Saiki Keitarou, Konishi Kiyoshi
Department of Microbiology, Nippon Dental University School of Life Dentistry at Tokyo, Chiyoda-ku, Tokyo 102-8159, Japan.
Microbiol Immunol. 2007;51(5):483-91. doi: 10.1111/j.1348-0421.2007.tb03936.x.
Gingipains are extracellular proteases important for the virulence of Porphyromonas gingivalis; however, the mechanism for the secretion of gingipains is poorly understood. In this report, we found that insertion mutants for PG0809 (83K1 and 83K2) were defective in black pigmentation and hemolysis. We cloned and sequenced PG0809 and found that PG0809 contains two additional nucleotides that are not deposited in the W83 genome database. The revised sequence reveals an in-frame fusion of PG0810 and PG0809 and is designated the sov gene. We constructed a sov deletion mutant (83K3) and showed that 83K3 was defective in the activities of black pigmentation, hemolysis, and hemagglutination. Furthermore, in 83K3, the activities of gingipains were severely reduced whereas those of other secreted proteases DPPIV, DPP-7, and PtpA were not affected. Immunoblot analysis using anti-RgpB antiserum showed that Arg-gingipains were poorly secreted in an outer membrane or into an extracellular portion but accumulated within the cells of 83K3, suggesting the secretion of gingipains is defected in 83K3. Taken together, our findings indicated that Sov is a novel protein required for the secretion of gingipains and suggested that the secretion system for gingipains is different from the conserved secretion systems.
牙龈蛋白酶是牙龈卟啉单胞菌毒力的重要胞外蛋白酶；然而，牙龈蛋白酶的分泌机制却知之甚少。在本报告中，我们发现PG0809的插入突变体（83K1和83K2）在黑色素生成和溶血方面存在缺陷。我们克隆并测序了PG0809，发现PG0809含有两个未存入W83基因组数据库的额外核苷酸。修订后的序列显示PG0810和PG0809发生了读框内融合，被命名为sov基因。我们构建了一个sov缺失突变体（83K3），发现83K3在黑色素生成、溶血和血凝活性方面存在缺陷。此外，在83K3中，牙龈蛋白酶的活性严重降低，而其他分泌性蛋白酶DPPIV、DPP - 7和PtpA的活性未受影响。使用抗RgpB抗血清进行的免疫印迹分析表明，精氨酸牙龈蛋白酶在外膜或细胞外部分分泌不佳，但在83K3细胞内积累，这表明83K3中牙龈蛋白酶的分泌存在缺陷。综上所述，我们的研究结果表明Sov是牙龈蛋白酶分泌所需的一种新蛋白，并提示牙龈蛋白酶的分泌系统不同于保守的分泌系统。
