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Infection and Immunity, November 2009, p. 5130-5138, Vol. 77, No. 11
0019-9567/09/$08.00+0     doi:10.1128/IAI.00439-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of the Binding Domain of Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase for Porphyromonas gingivalis Major Fimbriae{triangledown}

Hideki Nagata,1* Mio Iwasaki,1 Kazuhiko Maeda,1 Masae Kuboniwa,1 Ei Hashino,1 Masahiro Toe,1 Naoto Minamino,2 Hiromiki Kuwahara,2 and Satoshi Shizukuishi1

Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, Osaka 565-0871,1 Department of Pharmacology, National Cardiovascular Center Research Institute, Osaka 565-8565, Japan2

Received 20 April 2009/ Returned for modification 18 June 2009/ Accepted 1 September 2009

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.

* Corresponding author. Mailing address: Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-2922. Fax: 81-6-6879-2925. E-mail: nagatah{at}dent.osaka-u.ac.jp

{triangledown} Published ahead of print on 8 September 2009.

Editor: V. J. DiRita

Infection and Immunity, November 2009, p. 5130-5138, Vol. 77, No. 11
0019-9567/09/$08.00+0     doi:10.1128/IAI.00439-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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