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

精氨酸特异性牙龈蛋白酶和赖氨酸特异性牙龈蛋白酶是牙龈卟啉单胞菌产生的两种主要的半胱氨酸蛋白酶。为了阐明牙龈痛在牙龈卟啉卟啉与先天免疫系统相互作用中的作用，我们纯化了一种对牙龈痛敏感的先天免疫激活因子。在我们之前的研究中，已经发现该因子可以以toll样受体（TLR） 2和tlr4独立的方式激活先天免疫系统。对牙龈卟啉卟啉缺乏突变体KDP136 （rgpA rgpB kgp）进行厌氧培养和收获，并用1% triton X-114提取细胞表面成分。对细胞表面组分进行了离子交换色谱分析。利用中国仓鼠卵巢（CHO） NF-kB依赖性报告细胞系7.19测定了诱导核因子- κ B （NF-kB）激活的活性，该细胞系在TLR2-和tlr4依赖性信号通路中都存在缺陷。然后，对含有诱导7.19细胞活化活性的部分进行凝胶过滤层析。对含有诱导7.19个细胞活化活性的部分进行SDS-PAGE分析，检测到一个123 kD条带。经质谱分析，该纯化组分为牙龈卟啉卟啉ATCC33277基因组中由ORF PGN0748编码的蛋白。接下来，我们设计了牙龈卟啉卟啉菌KDP136的等基因突变体，其中ORF PGN0748被耐氨吡啶青霉素基因cepA取代。当7.19个细胞暴露于ORF PGN0748突变体时，NF-kB未被激活。这些结果表明，牙龈卟啉卟啉的牙龈敏感先天免疫激活因子是由ORF PGN0748编码的。该因子的失活可能有助于牙龈假单胞菌从先天免疫系统中逃脱。

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.