High resolution structure analysis of Porphyromonas gingivalis membrane proteins in the biofilm.

本研究的目的是鉴定和表征与口腔链球菌GAPDH相互作用的新的牙龈卟啉单胞菌成分。采用下拉法检测牙龈假单胞菌客户蛋白与口腔假单胞菌重组GAPDH之间的潜在相互作用。通过2d凝胶电泳对蛋白进行分离，得到5种蛋白；RagA4、AbfD、GAPDH、GDH和MDH采用蛋白质组学串联质谱法进行鉴定。通过生物分子相互作用分析系统分析龈假单胞菌客户蛋白与口腔假单胞菌GAPDH的相互作用。口腔链球菌GAPDH与5种客户蛋白有较高的亲和力。采用比浊法和荧光显微镜检测牙龈假单胞菌与口腔假单胞菌的共聚集性。重组RagA4、AbfD和GDH增强了共聚集，而重组GAPDH和MDH以剂量依赖性方式抑制了共聚集。此外，重组RagA4、AbfD和GDH调节了luxS在牙龈假单胞菌中的表达。这些结果表明，牙龈假单胞菌的5种客户膜蛋白可能是口腔链球菌与牙龈假单胞菌形成生物膜的调节因子。

The purpose of this study is to identify and characterize new Porphyromonas gingivalis components that interact with Streptococcus oralis GAPDH. Pull-down assay was performed to detect potential interactions between P. gingivalis client proteins and S. oralis recombinant GAPDH. The proteins were separated by 2D-gel electrophoresis, and the five proteins ; RagA4, AbfD, GAPDH, GDH and MDH were identified by a proteomic-tandem mass spectrometry. Interaction between these P. gingivalis client proteins and S. oralis GAPDH was analyzed via a biomolecular interaction analysis system. S. oralis GAPDH demonstrated high affinity with five client proteins. Coaggregation between P. gingivalis and S. oralis was measured by turbidimetric method and fluorescence microscope. Recombinant RagA4, AbfD and GDH enhanced the coaggregation, whereas recombinant GAPDH and MDH inhibited in a dose-dependent manner. Furthermore, recombinant RagA4, AbfD and GDH regulated expression of luxS in P. gingivalis. These results indicate that five client membrane proteins of P. gingivalis might be regulators in P. gingivalis biofilm formation with oral streptococci.