Saliva-coated Hydroxyapatite Beads Research Articles

Human salivary protein-derived peptides specific-salivary SIgA antibodies enhanced by nasal double DNA adjuvant in mice play an essential role in preventing Porphyromonas gingivalis colonization: an in-vitro study

BackgroundWe previously showed that fimbriae-bore from Poryphyromonas gingivalis (Pg), one of the putative periodontopathogenic bacteria specifically bound to a peptide domain (stat23, prp21) shared on statherin or acidic proline-rich protein 1 (PRP1) molecule of human salivary proteins (HSPs). Here, we investigated whether the nasal administration of DNA plasmid expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotide 1826 as double DNA adjuvant (dDA) with stat23 and prpr21 induces antigen (Ag)-specific salivary secretory IgA (SIgA) antibodies (Abs) in mice. Further, we examined that stat23- and prpr21-specific salivary SIgA Abs induced by dDA have an impact on Pg-binding to human whole saliva-coated hydroxyapatite beads (wsHAPs).Material and methodsC57BL/6N mice were nasally immunized with dDA plus sta23 or/and prp21 peptide as Ag four times at weekly intervals. Saliva was collected one week after the final immunization and was subjected to Ag-specific ELISA. To examine the functional applicability of Ag-specific SIgA Abs, SIgA-enriched saliva samples were subjected to Pg binding inhibition assay to wsHAPs.ResultsSignificantly elevated levels of salivary SIgA Ab to stat23 or prp21 were seen in mice given nasal stat23 or prp21 with dDA compared to those in mice given Ag alone. Of interest, mice nasally given the mixture of stat23 and prp21 as double Ags plus dDA, resulted in both stat23- and prp21-specific salivary SIgA Ab responses, which are mediated through significantly increased numbers of CD11c+ dendritic cell populations and markedly elevated Th1 and Th2 cytokines production by CD4+ T cells in the mucosal inductive and effector tissues. The SIgA Ab-enriched saliva showed significantly reduced numbers of live Pg cells binding to wsHAPs as compared with those in mice given double Ags without dDA or naïve mice. Additionally, saliva from IgA-deficient mice given nasal double Ags plus dDA indicated no decrease of live Pg binding to wsHAPs.ConclusionThese findings show that HSP-derived peptides-specific salivary SIgA Abs induced by nasal administration of stat23 and prp21 peptides plus dDA, play an essential role in preventing Pg attachment and colonization on the surface of teeth, suggesting a potency that the SIgA may interrupt and mask fimbriae-binding domains in HSPs on the teeth.

Abiotrophia defectiva adhere to saliva-coated hydroxyapatite beads via interactions between salivary proline-rich-proteins and bacterial glyceraldehyde-3-phosphate dehydrogenase.

Abiotrophia defectiva is a species of nutritionally variant streptococci that is found in human saliva and dental plaques and that has been associated with infective endocarditis. In our previous study, it was found that A. defectiva could bind specifically to saliva-coated hydroxyapatite beads (SHA). This study identified a cell surface component of A. defectiva that promotes adherence to SHA beads. The binding of A. defectiva to SHA was reduced in the presence of antibodies against human proline-rich protein (PRP); these results suggested that PRP may be a critical component mediating interactions between A. defectiva and the salivary pellicle. Two-dimensional gel electrophoresis of whole A. defectiva cells followed by Far-Western blotting was conducted by probing with synthetic peptides analogous to the binding region of PRP known as PRP-C. The results indicate that an A. defectiva protein of 37 kDa interacts with PRP-C. The results of amino-terminal sequencing of the adhesive A. defectiva protein revealed significant similarity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Recombinant GAPDH bound to immobilized PRP-C in a dose-dependent manner and binding of A. defectiva to SHA or to PRP was reduced in the presence of anti-GAPDH antiserum. Western blotting or electron immunomicroscopic observations with anti-GAPDH antiserum revealed that this protein was expressed in both cytosolic and cell wall fractions. These results suggest that A. defectiva could specifically bind to PRP via interactions with cell surface GAPDH; the findings suggest a mechanism underlying A. defectiva-mediated adherence to saliva-coated tooth surfaces.

Contribution of different adherent properties of Granulicatella adiacens and Abiotrophia defectiva to their associations with oral colonization and the risk of infective endocarditis.

Granulicatella adiacens (G. adiacens) and Abiotrophia defectiva (A. defectiva) colonize the oral cavity and form part of the normal flora in the intestinal and genitourinary tracts. As reported previously, the frequency of isolation of G. adiacens from the oral cavity was much higher than that of A. defectiva. However, it has been reported that compared with G. adiacens, A. defectiva was isolated at considerably higher frequencies from the blood of patients with infective endocarditis (IE). Hence, in this study, the in vitro interaction of G. adiacens and A. defectiva strains with host surfaces and biofilm formation was examined to assess whether their different adhesive properties contribute to their associations with oral colonization and IE, respectively. G. adiacens exhibited an increased binding ability to saliva-coated hydroxyapatite beads than A. defectiva following the addition of CaCl2. Furthermore, biofilm formation was observed only for G. adiacens with the use of a polystyrene tube and scanning electron microscopy analysis. Conversely, A. defectiva displayed significantly greater adherence to human umbilical vein endothelial cells and immobilized fibronectin than G. adiacens. These findings suggest that differences in binding properties to host components imply specific binding mechanisms in G. adiacens and A. defectiva, which might mediate selective colonization in the oral cavity or are associated with the pathogenicity of endocarditis.

Potent In Vitro and In Vivo Activity of Plantibody Specific for Porphyromonas gingivalis FimA

Fimbrial protein fimbrillin (FimA), a major structural subunit of Porphyromonas gingivalis, has been suggested as a vaccine candidate to control P. gingivalis-induced periodontal disease. Previously, cDNAs encoding IgG monoclonal antibodies (MAbs) against purified FimA from P. gingivalis 2561 have been cloned, and the MAbs have been produced in rice cell suspension. Here we examined the biological activities of the plant-produced MAb specific for FimA (anti-FimA plantibody) of P. gingivalis in vitro and in vivo. The anti-FimA plantibody recognized oligomeric/polymeric forms of native FimA in immunoblot analysis and showed high affinity for native FimA (KD = 0.11 nM). Binding of P. gingivalis (10(8) cells) to 2 mg of saliva-coated hydroxyapatite beads was reduced by 53.8% in the presence of 1 μg/ml plantibody. Anti-FimA plantibody (10 μg/ml) reduced invasion of periodontal ligament cells by P. gingivalis (multiplicity of infection, 100) by 68.3%. Intracellular killing of P. gingivalis opsonized with the anti-FimA plantibody by mouse macrophages was significantly increased (77.1%) compared to killing of bacterial cells with irrelevant IgG (36.7%). In a mouse subcutaneous chamber model, the number of recoverable P. gingivalis cells from the chamber fluid was significantly reduced when the numbers of bacterial cells opsonized with anti-FimA plantibody were compared with the numbers of bacterial cells with irrelevant IgG, 66.7% and 37.1%, respectively. These in vitro and in vivo effects of anti-FimA plantibody were comparable to those of the parental MAb. Further studies with P. gingivalis strains with different types of fimbriae are needed to investigate the usefulness of anti-FimA plantibody for passive immunization to control P. gingivalis-induced periodontal disease.

Production of monoclonal antibody against FimA protein from Porphyromonas gingivalis in rice cell suspension culture

Major fimbriae (FimA) of Porphyromonas gingivalis play an important role in the adherence of the bacterium to oral surfaces, making them a target for the development of a vaccine against periodontal disease. In our previous study, monoclonal antibodies to FimA were expressed in tobacco plants using the agroinfiltration method. In this report, for easy purification, monoclonal antibodies to FimA were produced and accumulated in rice callus suspension culture. The accumulated antibodies were purified by protein G-affinity chromatography. The plant-produced monoclonal antibodies inhibited the binding of P. gingivalis to saliva-coated hydroxyapatite beads, as well as the invasion of oral epithelial KB cells due to the bacterium. The antibodies enhanced the intracellular killing of P. gingivalis by polymorphonuclear neutrophils. These results suggest that FimA-specific monoclonal antibodies produced in a rice suspension culture were easily purified and biologically active against P. gingivalis, and thus may be used for passive immunization to prevent P. gingivalis-induced periodontal disease.

The effect of eugenol on the cariogenic properties of Streptococcus mutans and dental caries development in rats

Eugenol has been widely used in medicine due to its antibacterial, anti-inflammatory, antioxidant, anticancer and analgesic properties. The present study was designed to investigate the effects of eugenol on the cariogenic properties of Streptococcus mutans and dental caries development in rats. Eugenol demonstrated significant inhibitory effects against acid production by S. mutans. The synthesis of water-insoluble glucans by glucosyltransferases was reduced by eugenol. Eugenol also markedly suppressed the adherence of S. mutans to saliva-coated hydroxyapatite beads. Furthermore, topical application of eugenol reduced the incidence and severity of carious lesions in rats. These results suggest that the natural compound eugenol may be a useful therapeutic agent for dental caries.

Inhibitory effects of the phenolic fraction from the pomace of Vitis coignetiae on biofilm formation by Streptococcus mutans

ObjectivesThe anti-cariogenic properties of the phenolic fraction from the pomace of Vitis coignetiae (VcPP) were evaluated by in vitro assays and compared with fruit juices from V. coignetiae and common grapes and with other phenolic fractions. The effects of VcPP against the biofilm of Streptococcus mutans were investigated. DesignSucrose-dependent biofilm formation by S. mutans cultured in the presence of VcPP was measured by crystal violet dye uptake. Inhibition of adhesion to the saliva-coated hydroxyapatite (sHA) beads was quantified using fluorescent-labelled cells. The MIC for S. mutans was determined by colony counting on agar plates containing VcPP. The ability of VcPP to inhibit glucan synthesis by three distinct recombinant glucosyltransferases (Gtfs) was assessed by quantifying the production of water-soluble and -insoluble polysaccharides in bacterial cultures. In addition, the buffering effect of VcPP in cultures of S. mutans was evaluated. ResultsVcPP reduced adhesion of S. mutans to sHA and biofilm formation in a dose-dependent manner. The MIC of VcPP was 7.50mg/ml. VcPP inhibited GtfB activity associated with the synthesis of water-insoluble glucans. It also inhibited GtfD activity associated with the synthesis of water-soluble glucans at a concentration which was lower than that used for inhibition of GtfB. VcPP had no effect on acidification associated with glucose utilization by S. mutans. ConclusionsThe current study supports the potential of VcPP as a food additive for reducing caries by inhibiting adhesion to the tooth surface and GtfD-mediated soluble glucan synthesis.

Essential Oil of Curcuma longa Inhibits Streptococcus mutans Biofilm Formation

Curcuma longa (C. longa) has been used as a spice in foods and as an antimicrobial in Oriental medicine. In this study, we evaluated the inhibitory effects of an essential oil isolated from C. longa on the cariogenic properties of Streptococcus mutans (S. mutans), which is an important bacterium in dental plaque and dental caries formation. First, the inhibitory effects of C. longa essential oil on the growth and acid production of S. mutans were tested. Next, the effect of C. longa essential oil on adhesion to saliva-coated hydroxyapatite beads (S-HAs) was investigated. C. longa essential oil inhibited the growth and acid production of S. mutans at concentrations from 0.5 to 4 mg/mL. The essential oil also exhibited significant inhibition of S. mutans adherence to S-HAs at concentrations higher than 0.5 mg/mL. S. mutans biofilm formation was determined by scanning electron microscopy (SEM) and safranin staining. The essential oil of C. longa inhibited the formation of S. mutans biofilms at concentrations higher than 0.5 mg/mL. The components of C. longa essential oil were then analyzed by GC and GC-MS, and the major components were α-turmerone (35.59%), germacrone (19.02%), α-zingiberene (8.74%), αr-turmerone (6.31%), trans-β-elemenone (5.65%), curlone (5.45%), and β-sesquiphellandrene (4.73%). These results suggest that C. longa may inhibit the cariogenic properties of S. mutans.

Inhibitory effect of Aralia continentalis on the cariogenic properties of Streptococcus mutans

Ethnopharmacological relevance Aralia continentalis has been used in traditional Korean medicine for dental diseases such as toothache, dental caries, periodontal disease and gingivitis, and also has been used for neuralgia, analgesia, sweating, and as an antirheumatic. Aim of the study The present study was designed to investigate the inhibitory effect of Aralia continentalis extract on cariogenic properties of Streptococcus mutans, which is one of the most important bacteria in the formation of dental caries and dental plaque. Materials and methods The inhibitory effects of Aralia continentalis extract on the growth, acid production, water-insoluble glucan synthesis, and adhesion were investigated in Streptococcus mutans. The biofilm formation of Streptococcus mutans was determined by scanning electron microscopy (SEM) and safranin staining. Results The ethanol extract of Aralia continentalis showed concentration dependent inhibitory activity on the growth of Streptococcus mutans and significant inhibition of acid production at the concentrations of 0.25, 0.5, 1, 2 and 4 mg/ml compared to the control group. The synthesis of water-insoluble glucan by glucosyltransferase (GTFase) was decreased in the presence of 0.5–4 mg/ml of the extract of Aralia continentalis. The extract markedly inhibited Streptococcus mutans adherence to saliva-coated hydroxyapatite beads (S-HAs). The extract of Aralia continentalis has an inhibitory effect on the formation of Streptococcus mutans biofilms at the concentrations higher than 2 mg/ml. Conclusions These results suggest that Aralia continentalis may inhibit cariogenic properties of Streptococcus mutans, and also may support the scientific rationale that native inhabitants used the extract for the treatment of dental diseases.

Funoran-Containing Xylitol Gum and Tablets Inhibit Adherence of Oral Streptococci

The aim of this study was to evaluate inhibition of adherence of oral streptococci by addition of funoran to xylitol-containing chewing gum or tablets. The inhibitory effect by addition of funoran on adherence was evaluated by scintillation counting using 3H-labeled oral streptococci and saliva-coated hydroxyapatite beads. In samples containing more than 0.1% funoran, the number of attached bacteria was significantly reduced in all tested microorganisms compared with funoran-free samples. This reduction in adherence was also observed by scanning electron microscopy. The results indicate that addition of funoran to xylitol chewing gum or tablets inhibits colonization by oral streptococci, including Streptococcus mutans.

Antiadhesion and Antibiofilm Activities of High Molecular Weight Coffee Components against Streptococcus mutans

In previous studies we demonstrated that green and roasted coffee contains low molecular weight (LMW) compounds capable of inhibiting the ability of Streptococcus mutans, the major causative agent of human dental caries, to adhere to hydroxyapatite (HA) beads. This study addressed the ability of the whole high molecular weight coffee fraction (cHMW) and of its melanoidin and non-melanoidin components (GFC1-5), applied at concentrations that occur in coffee beverages, to (i) inhibit S. mutans growth; (ii) affect S. mutans sucrose-dependent adhesion to and detachment from saliva-coated HA beads (sHA); and (iii) inhibit biofilm development on microtiter plates. The results indicated that only cHMW is endowed with antimicrobial activity. The cHMW fraction and each of the five GFC components inhibited S. mutans adhesion, the strongest effect being exerted by cHMW (91%) and GFC1 (88%). S. mutans detachment from sHA was four times greater (∼20%) with cHMW and the GFC1 and GFC4 melanoidins than with controls. Finally, biofilm production by S. mutans was completely abolished by cHMW and was reduced by 20% by the melanoidin components GFC2 and GFC4 and by the non-melanoidin component GFC5 compared with controls. Altogether these findings show that coffee beverage contains both LMW compounds and HMW melanoidin and non-melanoidin components with a strong ability to interfere in vitro with the S. mutans traits relevant for cariogenesis.

Inhibition of Streptococcus mutans adherence and biofilm formation using analogues of the SspB peptide

Objective Streptococcus gordonii is a pioneer colonizer of the enamel salivary pellicle that forms biofilm on the tooth surfaces. Recent reports show the surface protein analogue peptide {400 (T) of SspB 390–402 is substituted to K forming SspB (390–T400K–402)} from S. gordonii interacts strongly with salivary receptors to cariogenic bacteria, Streptococcus mutans. To characterize the analogue peptide biological activities, we investigated its binding and inhibiting effects, and the role of its amino acid moities. Methods We measured binding activity of analogue peptides to salivary components using the BIAcore assay; assayed inhibition activities of peptides for bacterial binding and growth on saliva-coated hydroxyapatite beads (s-HA); and describe the peptides interfering with biofilm formation of S. mutans on polystyrene surfaces . Results The SspB (390–T400K–402 and –401) peptides significantly bound with salivary components and inhibited the binding of S. mutans and S. gordonii to s-HA without bactericidal activity; but did not inhibit binding of Streptococcus mitis, a beneficial commensal . Further, the lack of D and E-L at position 390 and 401–402 in the peptide, and substituted peptide SspB (D390H– or D390K–T400K–402) did not bind to salivary components or inhibit binding of S. mutans. The SspB (390–T400K–402) peptide inhibited biofilm formation on salivary components-coated polystyrene surfaces in absence of conditioned planktonic cells. Conclusions We found constructing the peptide to include positions 390(D), 400(K) and 401(E), two surface positive and negative connective charges, and at least 12 amino acids are required to bind salivary components and inhibit the binding of S. mutans and S. gordonii.

Isolation of red wine components with anti-adhesion and anti-biofilm activity against Streptococcus mutans

Abstract Red wine is a widely consumed beverage with multiple beneficial effects on human health. In the present paper, the anticaries properties of red wine were studied in vitro and ex vivo. Our in vitro findings shows that dealcoholised red wine, besides exerting antibacterial activity, strongly interferes with Streptococcus mutans adhesion to saliva-coated hydroxyapatite (sHA) beads, promotes its detachment from sHA, and powerfully inhibits in vitro biofilm formation. The main components responsible for such activities were found to be proanthocyanidins. The ability of red wine to inhibit ex vivo S. mutans biofilm formation on the occlusal surface of natural human teeth also was demonstrated. Our data indicates that protection of the oral cavity from the cariogenic action of S. mutans may be another beneficial effect of the moderate consumption of red wine.

Anti-cariogenic effects of polyphenols from plant stimulant beverages (cocoa, coffee, tea)

Polyphenols occurring in cocoa, coffee and tea can have a role in the prevention of cariogenic processes, due to their antibacterial action. Cocoa polyphenol pentamers significantly reduce biofilm formation and acid production by Streptococcus mutans and S. sanguinis. In the same way, trigonelline, caffeine and chlorogenic acid occurring in green and roasted coffee interfere with S. mutans adsorption to saliva-coated hydroxyapatite beads. Studies carried out on green, oolong and black tea indicate that tea polyphenols exert an anti-caries effect via an anti-microbial mode-of-action, and galloyl esters of (-)-epicatechin, (-)-epigallocatechin and (-)-gallocatechin show increasing antibacterial activities. The anti-cariogenic effects against alpha-haemolytic streptococci showed by polyphenols from cocoa, coffee, and tea suggest further studies to a possible application of these beverages in the prevention of pathogenesis of dental caries.

In vitro evaluation of yoghurt starter lactobacilli and Lactobacillus rhamnosus GG adhesion to saliva‐coated surfaces

The aim of the study was to evaluate the adhesion of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus rhamnosus strain GG to saliva-coated surfaces in vitro. Fifteen radiolabeled dairy L. delbrueckii subsp. bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated hydroxyapatite beads and polystyrene microtiter plates and the radioactivity was measured by liquid scintillation counter. The effects of lysozyme on the adhesion of lactobacilli and of pretreatment with lactobacilli on the adhesion of Streptococcus sanguinis were also assessed. All strains tested adhered to saliva-coated surfaces but with significantly different binding frequencies. The adhesion of the L. delbrueckii subsp. bulgaricus strains remained lower in comparison to L. rhamnosus strain GG. One L. delbrueckii subsp. bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment of the samples significantly increased lactobacillus adhesion to saliva-coated surfaces. The present results showed significant variations in the adhesion capacity of the Lactobacillus strains studied. Adhesion to oral surfaces is of primary importance for bacterial colonization in the mouth. Only one of the L. delbrueckii subsp. bulgaricus dairy starter culture strains investigated had a high adhesion percentage. This strain might then be considered for further investigations in the oral environment.

Production of Monoclonal Antibodies Specific to FimA of Porphyromonas gingivalis and Their Inhibitory Activity on Bacterial Binding

BackgroundThe FimA of Porphyromonas gingivalis is a crucial pathogenic component of the bacteria and has been implicated as a target for vaccine development against the periodontal diseases.MethodsIn this study, the purified fimbriae (FimA subunit polymers) protein was used for immunization in their native form and B hybridoma clones producing antibodies specific to FimA were established.ResultsThe monoclonal antibodies prepared from selected two clones, designated #123 (IgG2b/ kappa) and #265 (IgG1/kappa), displayed different patterns of binding activity against the cognate antigen. Both antibodies reacted with conformational epitopes expressed by partially dissociated oligomers, but not with monomer as elucidated by Western blot analysis. Ascites fluid containing the monoclonal antibodies showed the inhibitory activity against P. gingivalis to saliva-coated hydroxyapatite beads, an in vitro model for the pellicle-coated tooth surface.ConclusionThese results suggest that the monoclonal antibodies could be used as vaccine material against the periodontal diseases through passive immunization.

Effects of antibodies against a fusion protein consisting of parts of cell surface protein antigen and glucosyltransferase of Streptococcus sobrinus on cell adhesion of mutans streptococci

The cell surface protein antigen (PAg) and glucosyltransferases (GTFs) produced by Streptococcus sobrinus are considered to be major colonization factors of the organism. We constructed a fusion gene encoding a protein composed of the alanine-rich region of PAg (PAgA) and the glucan-binding domain (GB) of GTF-I, which catalyzes the synthesis of water-insoluble glucan in S. sobrinus. The fusion protein PAgA-GB was purified from cell extracts of Escherichia coli harboring the fusion gene, and antibodies against the fusion protein were prepared in rabbits. In the presence of sucrose, the antibody against PAgA-GB significantly inhibited the adhesion of both S. sobrinus MT8145 and Streptococcus mutans Xc to saliva-coated hydroxyapatite beads, and the inhibitory effect on S. sobrinus was stronger than that on S. mutans. In the absence of sucrose, the antibody against PAgA-GB significantly inhibited the adhesion of both S. sobrinus and S. mutans, however the inhibitory effect on S. sobrinus was unexpectedly weaker than that on S. mutans. A similar result was observed with the antibody against the intact recombinant PAg protein (rPAg), while the same antibody reacted more strongly against S. sobrinus than against S. mutans cells. Taken together, these results show that the antibody against S. sobrinus GTF-I may be useful for effective inhibition of the sucrose-dependent adhesion of S. sobrinus. However, PAg of S. sobrinus may not function primarily as a receptor for acquired pellicles, and other cell surface proteins may be involved in the sucrose-independent adhesion of S. sobrinus.

Anticariogenic Properties of the Extract ofCyperus rotundus

Streptococcus mutans (S. mutans) is known as the causative bacteria in the formation of dental plaque and dental caries. The aim of this experiment was to investigate the effects of Cyperus rotundus (C. rotundus) tuber extract on the growth, acid production, adhesion, and water-insoluble glucan synthesis of S. mutans. The growth and acid production were reduced by the extract of C. rotundus in a dose dependent manner. The extract of C. rotundus markedly inhibited the adherence of S. mutans to saliva-coated hydroxyapatite beads (HAs). The adherence was repressed by more than 50% at the concentration of 0.5 mg/ml of the extract and complete inhibition was observed at the concentration of 4 mg/ml of the extract. On the activity of glucosyltransferase (GTFase) which synthesizes water-insoluble glucan from sucrose, the extract of C. rotundus showed more than 10% inhibition at a concentration of 2 mg/ml. These results suggest that C. rotundus may inhibit cariogenic properties of S. mutans. Further studies are necessary to clarify the active constituents of C. rotundus responsible for such biomolecular activities.

Effect of Barley Coffee on the Adhesive Properties of Oral Streptococci

Some beverages and foods protect tooth surfaces against Streptococcus mutans colonization. Adhesion of S. mutans is a crucial step in the initiation and development of dental caries. In this study, we showed that barley coffee (BC), a beverage made from roasted barley, interferes with S. mutans adsorption to hydroxyapatite (HA), and we identified its antiadhesive components. The effects of sublethal concentrations (sub-MICs) of BC on the adhesion of S. mutans to saliva-coated HA beads were assessed using three experimental approaches: (A) Beads were pretreated with BC before adding bacteria, (B) BC and bacteria were added to the beads simultaneously, and (C) streptococci grown in the presence of sub-MICs of BC were added to the beads. All treatments induced variable but significant inhibition of S. mutans sucrose-dependent and -independent adherence to HA. Similar results were obtained with other oral streptococci. BC components were fractioned by dialysis and gel filtration chromatography; the <1000 Da molecular mass (MM) fraction, which contains polyphenols, zinc, and fluoride ions, and the >1000 kDa MM fraction, which consists of a potent brown antioxidant, melanoidin, both displayed antiadhesive properties. High-MM melanoidin was not detected in unroasted barley, indicating that it forms during the roasting process. Results suggest that BC consumption may influence the colonization of tooth surfaces by cariogenic bacteria.

Role of Streptococcus sanguinis sortase A in bacterial colonization

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, has low cariogenicity, though colonization on tooth surfaces by this bacterium initiates aggregation by other oral bacteria and maturation of dental plaque. Additionally, S. sanguinis is frequently isolated from infective endocarditis patients. We investigated the functions of sortase A (SrtA), which cleaves LPXTG-containing proteins and anchors them to the bacterial cell wall, as a possible virulence factor of S. sanguinis. We identified the srtA gene of S. sanguinis by searching a homologous gene of Streptococcus mutans in genome databases. Next, we constructed an srtA-deficient mutant strain of S. sanguinis by insertional inactivation and compared it to the wild type strain. In the case of the mutant strain, some surface proteins could not anchor to the cell wall and were partially released into the culture supernatant. Furthermore, adherence to saliva-coated hydroxyapatite beads and polystyrene plates, as well as adherence to and invasion of human epithelial cells were reduced significantly in the srtA-deficient strain when compared to the wild type. In addition, antiopsonization levels and bacterial survival of the srtA-deficient mutant were decreased in human whole blood. This is the first known study to report that SrtA contributes to antiopsonization in streptococci. Our results suggest that SrtA anchors surface adhesins as well as some proteins that function as antiopsonic molecules as a means of evading the human immune system. Furthermore, they demonstrate that SrtA of S. sanguinis plays important roles in bacterial colonization.