Identification of the binding domain of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase for Porphyromonas gingivalis major fimbriae

Abstract

Porphyromonas gingivalis forms communities with antecedent oral biofilm constituent streptococci. P. gingivalis major fimbriae bind to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) present on the streptococcal surface, and this interaction plays an important role in P. gingivalis colonization. This study identified the binding domain of Streptococcus oralis GAPDH for P. gingivalis fimbriae. S. oralis recombinant GAPDH (rGAPDH) was digested with lysyl endopeptidase. Cleaved fragments of rGAPDH were applied to a reverse-phase high-pressure liquid chromatograph equipped with a C18 column. Each peak was collected; the binding activity toward P. gingivalis recombinant fimbrillin (rFimA) was analyzed with a biomolecular interaction analysis system. The fragment displaying the strongest binding activity was further digested with various proteinases, after which the binding activity of each fragment was measured. The amino acid sequence of each fragment was determined by direct sequencing, mass spectrometric analysis, and amino acid analysis. Amino acid residues 166 to 183 of S. oralis GAPDH exhibited the strongest binding activity toward rFimA; confocal laser scanning microscopy revealed that the synthetic peptide corresponding to amino acid residues 166 to 183 of S. oralis GAPDH (pep166-183, DNFGVVEGLMTTIHAYTG) inhibits S. oralis-P. gingivalis biofilm formation in a dose-dependent manner. Moreover, pep166-183 inhibited interbacterial biofilm formation by several oral streptococci and P. gingivalis strains with different types of FimA. These results indicate that the binding domain of S. oralis GAPDH for P. gingivalis fimbriae exists within the region encompassing amino acid residues 166 to 183 of GAPDH and that pep166-183 may be a potent inhibitor of P. gingivalis colonization in the oral cavity.

FIG. 1.

(A) Results of SDS-PAGE analysis and Western blot assay of purified S. oralis ATCC 9811 rGAPDH. The sample was subjected to SDS-PAGE (5 to 15% gel) and electrotransferred onto a nitrocellulose membrane. After being blocked with Block Ace, the membrane was incubated with penta-His conjugate. Bound antibodies were visualized by employing an HRP conjugate substrate kit. (Left) SDS-PAGE gel with CBB staining; (right) Western blot. Lanes: 1, molecular mass standard proteins; 2, S. oralis ATCC 9811 rGAPDH. (B) HPLC profile and binding activity of lysyl endopeptidase-digested S. oralis rGAPDH with P. gingivalis rFimA. S. oralis rGAPDH was digested with lysyl endopeptidase and subjected to HPLC involving a Symmetry300 C₁₈ column. The elution of rGAPDH fragments was effected with a linear gradient of 0 to 60% acetonitrile at a flow rate of 1 ml/min. Fractionated fragments were collected manually by monitoring the absorbance at 210 nm. The binding activity of each peak toward P. gingivalis rFimA was measured by a BIAcore apparatus. 1, absorbance peak eluted at 41.5 min; 2, absorbance peak eluted at 46 min; 3, absorbance peak eluted at 47.5 min.

FIG. 2.

Inhibitory effects of pep166-183 on biofilm formation by P. gingivalis ATCC 33277 with various streptococci as determined by CLSM. HI-stained streptococci (5 × 10⁷ CFU/well; red) were inoculated into individual chambers coated with human whole saliva and cultured anaerobically at 37°C for 16 h. FITC-stained P. gingivalis ATCC 33277 (5 × 10⁶ CFU/well; green) was added to the wells in which streptococcal biofilm was observed. The mixtures were incubated anaerobically at 37°C for 24 h and analyzed by CLSM. In experiments evaluating the inhibition of biofilm formation, P. gingivalis ATCC 33277 was preincubated aerobically with pep166-183 or the control peptide at room temperature for 30 min. Magnification, ×40. The following streptococci were tested: S. oralis ATCC 9811 (A), S. oralis ATCC 10557 (B), S. gordonii G9B (C), S. sanguinis ATCC 10556 (D), and S. parasanguinis ATCC 15909 (E). Representative photographs are shown. In each panel, the upper section displays the monospecies streptococcal biofilm, and the lower section displays the biofilm formed by various streptococci and P. gingivalis ATCC 33277. Data are the means and standard errors of results for six fields in CLSM images. *, P < 0.001 for comparison to results obtained for the control peptide; **, volume of streptococci.

FIG. 3.

Inhibitory effects of pep166-183 on biofilm formation by S. oralis ATCC 9811 and P. gingivalis strains with different types of fimbriae as determined by CLSM. HI-stained S. oralis ATCC 9811 (5 × 10⁷ CFU/well; red) was inoculated into individual chambers coated with human whole saliva and cultured anaerobically at 37°C for 16 h. FITC-stained P. gingivalis OMZ 314 cells (A) and 6/26 cells (B) (5 × 10⁶ CFU/well; green) were added to the wells with S. oralis ATCC 9811 biofilm. The mixtures were incubated anaerobically at 37°C for 24 h and analyzed by CLSM. In experiments evaluating the inhibition of biofilm formation, P. gingivalis was preincubated with pep166-183 or control peptide aerobically at room temperature for 30 min. In each panel, the upper section displays the S. oralis ATCC 9811 monospecies biofilm and the lower section exhibits the biofilm formed by S. oralis ATCC 9811 and P. gingivalis. Magnification, ×40. Representative photographs are shown. Data are the means and standard errors of results for six fields in CLSM images. *, P < 0.001 for comparison to results obtained for the control peptide; **, volume of streptococci.