Identification and Characterization of Porphyromonas gingivalis Client Proteins That Bind to Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase
Identification and Characterization of Porphyromonas gingivalis Client Proteins That Bind to Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase
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DOI：
10.1128/iai.00875-12
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发表时间：
2013-03-01
影响因子：
3.1
通讯作者：
Amano, Atsuo
中科院分区：
文献类型：
--
作者：
Maeda, Kazuhiko;Nagata, Hideki;Amano, Atsuo
牙龈卟啉单胞菌和口腔链球菌的共聚集被认为在牙龈卟啉单胞菌定植中发挥重要作用。此前，我们报道了牙龈卟啉单胞菌菌毛与口腔链球菌甘油醛-3-磷酸脱氢酶（GAPDH）相互作用，并且GAPDH的氨基酸残基166至183对牙龈卟啉单胞菌菌毛表现出强结合活性（H. Nagata，M. Iwasaki，K. Maeda，M. Kuboniwa，E. Hashino，M. Toe，N. Minamino，H. Kuwahara，和 S. Shizukuishi，《感染》77：5130 - 5138，2009）。本研究旨在鉴定和表征除菌毛外与口链球菌 GAPDH 相互作用的牙龈卟啉单胞菌成分。进行pulldown测定以检测牙龈卟啉单胞菌客户蛋白与通过定点诱变删除氨基酸残基166至183的口链球菌重组GAPDH之间的潜在相互作用。通过二维凝胶电泳和串联质谱进行蛋白质组学分析，鉴定了 7 种蛋白质，即 tonB 依赖性受体蛋白 (RagA4)、精氨酸特异性蛋白酶 B、4-羟基丁酰辅酶 A 脱水酶 (AbfD)、赖氨酸特异性蛋白酶、GAPDH、NAD 依赖性谷氨酸脱氢酶 (GDH) 和苹果酸脱氢酶 (MDH)。光谱测定法。使用生物分子相互作用分析系统分析了这些客户蛋白与口链球菌 GAPDH 之间的相互作用。口链球菌 GAPDH 对七种客户蛋白中的五种（RagA4、AbfD、GAPDH、GDH 和 MDH）表现出高亲和力。通过比浊法和荧光显微镜测量牙龈卟啉单胞菌和口腔链球菌之间的相互作用。 RagA4、AbfD 和 GDH 增强共聚，而 GAPDH 和 MDH 抑制共聚。此外，牙龈卟啉单胞菌中luxS的表达被RagA4、AbfD和GDH上调，但被MDH下调。这些结果表明，五种牙龈卟啉单胞菌客户蛋白在口腔链球菌的牙龈卟啉单胞菌生物膜形成中起到调节剂的作用。
Coaggregation of 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 Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and that amino acid residues 166 to 183 of GAPDH exhibited strong binding activity toward P. gingivalis fimbriae (H. Nagata, M. Iwasaki, K. Maeda, M. Kuboniwa, E. Hashino, M. Toe, N. Minamino, H. Kuwahara, and S. Shizukuishi, Infect. Immun. 77: 5130 - 5138, 2009). The present study aimed to identify and characterize P. gingivalis components other than fimbriae that interact with S. oralis GAPDH. A pulldown assay was performed to detect potential interactions between P. gingivalis client proteins and S. oralis recombinant GAPDH with amino acid residues 166 to 183 deleted by site-directed mutagenesis. Seven proteins, namely, tonB-dependent receptor protein (RagA4), arginine-specific proteinase B, 4-hydroxybutyryl-coenzyme A dehydratase (AbfD), lysine-specific proteinase, GAPDH, NAD-dependent glutamate dehydrogenase (GDH), and malate dehydrogenase (MDH), were identified by two-dimensional gel electrophoresis followed by proteomic analysis using tandem mass spectrometry. Interactions between these client proteins and S. oralis GAPDH were analyzed with a biomolecular interaction analysis system. S. oralis GAPDH showed high affinity for five of the seven client proteins (RagA4, AbfD, GAPDH, GDH, and MDH). Interactions between P. gingivalis and S. oralis were measured by a turbidimetric method and fluorescence microscopy. RagA4, AbfD, and GDH enhanced coaggregation, whereas GAPDH and MDH inhibited coaggregation. Furthermore, the expression of luxS in P. gingivalis was upregulated by RagA4, AbfD, and GDH but was downregulated by MDH. These results indicate that the five P. gingivalis client proteins function as regulators in P. gingivalis biofilm formation with oral streptococci.