Functional Variation of the Antigen I/II Surface Protein in Streptococcus mutans and Streptococcus intermedius

Although Streptococcus intermedius and Streptococcus mutans are regarded as members of the commensal microflora of the body, S. intermedius is often associated with deep-seated purulent infections, whereas S. mutans is frequently associated with dental caries. In this study, we investigated the roles of the S. mutans and S. intermedius antigen I/II proteins in adhesion and modulation of cell surface characteristics. By using isogenic mutants, we show that the antigen I/II in S. mutans, but not in S. intermedius, was involved in adhesion to a salivary film under flowing conditions, as well as in binding to rat collagen type I. Binding to human fibronectin was a common function associated with the S. mutans and S. intermedius antigen I/II. Adhesion of S. mutans or S. intermedius to human collagen types I or IV was negligible. Hydrophobicity, as measured by water contact angles, and zeta potentials were unaltered in the S. intermedius mutant. The S. mutans isogenic mutants, on the other hand, exhibited more positive zeta potentials at physiological pH values than did the wild type. The results indicate common and species-specific roles for the antigen I/II in mediating the attachment of S. mutans and S. intermedius to host components and in determining cell surface properties.     

Interactions of Streptococcus mutans Fimbria-Associated Surface Proteins with Salivary Components

Streptococcus mutans has been implicated as the major causative agent of human dental caries. S. mutans binds to saliva-coated tooth surfaces, and previous studies suggested that fimbriae may play a role in the initial bacterial adherence to salivary components. The objectives of this study were to establish the ability of an S. mutans fimbria preparation to bind to saliva-coated surfaces and determine the specific salivary components that facilitate binding with fimbriae. Enzyme-linked immunosorbent assay (ELISA) established that the S. mutans fimbria preparation bound to components of whole saliva. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot techniques were used to separate components of whole saliva and determine fimbria binding. SDS-PAGE separated 15 major protein bands from saliva samples, and Western blot analysis indicated significant binding of the S. mutans fimbria preparation to a 52-kDa salivary protein. The major fimbria-binding salivary protein was isolated by preparative electrophoresis. The ability of the S. mutans fimbria preparation to bind to the purified salivary protein was confirmed by Western blot analysis and ELISA. Incubation of the purified salivary protein with the S. mutans fimbria preparation significantly neutralized binding of the salivary protein-fimbria complex to saliva-coated surfaces. The salivary protein, whole saliva, and commercial amylase reacted similarly with antiamylase antibody in immunoblots. A purified 65-kDa fimbrial protein was demonstrated to bind to both saliva and amylase. These data indicated that the S. mutans fimbria preparation and a purified fimbrial protein bound to whole-saliva-coated surfaces and that amylase is the major salivary component involved in the binding.     

Selective Binding of Blood Group-Reactive Salivary Mucins by Streptococcus mutans and Other Oral Organisms

Strains of Streptococcus mutans of four genetic groups and five serotypes, and strains of S. sanguis, S. mitis, S. salivarius, Actinomyces naeslundii, and A. viscosus, were found to bind blood group-reactive (BGR) mucin isolated from whole human saliva. The bacteria studied bound mucins with blood type A or B reactivity to a similar extent, suggesting that the carbohydrate moieties responsible for the A and B antigenic determinants were not involved. The organisms studied appeared to bind different fractions of BGR mucin molecules because preparations absorbed with cells of a given oral species no longer contained BGR molecules which bound to homologous organisms but still possessed BGR components which bound to varying degrees to other bacteria. Differences were even noted among S. mutans strains belonging to different genetic groups and serological types. Immunoglobulins could not be detected in the mucin preparations, and addition of anti-human immunoglobulin A (IgA), IgG, or IgM serum to reaction mixtures did not affect binding. Mucin pretreated with periodate or iodoacetate no longer bound to S. mutans H12 cells, suggesting that carbohydrate moieties and sulfhydryl groups played an essential role. Active cell metabolism was not required for BGR mucin binding; however, pretreatment of H12 cells with periodate or heat (100 degrees C for 15 min) reduced binding. Mucin labeled with [(14)C]phenyl isothiocyanate appeared to bind to S. mutans H12 cells comparably to untreated mucin; the binding also appeared to be specific because less than 15% of the labeled material became bound when incubated with an excess of streptococci. Binding of [(14)C]phenyl isothiocyanate-labeled mucin was not affected by neutral sugars tested or by preparations of c antigen, glycerol teichoic acid, dextran, or crude glucosyltransferase. However, binding was inhibited by several amines. BGR salivary mucins are present in the acquired pellicle covering teeth; the ability of bacteria to selectively bind such components suggest that they may serve as receptor molecules involved in the attachment of bacteria to teeth.     

The Collagen Binding Protein Cnm Contributes to Oral Colonization and Cariogenicity of Streptococcus mutans OMZ175

Streptococcus mutans is the etiological agent of dental caries and one of the many bacterial species implicated in infective endocarditis. The expression of the collagen-binding protein Cnm by S. mutans has been associated with extraoral infections, but its relevance for dental caries has only been theorized to date. Due to the collagenous composition of dentinal and root tissues, we hypothesized that Cnm may facilitate the colonization of these surfaces, thereby enhancing the pathogenic potential of S. mutans in advancing carious lesions. As shown for extraoral endothelial cell lines, Cnm mediates the invasion of oral keratinocytes and fibroblasts by S. mutans. In this study, we show that in the Cnm⁺ native strain, OMZ175, Cnm mediates stringent adhesion to dentinal and root tissues as well as collagen-coated surfaces and promotes both cariogenicity and carriage in vivo. In vitro, ex vivo, and in vivo experiments revealed that while Cnm is not universally required for S. mutans cariogenicity, it contributes to (i) the invasion of the oral epithelium, (ii) enhanced binding on collagenous surfaces, (iii) implantation of oral biofilms, and (IV) the severity of caries due to a native Cnm⁺ isolate. Taken together, our findings reveal that Cnm is a colonization factor that contributes to the pathogenicity of certain S. mutans strains in their native habitat, the oral cavity.     

Inhibitory Effects of MoAbs against a Surface Protein Antigen in Real-Time Adherence In vitro and Recolonization In vivo of Streptococcus mutans

A surface protein antigen (PAc) of Streptococcus mutans, particularly the A-region of the molecule, has been reported to interact with salivary components on the tooth surface. It might be a candidate antigen inducing the production of antibodies against the adherence of S. mutans to the tooth surface. We investigated the effects of monoclonal antibodies (MoAbs) obtained by immunization of synthetic PAc peptides that completely correspond to the amino acid sequence of part of the A-region. These MoAbs recognize several core B-cell epitopes in the sequence. Two (KH5 and SH2) of these antibodies reacted with both S. mutans and Streptococcus sobrinus, but not with Streptococcus sanguis, Streptococcus salivarius, Porphyromonas gingivalis or Lactobacillus casei. They clearly inhibited the real-time adherence of S. mutans to salivary components in a biosensor. KH5, which showed a real-time inhibition (71%), also significantly prevented the recolonization of S. mutans on the tooth surface in rats. These results suggested that the core B-cell epitope (-Y---L--Y----) recognized by KH5 was the essential sequence in the antigenic epitopes of PAc protein recognized specifically by the inhibitory antibody. Therefore, the amino acid residues were found to be important in the initial attachment of S. mutans to the tooth surface. These results provide for the mechanism of PAc molecule in the initial attachment of S. mutans on the tooth surface and more effective designs for the removal of S. mutans and S. sobrinus from the oral cavity.     

Salivary immunoglobulin A antibodies and recovery from challenge of Streptococcus mutans after oral administration of Streptococcus mutans vaccine in humans.

Heat-killed Streptococcus mutans was administered orally in two periods of 1 week to six subjects in an attempt to affect the salivary immunoglobulin A (IgA) response to this bacterium. Enzyme-linked immunosorbent assays were used to detect specific IgA antibody activity, and an immunofluorescent assay was used for measurement of total IgA in parotid saliva. The salivary IgA response to S. mutans was compared with that against a noncross-reacting antigen preparation from Escherichia coli and with antibody responses in five sham-immunized subjects. No change in salivary IgA response to S. mutans was observed after oral administration of this organism. Significantly less streptomycin-resistant S. mutans could be recovered from the six test subjects than from the five controls after the first of two challenges with streptomycin-resistant microorganisms. At the day of the first challenge, a significantly higher IgA antibody response to all tested antigens was observed in the test group than in the control group. The data show that this difference was not related to the oral administration of S. mutans but rather was an occasional finding. The coincidence of a rapid elimination of the challenge strain and a high IgA antibody response to S. mutans supports the concept that salivary IgA antibodies could have a biological significance in the human defense against cariogenic microorganisms.     

Aggregation and Adherence of Streptococcus sanguis: Role of Human Salivary Immunoglobulin A

Fourteen freshly isolated strains of Streptococcus sanguis were obtained from the dental plaque of five healthy adults. Whole saliva was collected concomitant with the plaque isolates from the five subjects, and a second whole saliva sample was collected 10 weeks later. All possible combinations of the first five saliva samples, the second five saliva samples, and 14 strains of bacteria were tested for aggregation. Of the 140 combinations examined, 108 of 140 (77%) of the strains aggregated with the first saliva samples and 95 of 140 (68%) aggregated with the second saliva samples. Overall, 72% of the strains aggregated with both the first and second saliva samples. Removal of immunoglobulin A (IgA) from these same salivas resulted in 38 of 108 (35%) of the aggregates decreasing in intensity with the first saliva samples and 27 of 95 (29%) of the aggregates decreasing in intensity with the second saliva samples. No aggregates increased in intensity with saliva samples when IgA had been removed. Removal of IgA from saliva also resulted in a mean decrease of 46% in adherence of S. sanguis to hydroxyapatite coated with the IgA-deficient saliva. Several strains of S. sanguis were shown to aggregate strongly with human salivary and colostral IgA. In addition, S. sanguis strain S7 showed a 31% stimulation of adherence to hydroxyapatite precoated with human salivary IgA over the uncoated controls. Stepwise removal of IgA from saliva resulted in a decrease in aggregation intensity from strong (4+) to weak (1+ to 2+). Similarly, the adherence of S. sanguis to hydroxyapatite coated with these saliva samples decreased linearly as the salivary IgA was depleted. Alternatively, the addition of a small quantity of salivary IgA (20 mug/ml) to progressively diluted saliva maintained a high level of adherence and strong aggregation until the saliva dilutions reached between 1:8 in the adherence experiments and 1:32 for the aggregations. These data indicate that salivary IgA may play an important role in the microbial ecology of human dental plaque formation.     

Binding of Todd-Hewitt broth antigens by Streptococcus mutans.

Streptococcus mutans 10449, grown in chemically defined culture medium, was tested for its ability to bind 3H-labeled Todd-Hewitt broth components (greater than 12,000 Mr). Maximum adsorption of radioactivity occurred within 5 min at room temperature, and cell-bound material was not completely removed by extended washing with buffer. Heat-killed, arsenate-inhibited, and viable bacteria bound similar quantities. Only 0.09% of the radioactivity in the preparation of high Mr Todd-Hewitt broth components was removed by absorption with excess numbers of S. mutans 10449 cells. Binding followed saturation kinetics and was competitively inhibited by unlabeled medium components, both the dialyzable and nondialyzable fractions. Other oral streptococci were also found to bind these complex medium components. Rabbit antiserum elicited to the high-molecular-weight Todd-Hewitt broth components reacted with monkey cardiac muscle and with S. mutans coated with medium components. Absorption of the anti-Todd-Hewitt broth serum with homogenized heart removed antibodies that reacted with Todd-Hewitt broth-coated S. mutans. Therefore, the tissue-specific antigens of this beef heart infusion medium that adsorb to S. mutans can interfere with the detection and characterization of antigens shared by these bacteria and animal tissues.     

Binding of Streptococcus mutans antigens to heart and kidney basement membranes.

Using indirect immunofluorescence, alkali-extracted components of Streptococcus mutans were found to bind in vitro to capillary walls and sarcolemmal sheaths of monkey cardiac muscle and to glomerular and tubular basement membranes of monkey kidney. Adsorption of S. mutans components to tissue fragments was also detected by indirect radioimmunoassay and immunoblotting on nitrocellulose paper. Antibodies did not bind to untreated, control tissues in these experiments, proving that antigens shared by S. mutans and tissue components were not involved. Rabbit and monkey heart and kidney components bound S. mutans antigens of 24,000, 35,000, and 65,000 Mr. Monkey heart also bound molecules of 90,000 and 120,000 Mr. Rabbits immunized by intravenous injection of disrupted S. mutans cells developed severe nephritis that was characterized by the deposition of immunoglobulins, complement component C3, and S. mutans antigens in the glomeruli. Immunoglobulin G eluted from nephritic kidneys reacted in immunoblots with the 24,000, 35,000, and 65,000 Mr components of S. mutans extract, indicating that the antigens that bound to tissue in vitro also bound in vivo and reacted with antibodies in situ. Antibodies to other S. mutans antigens were not detected in the kidney eluate, although they were present in the serum of the same rabbit.     

Binding of a Streptococcus mutans cationic protein to kidney in vitro.

An 8-kDa protein, with binding activity for heparin and heparan sulfate of basal laminae of animal tissues, was isolated from Streptococcus mutans MT703 and purified to homogeneity. Binding of radioiodinated 8-kDa protein to rabbit kidney tissue in vitro showed a high degree of specificity, as indicated by saturation kinetics, time dependence, and competitive inhibition by unlabeled protein. Binding activity for kidney tissue was competitively inhibited by selected glycosaminoglycans and polyanions in the following order: heparin greater than dextran sulfate greater than heparan sulfate greater than chondroitin sulfate greater than lipoteichoic acid greater than keratan sulfate greater than hyaluronic acid. Binding of the streptococcal protein to rabbit kidney tissue was also strongly inhibited by protamine sulfate, polylysine, and a random copolymer of lysine and alanine. Among the monosaccharides tested at 50 mM, glucosamine 2,3- or 2,6-disulfate, glucuronic acid, glucose 6-phosphate, and glucose 6-sulfate inhibited 50% or more of the binding activity, whereas N-acetylglucosamine 3-sulfate, glucosamine 6-sulfate, N-acetyl-glucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and a selection of neutral sugars were not inhibitory. The heparin-binding protein was detected on the cell wall of S. mutans and in the culture medium following growth. Several other species of streptococci produce an immunologically related protein of similar size.     

Interference of Salivary immunoglobulin A antibodies and other salivary fractions with adherence of Streptococcus mutans to hydroxyapatite.

The adherence of Streptococcus mutans to hydroxyapatite was studied in the presence of salivary fractions with varying activity of naturally occurring immunoglobulin A (IgA) antibodies. Human parotid saliva from different donors was fractionated by chromatography and compared. Salivary IgA antibodies had no decisive effect on the adherence of the S. mutans strain used. High-molecular-weight salivary components from some subjects had an adherence-promoting effect, whereas fractions collected after the void volume of a Sepharose 2B column always inhibited adherence. The data indicate that the influence of unfractionated saliva on adherence is dependent on the net effect of adherence-promoting and adherence-inhibiting components. This principle has to be considered when the effect of human saliva on microbial adherence is studied.     

Binding of Antigen to Surface to on Isolated Rat Mast Cells

This study concerns observations on isolated rat mast cells sensitized to specific antigen. Specific binding of antigen to the surface immunoglobulins of these cells could be demonstrated by immunofluorescence technique as well as by radiolabelled antigen binding and the degree of binding parallelled the allergic reaction as judged by histamine release. Exposure of mast cells to antigen at both low and high antigen concentrations did not change the distribution of the surface immunoglobulins. Furthermore, neither capping not shedding could be induced, even by excessive antigen stimulation. The amount of antigen molecules bound to surface Ig was linearly correlated to the allergic histamine release. When mast cells were sensitized to two antigens of different molecular weight, the cells showed the highest sensitivity to the antigen having the highest molecular weight. These results indicate that both the amount of antigen and the molecular weight of the antigen bound to surface Ig are important factors in the allergic histamine release from mast cells, whereas surface Ig redistribution is not.     

Immunoglobulin Binding Specificities of the Homology Regions (Domains) of Protein A

Protein A binds immunoglobulins and it has two target structures, one in Feγ (C_H) and the other in selected V_H regions. The protein has five homology regions (domains). A, B, C, D, and E. Fc-binding and V_H-binding have been reported to be non-competitive, suggesting that different domains are responsible for the binding of the two ligands. On the other hand, all five domains have been reported to bind Fc. I studied binding of different immunoglobulins by protein A or its domain B (rBB). The results show that separate domains bind V_H and Fc. If all five domains are capable of binding Fc, the ones that bind V_h have low affinity for Fc. Furthermore, the number of Fc-binding domains varies depending on the type of the IgG being bound. Human IgG1 or lgG2 or rabbit IgG (Fc) seem to be bound by several domains (possibly four), and domain B is one of them. Mouse IgG1 or lgG2b are bound by fewer domains not including B. Murine lgG2a is also bound by fewer domains but B is one of them.     

Salivary immunoglobulin A and serum antibodies to Streptococcus mutans ribosomal preparations in dental caries-free and caries-susceptible human subjects

Caries-free subjects or individuals with low caries susceptibility exhibited significantly higher (P less than 0.001) levels of naturally occurring salivary immunoglobulin A (IgA) and serum IgG, IgA, and IgM antibodies to a Streptococcus mutans ribosomal preparation than subjects with high caries susceptibility. Absorption of saliva and serum samples with S. mutans ribosomal preparations, but not with other S. mutans antigens or with Escherichia coli and Neisseria gonorrhoeae ribosomal preparations, removed the antibody activity. Absorption with Streptococcus sanguis ribosomes and NH4Cl-washed S. mutans ribosomes partially removed the anti-S. mutans ribosome antibody activity. These results provide evidence that naturally occurring salivary and serum antibodies to the S. mutans ribosomal preparation correlate with susceptibility to dental caries.     

Structure and Immunological Specificity of the Streptococcus mutans Group b Cell Wall Antigen

The Streptococcus mutans group b antigen of strain FA1 has been defined as to chemical composition and immunological specificity. The antigen in cold trichloroacetic acid extracts was fractionated on diethylaminoethyl-Sephadex A-25 at pH 8.5. Two forms were isolated: a polysaccharide and a mucoprotein. The two polymers reacted as a single substance in agar gel diffusion against specific adsorbed FA1 rabbit antisera but were separated by gel immunoelectrophoresis. No reaction with any other S. mutans or streptococcal group sera occurred. Galactose composed about one-third and galactosamine about 3% of the total weight of each polymer. Rhamnose was a major component of the polysaccharide (47%) but was present only in traces in the mucoprotein. The protein content of the latter was about 40%. No significant quantities of glycerol, phosphorus, or muramic acid were present in either case. Pepsin and trypsin had no effect on the serological specificity of the mucoprotein. d-Galactose and d-galactosamine were strong inhibitors (70%) of the precipitin reaction, whereas d-glucose, d-glucosamine, and N-acetyl-d-glucosamine inhibited between 25 and 35%. The results indicate that the antigen is a major antigenic component of the cell wall and that the specificity of the antigen resides in binding sites which contain both d-galactose and d-galactosamine. Agglutination of whole cells by specific group b antiserum indicates the antibody receptor sites of the polysaccharide antigen are at the surface of the streptococcal cell. The mucoprotein, but not the polysaccharide, was released from the cell by lysozyme. Lysis did not occur. The immunological specificity and other characteristics of the antigen establishes it as the identifying antigen of S. mutans group b.     

Five Homologous Repeats of the Protein G-Related Protein MIG Cooperate in Binding to Goat Immunoglobulin G

Protein MIG, from Streptococcus dysgalactiae, binds α₂-macroglobulin and immunoglobulin G (IgG). MIG-derived fusion proteins with one to five IgG-binding repeats differed up to 72,000-fold in avidity for goat IgG, indicating a considerable cooperativity of the repeats. Significant sequence variation in the IgG-binding repeats was recognized. Protein MIG interacted with goat IgG1 via both the Fc and Fab parts.     

Chemical Characterization of a Cell Wall Antigen from Streptococcus mutans FA1

The cell wall antigen from Streptococcus mutans (FA1) was extracted from cell walls and whole cells and was found to contain rhamnose, galactose, and glucosamine in a ratio of approximately 5:2:1.     

Mechanism of Adherence of Streptococcus mutans to Smooth Surfaces II. Nature of the Binding Site and the Adsorption of Dextran-Levan Synthetase Enzymes on the Cell-Wall Surface of the Streptococcus

The mechanism of adsorption of the Streptococcus mutans enzymes responsible for the synthesis of insoluble dextran-levan to the S. mutans cell-wall binding sites has been studied. Certain characteristics of these binding sites are presented. The adsorption of these enzymes to the cell surface occurred rapidly without the addition of a source of energy and over a pH range of 3 to 11. The adsorption was inhibited by soluble dextran, probably due to the strong affinity of the polymer to the enzyme. All other polymers and sugars studied showed little or no inhibition. The adsorption was also inhibited by antibody globulin to the a-d immunologically specific group antigen surface polysaccharide of S. mutans and by anti-dextran globulin. The inhibition by anti-a-d globulin is considered to be due to a restriction of access of enzyme to the binding site of the enzyme which may be located in close proximity to the group antigen. On the other hand, anti-dextran globulin appeared to directly inhibit the adsorption by covering the binding site. Dextranase destroyed the binding site and released glucose from the S. mutans cells. These data indicate that S. mutans grown in media containing glucose possesses a small amount of dextran on the cell surface, and that this dextran is, or is a part of, the binding site for enzymes which synthesize the insoluble dextran-levan polymer. Trypsin inhibited the synthesis of insoluble polysaccharide and the adherence of cells. It is not clear in this case that destruction of the binding sites occurred. These data present a partial explanation of the processes which may be concerned in the formation of dental plaque on the smooth surfaces of teeth.     

Identification of Core B Cell Epitope in the Synthetic Peptide Inducing Cross-Inhibiting Antibodies to a Surface Protein Antigen of Streptococcus Mutans

A surface protein antigen (PAc) of Streptococcus mutans, in particular, A—region of the molecule, has been considered as a possible target for the development of an effective anticaries vaccine. This region might be implicated in the induction of dental caries via interaction with salivary components. We have recently specified a unique peptide, TYEAALKQYEADL, as one of the minimum peptides that completely corresponds to the amino acid sequence of a part of the A—region. The unique peptide contains both T and B cell epitopes for the induction of cross—reacting antibodies to the PAc. In this study, we synthesized valine or glycine—substituted peptide analogs of this peptide and examined core B cell epitopes of this unique peptide by using ELISA inhibition assay. As a result, the core amino acid residues of—Y—Y—for B cell recognition were found to likely be not only important amino acids stabilizing the structure, but also might be essential for induction of the cross-inhibiting antibodies against PAc. These results will hopefully provide us with useful information for the design of an effective anticaries peptide vaccine.     

Immunoglobulin A Antibodies Reactive with Streptococcus mutans in Saliva of Adults, Children, and Predentate Infants

Immunoglobulin A (IgA) antibodies reactive with Streptococcus mutans MT3 cells (serotype c) were sought, using an indirect enzyme-linked immunosorbent assay, in the saliva of humans who either harbored or did not harbor detectable levels of this organism. Samples of unstimulated whole saliva from three adults and one child who were infected with S. mutans contained IgA which bound to MT3 cells. Saliva samples of two adults studied also contained IgA which reacted with S. mutans strains of serotypes e, g, a, and b, the latter two of which are rarely isolated from humans. The saliva of three children who did not harbor detectable levels of S. mutans and of three of five predentate infants also contained IgA reactive with MT3 cells. The latter observation is of special interest since S. mutans does not colonize the mouth before eruption of teeth. Thus, the presence of salivary IgA reactive with S. mutans cells is not necessarily related to present or past infection by this organism. Absorption with MT3 cells markedly reduced the reactivity of adult saliva without greatly altering the total concentration of IgA present; this suggests that the IgA was not binding to S. mutans MT3 cells via Fc receptors. The possibility that the antibodies which reacted with S. mutans MT3 may have been induced to other bacteria with cross-reactive antigens was supported by the finding that absorption of saliva with mixed bacterial growth derived from common dairy products significantly reduced its reactivity. Absorption experiments further suggested that a significant portion of the salivary IgA antibodies was binding to glucans on the cell surface.