The TA treated collagen exhibited 64% resistance against collagenolytic hydrolysis by ChC, whereas direct interaction of TA with ChC exhibited 99% inhibition against degradation of collagen and the inhibition was found to be concentration dependant. The kinetic inhibition of ChC has been deduced from the extent of hydrolysis of N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA). This data provides a selective competitive mode of inhibition on ChC activity seems to be influenced strongly by the nature and structure of TA. TA showed inhibitor activity against the ChC by molecular docking method. This result demonstrated that TA containing digalloyl radical possess the ability to inhibit the ChC. The inhibition of ChC in gaining new insight into the mechanism of stabilization of collagen by TA is discussed.     

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis

Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.     

Anti‐biofilm and bactericidal effects of magnolia bark‐derived magnolol and honokiol on Streptococcus mutans

Dental caries affects people of all ages and is a worldwide health concern. Streptococcus mutans is a major cariogenic bacterium because of its ability to form biofilm and induce an acidic environment. In this study, the antibacterial activities of magnolol and honokiol, the main constituents of the bark of magnolia plants, toward planktonic cell and biofilm of S. mutans were examined and compared with those of chlorhexidine. The minimal inhibitory concentrations of magnolol, honokiol and chlorhexidine for S. mutans were 10, 10 and 0.25 µg/mL, respectively. In addition, each agent showed bactericidal activity against S. mutans planktonic cells and inhibited biofilm formation in a dose‐ and time‐dependent manner. Magnolol (50 µg/mL) had greater bactericidal activity against S. mutans biofilm than honokiol (50 µg/mL) and chlorhexidine (500 µg/mL) at 5 min after exposure, while all showed scant activity against biofilm at 30 s. Furthermore; chlorhexidine (0.5–500 µg/mL) exhibited high cellular toxicity for the gingival epithelial cell line Ca9‐22 at 1 hr, whereas magnolol (50 µg/mL) and honokiol (50 µg/mL) did not. Thus; it was found that magnolol has antimicrobial activities against planktonic and biofilm cells of S. mutans. Magnolol may be a candidate for prevention and management of dental caries.     

Enhancement of fluoride activity against Streptococcus mutans biofilms by a substance separated from Polygonum cuspidatum

Polygonum cuspidatum is a plant with spreading rhizomes and numerous reddish-brown stems that has been used in Korean folk medicine to improve oral hygiene. Nevertheless, there are no reports related to its possible effect on the virulence of dental biofilms. In this study, the ability of a fraction (F1) separated from P. cuspidatum, alone or in combination with fluoride, to disrupt virulence factors and the composition of Streptococcus mutans biofilms was examined. F1 was mainly composed of resveratrol, emodin and physcion (approximately 16.2%, 18.9% and 2.07% of the weight of F1, respectively). F1 showed inhibitory effects on acid production and F-ATPase activity of S. mutans in biofilms, and could enhance fluoride activity against acid production and acid tolerance of S. mutans in biofilms. When S. mutans biofilms were briefly treated with F1 (10 min, a total of five times), the biomass accumulation, water-insoluble polysaccharides and intracellular iodophilic polysaccharides were reduced. Furthermore, the fluoride activity against biomass accumulation was enhanced by F1. These results suggest that F1 may be useful in the control of dental biofilms and in improving the cariostatic properties of fluoride without increasing its exposure.     

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.     

Damage of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> biofilms by carolacton,a secondary metabolite from the myxobacterium <Emphasis Type="Italic">Sorangium cellulosum</Emphasis>

Background  

Streptococcus mutans is a major pathogen in human dental caries. One of its important virulence properties is the ability to form biofilms (dental plaque) on tooth surfaces. Eradication of such biofilms is extremely difficult. We therefore screened a library of secondary metabolites from myxobacteria for their ability to damage biofilms of S. mutans.     

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.     

The anti-biofouling effect of Lactobacillus fermentum-derived biosurfactant against Streptococcus mutans

Biofouling in the oral cavity often causes serious problems. The ability of Streptococcus mutans to synthesize extracellular glucans from sucrose using glucosyltransferases (gtfs) is vital for the initiation and progression of dental caries. Recently, it was demonstrated that some biological compounds, such as secondary metabolites of probiotic bacteria, have an anti-biofouling effect. In this study, S. mutans was investigated for the anti-biofouling effect of Lactobacillus fermentum (L.f.)-derived biosurfactant. It was hypothesized that two enzymes produced by S. mutans, glucosyltransferases B and C, would be inhibited by the L.f.-biosurfactant. When these two enzymes were inhibited, fewer biofilms (or none) were formed. RNA was extracted from a 48-h biofilm of S. mutans formed in the presence or absence of L.f. biosurfactant, and the gene expression level of gtfB/C was quantified using the real-time polymerase chain reaction (RT-PCR). L.f. biosurfactant showed substantial anti-biofouling activity because it reduced the process of attachment and biofilm production and also showed a reduction in gtfB/C gene expression (P value < 0.05).     

Antibacterial activity of phellodendron bark against Streptococcus mutans

Streptococcus mutans is a major cause of tooth decay due to its promotion of biofilm formation and acid production. Several plant extracts have been reported to have multiple biological activities such as anti-inflammation and antibacterial effects. This study investigated the antibacterial activity of three plant extracts, phellodendron bark (PB), yucca, and black ginger, and found that PB had a stronger effect than the other extracts. Then, the minimum inhibitory concentration (MIC) of PB against 100 S. mutans strains was investigated. The MIC range of PB was 9.8–312.5 µg/mL. PB suppressed the growth kinetics of S. mutans in a dose-dependent manner, even at sub-MICs of PB. Then, we investigated the effect of PB on S. mutans virulence. The PB suppressed biofilm formation at high concentrations, although PB did not affect the expression of glucosyltransferase genes. Additionally, PB suppressed the decrease in pH from adding an excess of glucose. The expression of genes responsible for acid production was increased by the addition of excess glucose without PB, whereas their expression levels were not increased in the presence of 1× and 2× MIC of PB. Although PB showed a bacteriostatic effect on planktonic S. mutans cells, it was found that more than 2× MIC of PB showed a partial bactericidal effect on biofilm cells. In conclusion, PB not only showed antibacterial activity against S. mutans but also decreased the cariogenic activity in S. mutans.     

Inhibition of collagenase by Cr(III): its relevance to stabilization of collagen

Bacterial collagenase has now been reacted with a select series of Cr(III) complexes and modifications in the activity of chromium-modified collagenase has been deduced from the extent of hydrolysis of (2-furanacryloyl-L-leucyl-glycyl-L-prolyl-L-alanine), FALGPA. A homologous series of Cr(III) complexes with dimeric, trimeric and tetrameric structures as in 1, 2 and 3 respectively has been investigated for their ability to inhibit the action of collagenase against FALGPA. Whereas competitive and non-competitive modes of inhibition of collagenase are expressed by 1, (dimer) and 2, (trimer) respectively, the tetramer, 3, exhibits poor affinity to collagenase and the inhibition of the enzyme activity is uncompetitive. Evidence for different modes of inhibition of collagenase depending on the nature of Cr(III) species has been presented in this work. Circular dichroism and gel electrophoresis data on Cr(III) modified collagenase corroborate the hypothesis that the inhibition of collagenase by the heavy metal ion arises from secondary and quaternary structural changes in the enzyme. The implications of the observed Cr(III) species specific inhibition of collagenase in gaining new insight into the mechanism of stabilization of collagen by Cr(III) are discussed.     

Antimicrobial Activity of Propolis on Oral Microorganisms

Formation of dental caries is caused by the colonization and accumulation of oral microorganisms and extracellular polysaccharides that are synthesized from sucrose by glucosyltransferase of Streptococcus mutans. The production of glucosyltransferase from oral microorganisms was attempted, and it was found that Streptococcus mutans produced highest activity of the enzyme. Ethanolic extracts of propolis (EEP) were examined whether EEP inhibit the enzyme activity and growth of the bacteria or not. All EEP from various regions in Brazil inhibited both glucosyltransferase activity and growth of S. mutans, but one of the propolis from Rio Grande do Sul (RS2) demonstrated the highest inhibition of the enzyme activity and growth of the bacteria. It was also found that propolis (RS2) contained the highest concentrations of pinocembrin and galangin. Received: 8 June 1997 / Accepted: 7 July 1997     

Improving the Efficiency of the Modified Hodge Test in KPC-Producing Klebsiella pneumoniae Isolates by Incorporating an EDTA Disk

Streptococcus mutans (S. mutans) is the main etiological agent of dental caries, and adheres to the tooth surface through the sortase A (SrtA)-mediated cell wall-anchored protein Pac. Inhibition of SrtA activity results in a marked reduction in the adhesion potential of S. mutans, and the frequency of dental caries. Morin is a natural plant extract that was previously reported to inhibit Staphylococcus aureus SrtA activity. Here, we demonstrate that morin has an inhibitory effect against S. mutans UA159 SrtA, with an IC₅₀ of 27.2 ± 2.6 μM. Western blotting demonstrated that 30 μM morin induced the partial release of the Pac protein into the supernatant. The biofilm mass of S. mutans was reduced in the presence of 30 μM morin, which was not caused by a decrease in S. mutans viability. These results indicate that morin might be important as a new agent to prevent caries.     

Peptide Inhibitors of Streptococcus mutans in the Control of Dental Caries

Peptides have been investigated as potential inhibitors of Streptococcus mutans, the main cause of dental caries, and have demonstrated considerable promise. In a human trial, topical application to tooth surfaces of a synthetic peptide inhibitor (p1025) of S. mutans adhesion prevented recolonisation with the oral pathogen following treatment with chlorhexidine gluconate (a broad spectrum antiseptic compound). An important feature of this treatment is that the duration of protection extends well beyond the period in which p1025 is applied. The specific targeting of S. mutans which allows other members of the oral flora associated with health to recolonise the oral cavity and competitively exclude S. mutans may explain the extended protection. Further in vitro studies have identified several other peptides which may have potential as inhibitors of S. mutans. Of particular interest are studies that demonstrate that competence stimulating peptides of S. mutans act as inhibitors of S. mutans growth and that peptides derived from the competence stimulating peptides can be used as a means of specifically targeting broad spectrum antimicrobial peptides.     

The antimicrobial effects of deglycyrrhizinated licorice root extract on Streptococcus mutans UA159 in both planktonic and biofilm cultures

The objective of the study was to investigate the antimicrobial effects of deglycyrrhizinated licorice root extracts (DG-LRE) against Streptococcus mutans UA159 in both the planktonic and biofilm phases by determining the minimum inhibitory concentration and minimum bactericidal concentration, and by performing time-kill kinetic, growth, adhesion, and biofilm assays. The cell toxicity of DG-LRE on normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. This study showed that DG-LRE had strong antimicrobial activity against S. mutans in the planktonic phase with little cytotoxic effect on NHGF cells. In addition, DG-LRE significantly inhibited biofilm formation by S. mutans UA159 at concentrations over 4 μg/ml for glucose or 16 μg/ml for sucrose, respectively, regardless of the presence of saliva-coating. To the best of our knowledge, this is the first report to provide evidence that DG-LRE demonstrates antimicrobial activity against S. mutans. These results suggest that DG-LRE can be used in developing oral hygiene products, such as gargling solution and dentifrice to prevent human dental caries.     

Studies on collagen-tannic acid-collagenase ternary system: Inhibition of collagenase against collagenolytic degradation of extracellular matrix component of collagen

We report the detailed studies on the inhibitory effect of tannic acid (TA) on Clostridium histolyticum collagenase (ChC) activity against degradation of extracellular matrix component of collagen. The TA treated collagen exhibited 64% resistance against collagenolytic hydrolysis by ChC, whereas direct interaction of TA with ChC exhibited 99% inhibition against degradation of collagen and the inhibition was found to be concentration dependant. The kinetic inhibition of ChC has been deduced from the extent of hydrolysis of N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA). This data provides a selective competitive mode of inhibition on ChC activity seems to be influenced strongly by the nature and structure of TA. TA showed inhibitor activity against the ChC by molecular docking method. This result demonstrated that TA containing digalloyl radical possess the ability to inhibit the ChC. The inhibition of ChC in gaining new insight into the mechanism of stabilization of collagen by TA is discussed.     

Anti-biofilm activity of a novel pit and fissure self-adhesive sealant modified with metallic monomers

Abstract

Dental plaque is a biofilm composed of a complex oral microbial community. The accumulation of plaque in the pit and fissures of dental elements often leads to the development of tooth decay (dental caries). Here, potent anti-biofilm materials were developed by incorporating zinc methacrylates or di-n-butyl-dimethacrylate-tin into the light-curable sealant and their physical, mechanical, and biological properties were evaluated. The data revealed that 5% di-n-butyl-dimethacrylate-tin (SnM 5%) incorporated sealant showed strong anti-biofilm efficacy against various single-species (Streptococcus mutans or Streptococcus oralis or Candida albicans) and S. mutans-C. albicans cross-kingdom dual-species biofilms without either impairing the mechanical properties of the sealant or causing cytotoxicities against mouse fibroblasts. The findings indicate that the incorporation of SnM 5% in the experimental pit and fissure self-adhesive sealant may have the potential to be part of current chemotherapeutic strategies to prevent the formation of cariogenic oral biofilms that cause dental caries.     

A Role for Lewis a antigens on salivary agglutinin in binding to Streptococcus mutans

Streptococcus mutans is a major etiological agent in dental caries. Salivary agglutinin is one of the main salivary components binding to S.mutans. To learn more about the interaction of salivary agglutinin with S.mutans, parotid, submandibular, sublingual and palatal saliva samples were incubated with S. mutans suspension. Both depleted saliva samples and bacterial extracts were analyzed by SDS-PAGE and immunoblotting. Salivary agglutinin was present in all types of glandular saliva and in all cases bound to S.mutans, also to PC337C, a P1^– mutant of S.mutans. Agglutinin was separated by SDS-PAGE under reducing and non-reducing conditions and then transferred to nitrocellulose. Non-reduced agglutinin bound S.mutans, but reduced agglutinin did not. Adhesion of S.mutans to agglutinin-coated microplates was inhibited by amine-containing components, 1 M NaCl or KCl and EDTA. Adhesion decreased with decreasing pH with no adhesion below pH 5.0. These data suggest that calcium-dependent electrostatic interactions play a role in binding. By immunoblotting was demonstrated that blood group antigens and Lewis antigens were present on agglutinin. Synthetic blood group antigens and Lewis antigens covalently coupled to polyacrylamide were tested for binding to S.mutans. Only Le^a(Gal1,3(Fuc1,4)GlcNAc) bound to S.mutans, whereas the blood group antigens Le^b, Le^x, Le^y, H1, H2, A, B and sialylated Le^a did not. Le^a without galactose (Fuc1,4GlcNAc) still bound to S. mutans, but Le^a without fucose (Gal1,3GlcNAc) did not. Binding of agglutinin to S. mutans was not inhibited by Le^a. In conclusion, S. mutans can bind to Le^a carbohydrate epitopes in which the fucose is an essential residue. Le^a carbohydrate epitopes are present on salivary agglutinin but play no major role in binding.     

Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase

Inhibitory effect of green tea polyphenols viz., catechin and epigallocatechin gallate (EGCG) on the action of collagenase against collagen has been probed in this study. Catechin and EGCG treated collagen exhibited 56 and 95% resistance, respectively, against collagenolytic hydrolysis by collagenase. Whereas direct interaction of catechin and EGCG with collagenase exhibited 70 and 88% inhibition, respectively, to collagenolytic activity of collagenase against collagen and the inhibition was found to be concentration dependent. The kinetics of inhibition of collagenase by catechin and EGCG has been deduced from the extent of hydrolysis of (2-furanacryloyl-L-leucyl-glycyl-L-prolyl-L-alanine), FALGPA. Both catechin and EGCG exhibited competitive mode of inhibition against collagenase. The change in the secondary structure of collagenase on treatment with catechin and EGCG has been monitored using circular dichroism spectropolarimeter. CD spectral studies showed significant changes in the secondary structure of collagenase on treatment with higher concentration of catechin and EGCG. Higher inhibition of EGCG compared to catechin has been attributed to the ability of EGCG to exhibit better hydrogen bonding and hydrophobic interaction with collagenase.     

Collagen Binding of Bifidobacterium adolescentis

In this study, 13 bifidobacterial strains were tested for their ability to adhere to immobilized extracellular matrix (ECM) proteins. Only two Bifidobacterium adolescentis strains adhered to immobilized type I and type V collagens, but not to laminin, fibronectin, and type III and IV collagens. The adhesion of B. adolescentis BB-119 to type V collagen was inhibited by type I and V collagens and gelatin, and was diminished after protease treatment of the cells. Periodate treatment of immobilized collagen and the presence of galactose inhibited the adhesion of strain BB-119 to type V collagen. Two cell surface proteins with molecular masses of 36 kDa and 52 kDa from strain BB-119 were found to bind to horseradish peroxidase-conjugated type V collagen by ligand blotting. We concluded that B. adolescentis BB-119 binds to type V collagen at galactose chains as target via these two cell surface proteins by their lectin-like activity. Received: 15 October 1996 / Accepted: 20 November 1996