Toshihiko Koga

Molecular characterization of a surface protein antigen gene from serotype c Streptococcus mutans, implicated in dental caries

The complete nucleotide sequence of the gene for a cell‐surface protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was determined. The pac gene consisted of 4695 bp and coded for a 170 773 D protein. The pac gene product contained a putative 38 amino acid signal peptide, resulting in a 166817D mature protein. A potential promoter sequence and a putative Shine‐Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in the PAc. One repeating region located in the N‐terminal region was rich in alanine, and the other located in the central region was rich in proline. Southern blot analysis under the less stringent condition (allowing up to 35% base mismatch) revealed that the probe covering the prolinerich region hybridized to DNA preparations from strains of Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei as well as Streptococcus mutans.

Cloning of a surface protein antigen gene from serotype c Streptococcus mutans

The structural gene for a 190kD protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was cloned into the plasmid vector pUC118. SDS‐polyacrylamide gel electrophoresis and Western immunoblotting showed that the Escherichia coli harbouring the chimaeric plasmid produced multiple polypeptides of 190‐210kD. Immunodiffusion analysis revealed that the cloned PAc had the same specific determinants as S. mutans PAc. The cloned pac gene was mapped, and its transcriptional orientation was determined by characterizing deletion mutants of the chimaeric plasmid. Southern blot analysis with the cloned gene sequence as a probe revealed the presence of a homologous sequence in DNAs from sero‐types e and f S. mutans. PAc‐defective mutants were constructed by inserting an erythromycin‐resistance gene into the pac gene. The cell‐surface hydrophobicity of the mutants was lower than that of the parent strain.

Effect of subculturing on expression of a cell-surface protein antigen by Streptococcus mutans

Two freshly isolated strains, Xc and Yc, of Streptococcus mutans serotype c from human dental plaque were subcultured 100 times in Brain Heart Infusion broth. The cell-surface hydrophobicity of strain Xc markedly decreased after subculturing 60 times, but that of strain Yc remained unaltered. Radioimmunoassay showed a close correlation between surface hydrophobicity and the amount of a cell-surface protein antigen (PAc) of Mr 190,000. One hydrophilic variant (strain Xc100L), one relatively hydrophobic variant (strain Xc100H), and two hydrophobic variants (strains Yc100H1 and Yc100H2) were isolated from the 100-fold subcultures of hydrophobic strains Xc and Yc, respectively. SDS-PAGE showed that the amount of cell-associated and cell-free PAc of strain Xc100L was smaller than that of strains Xc and Xc100H. Strain Yc100H2 produced larger amounts of cell-associated PAc than strains Yc and Yc100H1. Resting cells of hydrophilic strain Xc100L attached in smaller numbers to saliva-coated hydroxyapatite than did other hydrophobic strains. RNA dot-blot analysis demonstrated a significant decrease in PAc-specific mRNA in strain Xc100L, as compared with strains Xc and Xc100H. Neither rearrangement nor deletion in the structural gene (pac) for PAc of these strains was observed by Southern blot analysis. These findings suggest that a mechanism which regulates the transcription of the pac gene participates in the quantitative variation of PAc after repeated subculturing.

Characterization of a Cell-surface Protein Antigen of Hydrophilic Streptococcus mutans Strain GS-5

Fourteen strains of Streptococcus mutans serotype c were examined for their cell-surface protein antigens in terms of hydrophobicity, Mr and immunochemical specificities. Thirteen strains were hydrophobic, while strain GS-5 was markedly hydrophilic as compared to the other strains tested. Cell-surface protein antigens were then analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting. A protein antigen of Mr 190,000 (PAc) was found in cell extracts and culture supernatants of all the hydrophobic strains. Neither culture supernatant nor cell extract of strain GS-5 contained PAc. Strain GS-5, however, produced extracellularly a large amount of a protein of Mr 155,000 (PAGS-5) which reacted with rabbit anti-PAc serum. Immunodiffusion analysis showed that PAGS-5 lacked a part of the antigenic moieties in the PAc molecule. SDS-PAGE and radioimmunoassay showed a small amount of PAGS-5 on the cell surface of strain GS-5. These findings suggest that PAGS-5 may correspond to PAc which lacks a region participating in binding of PAc to the streptococcal cell.

Homology between surface protein antigen genes of Streptococcus sobrinus and Streptococcus mutans

The structural gene (pag gene) for a 210 kDa protein antigen of Streptococcus sobrinus serotype g was cloned and compared with that (pac gene) of a 190 kDa protein antigen of Streptococcus mutans serotype c. Immunodiffusion analysis revealed that the product of the pag gene immunologically cross‐reacted with that of the pac gene. Southern blot and nucleotide sequence analyses revealed that a significant homology existed between the middle regions of the two structural genes.

Electron-microscopic observation of adherence of serotype c Streptococcus mutans to the enamel surface due to glucan synthesis

Cellular adherence of three strains with water-insoluble glucan (IG)-synthesizing ability and a strain lacking the ability of serotype c Streptococcus mutans to the saliva-coated human enamel surface was examined by scanning (SEM) and transmission (TEM) electron-microscopy. SEM revealed that organisms of all strains used adhered directly to the enamel surface in the absence of sucrose. Cell-to-cell attachment was scarcely observed in the absence of sucrose. Cell-to-cell attachment via amorphous substance on the cell surface was observed by SEM when the strains with IG-synthesizing ability were incubated with the saliva-coated enamel in the presence of sucrose. TEM revealed that cell-associated enzymes of these strains synthesized filamentous and double-stranded fibrillar structures from sucrose. The strain lacking IG-synthesizing ability was unable to induce cell-to-cell attachment in the presence of sucrose, nor was it able to synthesize the amorphous substance. These results indicate that production of IG by cell-associated glucosyltransferase participates in cellular accumulation of serotype c S. mutans.