Identification of Porphyromonas gingivalis client proteins with Streptococcus oralis GAPDH

Objective: Coaggregation between Porphyromonas gingivalis and oral streptococci is thought to play an important role in P. gingivalis colonization. Previously, we reported that P. gingivalis major fimbriae interacted with Streptococus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Infect Immun 72: 1341-8, 2004). The purpose of this study is to identify new P. gingivalis client components except fimbriae that interact with S. oralis GAPDH. Methods: Pull-down assay was performed to detect potential interactions between His-tagged recombinant GAPDH of S. oralis (rGAPDH) and P. gingivalis client proteins. P. gingivalis ATCC 33277 was cultured in TSB media with rGAPDH, and harvested by centrifugation. After washed, the pellets were lysed and mechanically broken by mild ultrasonication. After centrifugation at 10,000×g for 30 min to remove unbroken cells, the supernatant was applied to a Ni-NTA column. After washed with 10 mM Tris-HCl buffer, the components that bound to S. oralis rGAPDH were eluted with 10 mM Tris-HCl buffer containing 1 M sodium chloride (pH 7.5). The eluted proteins were separated by 2D -gel electrophoresis. Spots of interest were excised from duplicate gels, and digested with trypsin. A proteomic-mass spectrometry approach was adopted to identify the proteins. Results: Seven spots were separated by 2D-gel electrophoresis. Amino acid sequencing by tandem mass spectrometry revealed that the spots were identical to RagA4, arginine-specific proteinase B, 4-hydroxybutyryl-CoA dehydratase, lysine-specific proteinase, GAPDH, NAD-dependent glutamate dehydrogenase and malate dehydrogenase. Conclusion: This study indicates that seven client membrane proteins of P. gingivalis except fimbriae bound to S. oralis GAPDH and might play a role in P. gingivalis colonization.