Tosiki Nisizawa

Oral Streptococci Exhibit Diverse Susceptibility to Human β-Defensin-2: Antimicrobial Effects of hBD-2 on Oral Streptococci

We examined the antimicrobial effects of human β-defensin-2 (hBD-2) on 17 species of oral streptococci to investigate the involvement of antimicrobial peptide activity in oral microflora development and the clinical use of the antimicrobial peptide for oral microflora control. Oral streptococci exhibit diverse levels of susceptibility to human β-defensin-2 (hBD-2). Two major cariogenic bacterial species, Streptococcus mutans (S. mutans) and S. sobrinus, were found to be susceptible to the peptide, indicating that it is a potential therapeutic agent for preventing dental caries. S. mitis exhibited the lowest susceptibility to the peptide. S. mitis is a major indigenous bacterium in the oral microflora, and our results suggest that it might possess a certain resistance mechanism against hBD-2.

RGD motif enhances immunogenicity and adjuvanicity of peptide antigens following intranasal immunization

The use of peptides for various aspects of medical science has been a significant advance. Peptide-based vaccines are promising, but weak immunogenic potency is impeding the clinical application. We have remarkably enhanced the immunogenicity of peptide antigens by addition of motifs that bind to cell attachment proteins, such as arginine-glysine-aspartate (RGD), to the amino acid sequence. The modified peptides induced antigen-specific serum antibodies by intranasal immunization without adjuvants. RGD, an integrin-binding motif was the strongest, among several molecules tested in this experiment, giving an average of 10 times enhancement of antibody titers when incorporated into several peptide antigens. The peptides also acted as an efficient adjuvant following the intranasal immunization with protein antigens. Our data support the feasibility of developing peptide vaccines and peptide adjuvants for intranasal vaccination.

Purification and Characterization of Glucosyltransferases from Streptococcus mutans 6715

A water-soluble glucan-synthesizing glucosyltransferase (GTase-S) and a water-insoluble glucan-synthesizing glucosyltransferase (GTase-I) were purified from culture supernatant of Streptococcus mutans 6715 (serotype g) by ammonium sulphate precipitation, chromatofocusing on a Polybuffer exchanger PBE 94 column, and subsequent phenyl-Sepharose CL-4B or hydroxyapatite column chromatography. The GTase-S and GTase-I activities were purified 4019- and 4714-fold, respectively, and the molecular weights were calculated to be 160000 and 165000, respectively. GTase-S had a pH optimum of 5.0, a Km of 8.8 mM for sucrose in the presence of 20 microM-dextran T10, and an isoelectric point of pH 4.3. GTase-I had two pH optima of 5.0 and 7.0, Km values of 4.9 mM (at pH 5.0) and 7.0 mM (at pH 7.0), mM (at pH 7.0), and an isoelectric point of pH 4.9. Methylation analysis indicated that the water-soluble glucan produced by GTase-S was a highly branched 1,6-alpha-linked D-glucan with 1,3-linked glucose residues, and that the water-insoluble glucan synthesized by GTase-I was composed of 1,3-alpha-linked glucose units.

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.

Difference in mode of inhibition between α-d-xylosyl β-d-fructoside and α-isomaltosyl β-d-fructoside in synthesis of glucan by Streptococcus mutansd-glucosyltransferase

Abstract Both α-isomaltosyl β- d -fructoside and α- d -xylosyl β- d -fructoside show strong inhibition of the synthesis of water-insoluble and water-soluble d -glucans from sucrose by a partially purified preparation of a d -glucosyltransferase (GTase) from Streptococcus mutans 6715; however, the inhibitory modes differ substantially. In the presence of α-isomaltosyl β- d -fructoside, the production of reducing sugars and the consumption of sucrose are remarkably enhanced, compared with a control of sucrose alone. Under these conditions, a large proportion of low-molecular-weight glycan (lmwg) and a series of nonreducing oligosaccharides (both containing d -fructosyl groups or residues) are produced. In contrast, in the presence of α- d -xylosyl β- d -fructoside, the production of reducing sugars and the sucrose consumption are strikingly suppressed, and no lmwg or oligosaccharides are produced. Thus, it may be concluded that α-isomaltosyl β- d -fructoside acts as an alternative acceptor for the d -glucosyl and/or d -glucanosyl transfer reactions of the enzyme, and serves to lessen the formation of insoluble and soluble d -glucan, although it stimulates the transferring activity of the enzyme. On the other hand, α- d -xylosyl β- d -fructoside competitively inhibits the sucrose-splitting activity of the enzyme as an analog to sucrose, and thereby diminishes the synthesis of d -glucan.

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.

INHIBITORY EFFECT OF A SELF-DERIVED PEPTIDE ON GLUCOSYLTRANSFERASE OF STREPTOCOCCUS MUTANS : POSSIBLE NOVEL ANTICARIES MEASURES

Glucosyltransferase (GTF) plays an important role in the development of dental caries. We examined the possible presence of self-inhibitory segments within the enzyme molecule for the purpose of developing anticaries measures through GTF inhibition. Twenty-two synthetic peptides derived from various regions presumably responsible for insoluble-glucan synthesis were studied with respect to their effects on catalytic activity. One of them, which is identical in amino acid sequence to residues 1176–1194, significantly and specifically inhibited both sucrose hydrolysis and glucosyl transfer to glucan by GTF-I. Double-reciprocal analysis revealed that the inhibition is noncompetitive. Scramble peptides, composed of the identical amino acids in randomized sequence, had no effect on GTF-I activity. Furthermore, the peptide is tightly bound to the enzyme once complexed, even in the presence of sodium dodecyl sulfate (SDS). Kinetic analysis using an optical evanescent resonant mirror cuvette system demonstrated that the enzyme-peptide interaction was biphasic. These results indicate that the peptide directly interacts with the enzyme with high affinity and inhibits its activity in a sequence-specific manner. This peptide itself could possibly be an effective agent for prevention of dental caries, although its effectiveness may be improved by further modification.

Ability of Various Oral Bacteria to Bind Human Plasma Fibronectin

The present study describes the ability of various oral bacteria to bind human plasma fibronectin (PFN). Avid binding of 125I‐PFN was found for Streptococcus mutans (serotypes a to h), Streptococcus sanguis, group A Streptococcus pyogenes and Staphylococcus aureus, while other gram‐positive bacterial species tested demonstrated only weak or negligible PFN binding ability. Two gram‐negative bacterial species, Bacteroides gingivalis and Escherichia coli, did not significantly bind PFN. 125I‐PFN binding to S. mutans 6715 cells was decreased by pretreatment with unlabeled PFN, and the radiolabeled PFN bound to the cell surface was released on addition of unlabeled PFN. Strong inhibition of 125I‐PFN binding to S. mutans 6715 cells was obtained by protease pretreatment, while partial inhibition was also observed following treatment with acid, alkali, lipase, and monoclonal anti‐polyglycerophosphate. These results suggest that PFN binding to S. mutans cells is reversible and that PFN receptors on the cell surface appear to be heat‐stable multiple proteins.

A novel method for enhancement of peptide vaccination utilizing T-cell epitopes from conventional vaccines

Peptide vaccines have two fundamental weak points, namely low antigenicity and MHC-restriction. In our previous study, we proposed the design of vaccine peptide to overcome these weakpoints. The vaccine was constructed in the following order, N-terminal, Arg-Gly-Asp (RGD), T-cell epitope peptide, di-lysine linker (KK) to B-cell epitope peptide. Although the vaccine peptide can basically induce B-cell epitope peptide specific antibodies to the host without immune adjuvants via intraperitoneal, subcutaneous and intranasal administration, some peptide antigens require adjuvants for antibody induction. In this study, we propose a novel protocol to enhance the immunogenicity of the peptide utilizing the host immune response to a conventional toxoid vaccine, which are lymphocyte activities to the T-cell epitope peptide. We selected multiagretope-type T-cell epitopes from diphtheria toxoid, a conventional vaccine antigen, and a part of amyloid-beta peptide (Aβ) as a B-cell epitope. The conventional toxoid vaccine was immunized before the peptide immunization. Using this protocol, we succeeded in the enhancement of the anti-Aβ antibodies induction by intranasal immunization without any immune adjuvants in C57BL/6 and Balb/c mice. Furthermore, the vaccine peptide induced the transformation of peripheral blood lymphocytes collected from healthy volunteers carrying immunities to diphtheria toxoid. These results suggested that our peptide vaccines with the novel protocol would provide an effective method for antibody induction.