Identification of antigenic epitopes in a surface protein antigen of Streptococcus mutans in humans.

The reactivities of antibodies in human serum and saliva to a cell surface protein antigen (PAc) of Streptococcus mutans and synthetic peptides covering the PAc molecule were examined. Both an enzyme-linked immunosorbent assay (ELISA) and Western blotting (immunoblotting) showed that all the serum samples from five adult subjects harboring serotype c S. mutans in their oral cavity reacted with recombinant PAc (rPAc). On the other hand, the serum from a 4-month-old infant did not react with rPAc in ELISA. The immunoglobulin A (IgA) antibodies in saliva samples from the five adult subjects reacted with rPAc. However, in saliva samples from these subjects, the titers of IgA antibody to rPAc did not correlate with the titers of serum antibody to the antigen. To map continuous antigenic epitopes in the PAc molecule, we synthesized 153 decapeptides covering the entire mature PAc molecule, 121 overlapping decapeptides covering the alanine-rich repeating region (A-region) of the PAc molecule, and 21 overlapping decapeptides covering the middle region (residues 824 to 853) according to multiple pin-coupled peptide synthesis technology. Of 153 decapeptides covering the mature PAc, 27 decapeptides showed a strong reaction with the antibodies in serum from the adult subjects. The epitope-scanning patterns in the serum samples from these subjects were also very similar to each other. The antigenic epitope patterns in the saliva resembled those in the serum. However, the ELISA titers of salivary IgA antibodies to these decapeptides differed from the titers of the serum antibody. Of the 121 overlapping decapeptides covering the A-region, 27 decapeptides showed a positive reaction with the antibodies in serum from the adult subjects. All of these 27 decapeptides had either one or two of the five common sequences YQAXL, NADAKA, VQKAN, NNAKNA, and IKKRNA. Six decapeptides of the 21 overlapping decapeptides covering the middle region reacted strongly with the serum antibodies from a high PAc responder, and each of the six decapeptides had one of the two common sequences KVTKEKP and VKPTAPTK. These epitopes might therefore be relevant to the humoral responses against the PAc protein during natural infection with S. mutans in humans.     

Identification of antigenic epitopes in an alanine-rich repeating region of a surface protein antigen of Streptococcus mutants.

A surface protein antigen (PAc) of Streptococcus mutans with a molecular mass of 190 kDa is considered to play an important role in the initial attachment of this streptococcus to the tooth surface. Two internal repeating amino acid sequences are present in the PAc molecule. One repeating region located in the N-terminal region is rich in alanine (A-region), and the other, located in the central region, is rich in proline (P-region). To identify antigenic epitopes on the A-region of the PAc protein, 82 sequential overlapping synthetic decapeptides covering one of the repetitive units of the A-region were synthesized. In the epitope scanning analyses using murine antisera raised against recombinant PAc (rPAc), multiple antigenic epitopes were found in the repetitive unit of the A-region, and some of them reacted with antisera to rPAc from BALB/c, B10, B10.D2, and B10.BR mice. In particular, a peptide YEAALKQY (residues 366 to 373) was recognized by anti-rPAc sera from all four strains of mice. The reactivities of anti-rPAc sera in the epitope scanning were confirmed by using a purified synthetic peptide, NAKATYEAALKQYEADLAA (corresponding to residues 361 to 379). Furthermore, antisera against a surface protein antigen PAg (SpaA) of Streptococcus sobrinus from BALB/c mice reacted strongly to residues 330 to 337, 362 to 369, and 366 to 373 of the PAc protein by the epitope scanning analysis. An AKATYEAALKQY (residues 362 to 373 of the PAc protein)-like sequence, AKANYEAKLAQY, was found within the A-region of S. sobrinus PAg, suggesting that the amino acid sequences AKA-YEA and YEA-L-QY may be major cross-reactive epitopes of the S. mutans PAc protein and the S. sobrinus PAg protein.     

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.     

An antigenic peptide inducing cross-reacting antibodies inhibiting the interaction of Streptococcus mutans PAc with human salivary components.

A 190-kDa surface protein antigen (PAc) of Streptococcus mutans, in particular the A region of this molecule, may be implicated in the induction of dental caries via an interaction with salivary components. For this reason, it was probably used successfully as an antigenic component for experimental vaccination to prevent dental caries in animals. While developing a synthetic peptide vaccine for dental caries, as reported herein, we have identified a unique peptide, TYEAALKQYEADL, as a candidate vaccinal immunogen. The amino acid sequence of this peptide completely corresponds to the sequence of a B-cell epitope in the A region of PAc and additionally contains its own T-cell epitope for B10.D2 mice within the molecule. This peptide strongly induces the production of only cross-reacting antibodies against PAc. In addition, as demonstrated by surface plasmon resonance analysis using the BIAcore system, these cross-reacting antibodies inhibit approximately 50% of the binding of fluid-phase salivary components to immobilized recombinant PAc.     

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.     

Contribution of the alanine-rich region of Streptococcus mutans P1 to antigenicity, surface expression, and interaction with the proline-rich repeat domain

Streptococcus mutans is considered to be the major etiologic agent of human dental caries. Attachment of S. mutans to the tooth surface is required for the development of caries and is mediated, in part, by the 185-kDa surface protein variously known as antigen I/II, PAc, and P1. Such proteins are expressed by nearly all species of oral streptococci. Characteristics of P1 include an alanine-rich repeat region and a centrally located proline-rich repeat region. The proline-rich region of P1 has been shown to be important for the translational stability and translocation of P1 through the bacterial membrane. We show here that (i) several anti-P1 monoclonal antibodies require the simultaneous presence of the alanine-rich and proline-rich regions for binding, (ii) the proline-rich region of P1 interacts with the alanine-rich region, (iii) like the proline-rich region, the alanine-rich region is required for the stability and translocation of P1, (iv) both the proline-rich and alanine-rich regions are required for secretion of P1 in Escherichia coli, and (v) in E. coli, P1 is secreted in the absence of SecB.     

A peptide domain of bovine milk lactoferrin inhibits the interaction between streptococcal surface protein antigen and a salivary agglutinin peptide domain

The peptide domain of salivary agglutinin responsible for its interaction with cell surface protein antigen (PAc) of Streptococcus mutans or bovine lactoferrin was found in the same peptide, scavenger receptor cysteine-rich domain peptide 2 (SRCRP2). Inhibition studies suggest that PAc and lactoferrin, of which residues 480 to 492 seem important, competitively bind to the SRCRP2 domain of salivary agglutinin.     

Immunogenicity of peptides coupled with multiple T-cell epitopes of a surface protein antigen of Streptococcus mutans.

A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of the molecule, has been noted as a possible target of effective dental caries vaccine. We have previously shown that two peptides of 19 amino acids (residues 361-379, NAKATYEAALKQYEADLAA, and residues 301-319, ANAANEADYQAKLTAYQTE), which correspond to parts of the A-region, contain both T- and B-cell epitopes for the induction of cross-reacting antibodies to the PAc. In this study, for development of an appropriate antigen as a peptide vaccine for use in prophylactic dentistry, we analysed in detail the localization of the T- and B-cell epitopes of PAc(361-379) peptide and the T-cell epitope of PAc(301-319) peptide in B10 congenic mice. In four murine major histocompatibility complex (MHC) haplotypes (H-2f,d,a and k), PAc(361-377) peptide showed T- and B-cell epitopes forming a cluster. It was found that the antibody which was induced by the immunization with the peptide was strongly cross-reactive with recombinant (r)PAc. Meanwhile, PAc(305-318) peptide, recognised by five strains of mice of different MHC haplotypes (H-2f,d,a,k and s), also bore multiple T-cell epitopes. PAc(361-377) peptide coupled to PAc(305-318) significantly elevated cross-reacting antibody levels compared to immunization with PAc(361-377) only in four H-2 haplotypes. Moreover, a peptide with PAc(305-318) coupled to the N-terminal region of PAc(361-377) produced significant cross-reacting antibody against rPAc, even in B10.S mice which had not responded to immunization with PAc(361-379) peptide. Therefore, it was suggested that coupling among the peptides forming a cluster might be effective in increasing immunogenicity. These results may provide us with a useful strategy for the design of peptide-based vaccines for S. mutans in the future.     

Role of peptide antigen for induction of inhibitory antibodies to Streptococcus mutans in human oral cavity

The alanine-rich repeating region (A-region) in the surface protein antigen (PAc) of Streptococcus mutans has received much attention as an antigenic component for vaccines against dental caries. The PAc (residue 361-386) peptide in the A-region possesses a multiple binding motif (L- -V-K- -A) to various HLA-DR molecules and a B-cell core epitope (- Y- - -L- -Y- - - -) that recognizes the inhibiting antibody to S. mutans. In the present study, we investigated the immunogenicity of the PAc (361-386) peptide in humans and regulators of induction of the anti-PAc (361-386) peptide IgA antibody (aPPA) in saliva. The PAc (361-386) peptide was confirmed as an ideal peptide antigen for induction of the inhibiting antibody to S. mutans in 151 healthy human subjects (36.6 +/- 12.6 years old) by quantitative analyses of oral bacteria and ELISA, as the aPPA titre in human saliva decreased significantly in an age-dependent manner. Homozygous DRB1*0405 and 1502, and heterozygous DRB1*0405/1502 showed a negative association with production of aPPA and tended to reduce the number of total streptococci in saliva. In contrast, the DRB1*1501 allele was significantly correlated with a high level of induction of the antibodies, and also tended to reduce lactobacilli and mutans streptococci. Further, peptide immunogenicity was confirmed in NOD-SCID mice grafted with human peripheral blood mononuclear cells. Our results indicate that the interplay between regulators such as age, DRB1 genotype, cytokines, and peptide immunogenicity may provide a potential means for developing a vaccine useful for the prevention of dental caries as well as their diagnosis.     

Binding of Salivary Glycoprotein-Secretory Immunoglobulin A Complex to the Surface Protein Antigen of Streptococcus mutans

The interaction between a surface protein antigen (PAc) of Streptococcus mutans and human salivary agglutinin was analyzed with a surface plasmon resonance biosensor. The major component sugars of the salivary agglutinin were galactose, fucose, mannose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid. Binding of salivary agglutinin to PAc was calcium dependent and heat labile and required a pH greater than 5. Binding was significantly inhibited by N-acetylneuraminic acid and α2,6-linked sialic acid-specific lectin derived from Sambucus sieboldiana in a dose-dependent manner. Pretreatment of the salivary agglutinin with sialidase reduced the binding activity of the agglutinin to the PAc molecule. The agglutinin was dissociated into high-molecular-mass glycoprotein and secretory immunoglobulin A (sIgA) components by electrophoretic fractionation in the presence of 1% sodium dodecyl sulfate and 1% 2-mercaptoethanol. Neither of the components separated by electrophoretic fractionation, high-molecular-mass glycoprotein or sIgA, bound to the PAc molecule. Furthermore, the high-molecular-mass glycoprotein strongly inhibited the binding of the native salivary complex to PAc. These results suggest that the complex formed by the high-molecular-mass salivary glycoprotein and sIgA is essential for the binding reaction and that the sialic acid residues of the complex play an important role in the interaction between the agglutinin and PAc of S. mutans.

Streptococcus mutans has been implicated as a prime causative organism of dental caries, one of the most common diseases in humans. Colonization of the surface of a tooth by S. mutans is thought to be initiated by attachment of the organism to acquired pellicles on tooth surfaces (21). A 190-kDa surface protein antigen, which has been variously designated as antigen I/II, B, IF, P1, SR, MSL-1, and PAc (37), is known to be one of the factors which mediates the binding of the organism to the tooth surface (3, 21, 23).Various salivary components, such as secretory immunoglobulin A (sIgA) (36), β₂-microglobulin (12), histidine-rich polypeptides (32), a 60-kDa glycoprotein (1), lysozyme (39), lactoferrin (43), and high-molecular-mass glycoproteins (4, 7, 13, 20), have been reported to bind to S. mutans and/or to induce agglutination of the organism. Much attention has also been focused on the interaction between PAc and salivary components (5, 8, 21, 27–29). Russell and Mansson-Rahemtulla (38) reported that antigen I/II binds to several salivary components, including basic proline-rich proteins with molecular weights of 28,000 and 38,000, lysozyme, and α-amylase separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Much remains to be learned about the interaction between native salivary agglutinin and PAc of S. mutans.In this study, using a surface plasmon resonance biosensor, we isolated and characterized a complex of high-molecular-mass salivary glycoprotein and sIgA which binds to PAc. The data presented in this report suggest that the complex of salivary glycoprotein and sIgA is an important component in the interaction between saliva and the PAc protein in a native condition and that sialic acid residues in the complex play a significant role in this interaction.     

Genetic control of immune responses in mice to synthetic peptides of a Streptococcus mutans surface protein antigen.

The immune responses to a cell surface protein antigen (PAc) of Streptococcus mutans and a peptide corresponding to residues 301 to 319 of the protein antigen [PAc(301-319)] in various strains of mice were studied, with attention being given to the haplotype of major histocompatibility complex (MHC) class II genes. Subcutaneous immunization of mice carrying the MHC class II I-Ad gene [BALB/c, B10.D2, B10.GD, and (B10.D2 x B10.G)F1 mice] with the peptide induced strong serum immunoglobulin G (IgG) responses to recombinant PAc (rPAc) and the peptide. Subcutaneous immunization of mice carrying the haplotype k or b of the H-2 I-A gene (C3H/HeN, C57BL/6, B10.BR, B10.A, or B10 mice) with the peptide induced intermediate serum IgG responses to rPAc and the peptide, and subcutaneous immunization of mice carrying the haplotype s or q of the H-2 I-A gene (DBA/1, B10.S, or B10.G mice) induced weak serum IgG responses to rPAc and the peptide compared with the responses of mice carrying the I-Ad gene. PAc(301-319) strongly induced PAc(301-319)-specific T-cell proliferation in B10.D2 mice but not in B10.G mice. The T-cell proliferation in B10.D2 mice was inhibited by treatment of antigen-presenting cells with anti-I-Ad monoclonal antibody but not with anti-I-Ab monoclonal antibody. These results indicate that the immune responses to the peptide in mice are genetically restricted or dominated by the MHC class II gene (I-Ad). To map antigenic epitopes in PAc(301-319) and PAc in mice bearing different H-2 haplotypes, 10 overlapping decapeptides covering PAc(301-319) and 153 decapeptides covering the entire mature PAc were synthesized. Of 10 decapeptides covering PAc(301-319), 6, 7, 1, and 1 decapeptides showed strong reactions with anti-PAc(301-319) sera from B10.D2 (H-2d), B10.GD (H-2g2), B10.BR (H-2k), and B10.A (H-2a) mice, respectively. None of these overlapping decapeptides reacted with anti-PAc(301-319) sera from B10.S (H-2s) and B10.G (H-2q) mice. Epitope-scanning analyses of the mature PAc molecule showed that antigenic epitopes scattered throughout the molecule and that antigenic epitope patterns differed in mice with different H-2 haplotypes. In addition, there was little overlap of immunogenic peptides among the mice with different haplotypes.     

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.     

A protein fragment of streptococcal cell surface antigen I/II which prevents adhesion of Streptococcus mutans.

Attachment of Streptococcus mutans to the tooth surface involves a cell surface protein with an M(r) of 185,000, termed streptococcal antigen (SA) I/II. Four overlapping fragments of the gene encoding SA I/II were amplified by polymerase chain reaction, cloned, and expressed in Escherichia coli. The recombinant polypeptides were assayed for adhesion-binding activity to salivary receptors and for recognition by a panel of monoclonal antibodies (MAbs) raised against SA I/II. Two of the MAbs which are known to prevent colonization of S. mutans in vivo bound the recombinant polypeptide comprising residues 816 to 1161. In vitro adhesion of S. mutans to saliva-coated hydroxyapatite beads was also inhibited specifically by a polypeptide (residues 816 to 1213) encompassing the same region. The evidence from the MAbs preventing colonization of S. mutans and the adherence inhibition assay suggests that an adhesion-binding activity resides within the portion of SA I/II comprising residues 816 to 1213, which is highly conserved among oral streptococcal species.     

Role of the charged tail in localization of a surface protein antigen of Streptococcus mutans.

To make clear the role of the C terminus of a surface protein antigen (PAc) of Streptococcus mutans, stepwise truncations beginning at the C terminus of PAc were performed by utilizing site-directed mutagenesis. A remarkable increase in the amount of cell-free PAc was observed upon deletion of four or more amino acid residues at the C terminus. On the other hand, the amount of cell surface PAc gradually decreased when increasing numbers (four or more) of amino acid residues were deleted at the C terminus, and deletion of six amino acids involving both the total charged tail and Leu, an amino acid residue immediately upstream of the charged tail, resulted in a drastic reduction in the amount of cell surface PAc. These results indicate that the cytoplasmic charged tail and Leu residue are required for cell surface localization of PAc in S. mutans.     

Sequence and structural analysis of surface protein antigen I/II (SpaA) of Streptococcus sobrinus.

Streptococcal antigen I/II or the surface protein antigen A (SpaA) of Streptococcus sobrinus is an adhesin which mediates binding of the organism to tooth surfaces. The complete sequence of the gene which encodes SpaA has been determined. The gene consists of 4,584 bp and encodes a protein of 1,528 amino acid residues. The deduced amino acid sequence shows extensive homology with those of the cell surface adhesins from Streptococcus mutans serotypes c and f and from Streptococcus sanguis. Structural analysis of the N-terminal region (residues 50 to 550), which is rich in alanine and includes four tandem repeats of an 82-residue sequence, suggests that it adopts an alpha-helical coiled-coil conformation. Cell surface hydrophobicity may be associated with this region. The C-terminal region is more conserved and includes two tandem repeats of a 39-residue proline-rich sequence. A further proline-rich sequence in this region is predicted to span the cell wall. Although a hydrophobic sequence is present in the C-terminal region, it appears to be too short to span the cell membrane. Anchoring of SpaA in the cell membrane may therefore require some form of posttranslational modification or association with another membrane protein.     

Oral immunization with recombinant Streptococcus lactis carrying the Streptococcus mutans surface protein antigen gene.

A recombinant Streptococcus lactis strain which carries the structural gene for a surface protein antigen (PAc) of 190,000 daltons from Streptococcus mutans serotype c was constructed for development of an oral vaccine against dental caries. The gene from S. mutans MT8148 joined to shuttle vector pSA3 was successfully transformed into S. lactis IL1403. A small amount of PAc was detected in the cell homogenate and cytoplasmic fraction of the recombinant S. lactis, but not in the culture supernatant of the recombinant, by Western immunoblotting and dot immunoblotting. The level of PAc-specific mRNA in the recombinant strain was lower than that in S. mutans MT8148. However, significant salivary immunoglobulin A and serum immunoglobulin G responses to PAc were induced in mice immunized orally with the recombinant S. lactis.     

Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis.

Fimbriae are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. It has been demonstrated that purified fimbriae bind to whole human saliva adsorbed to hydroxyapatite (HAP) beads, and the binding appears to be mediated by specific protein-protein interactions. Recently, we expressed the recombinant fimbrillin protein (r-Fim) of P. gingivalis corresponding to amino acid residues 10 to 337 of the native fimbrillin (A. Sharma, H.T. Sojar, J.-Y. Lee, and R.J. Genco, Infect. Immun. 61:3570-3573, 1993). We examined the ability of individual salivary components to promote the direct attachment of r-Fim to HAP beads. Purified r-Fim was radiolabeled with 125I and incubated with HAP beads which were coated with saliva or purified individual salivary components. Whole, parotid, and submandibular-sublingual salivas increased the binding of 125I-r-Fim to HAP beads. Submandibular-sublingual saliva was most effective in increasing the binding of 125I-r-Fim to HAP beads (1.8 times greater than that to uncoated HAP beads). The binding of 125I-r-Fim to HAP beads coated with acidic proline-rich protein 1 (PRP1) or statherin was four and two times greater, respectively, than that to uncoated HAP beads. PRP1 and statherin molecules were also found to bind 125I-r-Fim in an overlay assay. The binding of intact P. gingivalis cells to HAP beads coated with PRP1 or statherin was also enhanced, by 5.4 and 4.3 times, respectively, over that to uncoated HAP beads. The interactions of PRP1 and statherin with 125I-r-Fim were not inhibited by the addition of carbohydrates or amino acids. PRP1 and statherin in solution did not show inhibitory activity on 125I-r-Fim binding to HAP beads coated with PRP1 or statherin. These results suggest that P. gingivalis fimbriae bind strongly through protein-protein interactions to acidic proline-rich protein and statherin molecules which coat surfaces.     

Conservation of salivary glycoprotein-interacting and human immunoglobulin G-cross-reactive domains of antigen I/II in oral streptococci.

In this study we localized more precisely the salivary glycoprotein-interacting and the human immunoglobulin G (hIgG)-cross-reacting domains on the SR molecule, an antigen I/II-related protein from S. mutans serotype f. Mapping of the SR molecule with polypeptides expressed by subclones covering the entire molecule and with synthetic peptides demonstrates that the salivary glycoprotein-binding domain is located in the N-terminal alanine-rich repeats of the SR molecule. In order to investigate the degree of conservation of both regions in various oral streptococci, we tested the reactivity of 8 representative strains of the mutans group and 11 nonmutans oral Streptococcus strains (S. anginosus, S. milleri, S. constellatus, S. intermedius, S. mitis, S. sanguis, S. gordonii, S. salivarius, and S. mitis strains) with antipeptide antibodies in a whole-cell enzyme linked immunosorbent assay together with colony hybridization analysis using DNA probes designed to map these two regions. All the mutans group strains except S. rattus and the 11 nonmutans streptococcal strains showed a high conservation of the C-terminal part of the SR molecule, especially the hIgG-cross-reacting domain, and less homology for the N-terminal salivary glycoprotein-binding region. Almost all of the sera from patients with rheumatic disease reacted strongly with SR from S. mutans serotype f, P1 from S. mutans serotype c, and four peptides located in the hIgG-cross-reacting region and not with peptides located at the C and N termini and in the proline-rich repeats. These results confirm that epitopes located within this region are immunogenic in humans and could lead to the synthesis of natural anti-IgG antibodies.     

Identification of a salivary agglutinin-binding domain within cell surface adhesin P1 of Streptococcus mutans.

DNA encoding the alanine-rich region (A-region) of the cell surface adhesin, P1, from Streptococcus mutans was subcloned and expressed as a fusion protein with the maltose-binding protein (MBP) of Escherichia coli. The A-region fusion protein was shown to competitively inhibit both adherence of S. mutans to salivary agglutinin-coated hydroxyapatite and fluid-phase agglutinin-mediated aggregation of this organism. MBP alone or an MBP-paramyosin fusion protein was not inhibitory. Proteolytic cleavage of the fusion protein into its component moieties, MBP and A-region, resulted in breakdown of the A-region into three main fragments. Western immunoblot analysis of calcium-dependent agglutinin binding to this preparation revealed binding specificity for a 28-kDa fragment. Thus, the A-region of P1 is an important domain which interacts directly with salivary agglutinin, and this interaction interferes with both the aggregation and the adherence mechanisms in vitro.     

Effects of antibodies against cell surface protein antigen PAc-glucosyltransferase fusion proteins on glucan synthesis and cell adhesion of Streptococcus mutans.

Cell surface protein antigen (PAc) and glucosyltransferases (GTFs) produced by Streptococcus mutans are considered to be major colonization factors of the organism, and the inhibition of these two factors is predicted to provide protection against dental caries. In this study, we have constructed fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc with the glucan binding (GB) domain of GTF-I, an enzyme catalyzing the synthesis of water-insoluble glucan from sucrose, and fusion protein PAcA-SB, a fusion of PAcA with the sucrose binding (SB) domain of GTF-I. The recombinant fusion proteins were purified from cell extracts of Escherichia coli harboring the fusion genes, and rabbit antibodies against these fusion proteins were prepared. Water-insoluble glucan synthesis by cell-associated and cell-free GTF preparations from S. mutans as well as total glucan synthesis by GTF-I was markedly inhibited by anti-PAcA-GB immunoglobulin G (IgG) antibodies but not by anti-PAcA-SB IgG antibodies. Significant inhibition of the sucrose-independent and sucrose-dependent adhesion of S. mutans to saliva-coated hydroxyapatite beads was observed when anti-PAcA-GB antibodies were added to the reaction mixture. Anti-PAcA-SB antibodies inhibited the adhesion of S. mutans to the beads in the absence of sucrose but not in the presence of sucrose. Immunization with the fusion protein PAcA-GB may be useful for controlling the colonization of teeth by S. mutans.