Analysis of the mechanism for a periodontopathic bacteria P. gingivalis to escape from innate immune system

精氨酸特异性牙龈菌蛋白酶和赖氨酸特异性牙龈菌蛋白酶是牙龈卟啉单胞菌产生的两种主要半胱氨酸蛋白酶。为了阐明牙龈卟啉菌蛋白酶在牙龈卟啉菌与天然免疫系统相互作用中的作用，我们纯化了一种对牙龈卟啉菌蛋白酶敏感的天然免疫激活因子。本研究以牙龈卟啉单胞菌gingipain缺陷型KDP 136（rgpA rgpB kgp）为材料，在厌氧条件下培养，用1%triton X-114提取细胞表面成分。对细胞表面组分进行离子交换层析。使用中国仓鼠卵巢（CHO）NF-κ B依赖性报告细胞系7.19（TLR 2和TLR 4依赖性信号传导途径均存在缺陷）测定各组分诱导核因子-κ B（NF-κ B）活化的活性。然后，将含有诱导7.19细胞活化的活性的级分进行凝胶过滤层析。对含有诱导7.19细胞活化活性的级分进行SDS-PAGE分析，检测到123 kD条带。对纯化的组分进行质谱分析，并鉴定为由牙龈卟啉单胞菌ATCC 33277基因组中的ORF PGN 0748编码的蛋白质。接下来，我们设计了牙龈卟啉单胞菌KDP 136的同基因突变体，其中ORF PGN 0748被氨苄青霉素抗性基因cepA取代。结果表明，PGN 0748编码的天然免疫激活因子对牙龈卟啉单胞菌的牙龈蛋白酶敏感，而PGN 0748编码的天然免疫激活因子对NF-kB无激活作用。牙龈卟啉菌蛋白酶对该因子的失活将有助于牙龈卟啉菌逃避先天免疫系统。

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine protainases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, we purified an innate immune activating factor that is sensitive to the gingipain. In our previous study, it has been revealed that this factor can activate the innate immune system in a Toll-like receptor (TLR) 2 and TLR4-independent manner.A P. gingivalis gingipain-deficient mutant, KDP136 (rgpA rgpB kgp), were grown anaerobically and harvested and the cell surface components were extracted using 1% triton X-114. The cell surface components were subjected to ion exchange chromatography. The activity of each fraction to induce nuclear factor-kappa B (NF-kB) activation was determined using a Chinese hamster ovary (CHO) NF-kB-dependent reporter cell line, 7.19, that is defective in both TLR2- and TLR4-dependent signaling pathways. Then, the fraction containing the activity to induce the activation of 7.19 cells were subjected to, gel filtration chromatography. The fraction containing the activity to induce the activation of 7.19 cells were subjected to the SDS-PAGE analysis, and a 123 kD band was detected. The purified component was subjected to Mass spectrometry analysis, and identified as a protein encoded by the ORF PGN0748 in the genome of P. gingivalis ATCC33277. Next, we engineered an isogenic mutant of P. gingivalis KDP136 in which ORF PGN0748 was replaced by an ampicillin-resistant gene, cepA. When 7.19 cells w exposed to ORF PGN0748 mutant, NF-kB was not activated.These results indicated that gingipain-sensitive innate immune activating factor in P. gingivalis was encoded by ORF PGN0748. The inactivation of this factor by gingipain would be helpful for P. gingivalis to escape from the innate immune system.