In the course of developing a synthetic peptide vaccine for dental caries, we identified a unique 13-mer peptide named PAc(365-377), TYEAALKQYEADL, as a minimum peptide inducing cross-inhibiting antibodies to a cell surface protein antigen (PAc) of Streptococcus mutans. However, the peptide could hardly induce the production of antibody in the absence of adjuvant. Thus using this peptide as a unit peptide, tandem constructs of dimeric unit peptide with or without spacer amino acid residues were synthesized, and their antigenicities were examined in B10.D2 mice. Significant augmentation of antigenicity was obtained in all of the dimeric unit peptides with spacers, especially for lysine spacers. In addition, analysis for cross-reactivity of anti-construct antibodies against a set of double valine-substituted analogues of the unit peptide revealed that the di-lysine spacer might be more effective in inducing the cross-reacting antibodies to rPAc.
A cell surface protein antigen (PAc) of Streptococcus mutans may be involved in the binding of bacteria to the tooth surface, and has long been focused upon as a candidate for a preventive vaccine of dental caries. Previously the peptide PAc (365-377) was shown to raise an antibody in B10.D2 mice which inhibited the binding of salivary components to the PAc molecule. Using this peptide as a unit peptide, two constructs based on multiple antigenic peptides, and several types of tandem repeats of two or three copies were synthesized to estimate the immunogenicity of these peptides. Increase in the immunogenicity was observed for all constructs with the use of an adjuvant compared to the unit peptide alone. However, the tandem repeat constructs generally induced antibody production in the absence of adjuvant, while the multiple antigenic peptide constructs did not induce antibody production under the same condition. Although such a phenomenon may be restricted to this particular peptide sequence, these results may influence the strategy for the design of peptide vaccines.
BackgroundPorphyromonas gulae are black-pigmented anaerobic bacteria isolated from the gingival sulcus of various animal hosts and are distinct from Porphyromonas gingivalis originating in humans. We previously reported the antigenic similarities of 41-kDa fimbriae between P. gulae ATCC 51700 and P. gingivalis ATCC 33277. In this study, to clarify the presence of another type of fimbriae of P. gulae, we have purified and characterized the secondary fimbrial protein from P. gulae ATCC 51700.MethodsThe secondary fimbrial protein was purified from P. gulae ATCC 51700 using an immunoaffinity column coupling with antibodies against the 41-kDa fimbrial protein. The expression of fimbriae on the cell surface of P. gulae ATCC 51700 was investigated by transmission electron microscopy. The N-terminal amino acid sequence was determined by an amino acid sequencer system.ResultsThe molecular mass of this protein was approximately 53-kDa, as estimated by SDS-PAGE. The polyclonal antibodies against the 53-kDa protein did not react with the 41-kDa fimbrial protein of P. gulae ATCC 51700. Immunogold electron microscopy revealed that anti-53-kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. The amino acid sequence of the N-terminal 15 residues of the 53-kDa fimbrial protein showed only 1 of 15 residues identical to the 41-kDa fimbrial protein.ConclusionThe 53-kDa fimbriae are different in molecular weight and antigenicity from the 41-kDa fimbrial protein of P. gulae ATCC 51700. These results clearly suggest that the 41-kDa and the 53-kDa fimbriae are distinct types of fimbriae expressed simultaneously by this organism.
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