Nakayama K, Yoshimura F, Kadowaki T, Yamamoto K
Department of Microbiology, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
J Bacteriol. 1996 May;178(10):2818-24. doi: 10.1128/jb.178.10.2818-2824.1996.
Arginine-specific cysteine proteinase (Arg-gingipain [RGP], a major proteinase secreted from the oral anaerobic bacterium Porphyromonas gingivalis, is encoded by two separate genes (rgpA and rgpB) on the P. gingivalis chromosome and widely implicated as an important virulence factor in the pathogenesis of periodontal disease (K. Nakayama, T. Kadowaki, K. Okamoto, and K. Yamamoto, J. Biol. Chem. 270:23619-23626, 1995). In this study, we investigated the role of RGP in the formation of P. gingivalis fimbriae which are thought to mediate adhesion of the organism to the oral surface by use of the rgp mutants. Electron microscopic observation revealed that the rgpA rgpB double (RGP-null) mutant possessed very few fimbriae on the cell surface, whereas the number of fimbriae of the rgpA or rgpB mutant was similar to that of the wild-type parent strain. The rgpB+ revertants that were isolated from the double mutant and recovered 20 to 40% of RGP activity of the wild-type parent possessed as many fimbriae as the wild-type parent, indicating that RGP significantly contributes to the fimbriation of P. gingivalis as well as to the degradation of various host proteins, disturbance of host defense mechanisms, and hemagglutination. Immunoblot analysis of cell extracts of these mutants with antifimbrilin antiserum revealed that the rgpA rgpB double mutant produced small amounts of two immunoreactive proteins with molecular masses of 45 and 43 kDa, corresponding to those of the precursor and mature forms of fimbrilin, respectively. The result suggests that RGP may function as a processing proteinase for fimbrilin maturation. In addition, a precursor form of the 75-kDa protein, one of the major outer membrane proteins of P. gingivalis, was accumulated in the rgpA rgpB double mutant but not in the single mutants and the revertants, suggesting an extensive role for RGP in the maturation of some of the cell surface proteins.
精氨酸特异性半胱氨酸蛋白酶（精氨酸牙龈蛋白酶[RGP]，是口腔厌氧菌牙龈卟啉单胞菌分泌的一种主要蛋白酶，由牙龈卟啉单胞菌染色体上两个独立的基因（rgpA和rgpB）编码，在牙周病发病机制中被广泛认为是一种重要的毒力因子（K. 中山、T. 门胁、K. 冈本和K. 山本，《生物化学杂志》270:23619 - 23626，1995年）。在本研究中，我们利用rgp突变体研究了RGP在牙龈卟啉单胞菌菌毛形成中的作用，菌毛被认为介导该菌与口腔表面的黏附。电子显微镜观察显示，rgpA rgpB双突变体（RGP缺失）在细胞表面几乎没有菌毛，而rgpA或rgpB突变体的菌毛数量与野生型亲本菌株相似。从双突变体中分离出的rgpB +回复突变体恢复了野生型亲本20%至40%的RGP活性，其菌毛数量与野生型亲本一样多，这表明RGP对牙龈卟啉单胞菌的菌毛形成以及各种宿主蛋白的降解、宿主防御机制的干扰和血细胞凝集都有显著作用。用抗菌毛蛋白抗血清对这些突变体的细胞提取物进行免疫印迹分析表明，rgpA rgpB双突变体产生少量两种免疫反应性蛋白，分子量分别为45 kDa和43 kDa，分别对应菌毛蛋白的前体和成熟形式。结果表明RGP可能作为菌毛蛋白成熟的加工蛋白酶发挥作用。此外，牙龈卟啉单胞菌主要外膜蛋白之一的75 kDa蛋白的前体形式在rgpA rgpB双突变体中积累，而在单突变体和回复突变体中没有积累，这表明RGP在一些细胞表面蛋白的成熟中具有广泛作用。
