Zhang Rui, Yang Jie, Wu Juan, Sun Wei-Bin, Liu Yu
Department of Periodontology, Hospital of Stomatology, Medical School of Nanjing University, Nanjing, China.
J Dent Sci. 2016 Sep;11(3):279-286. doi: 10.1016/j.jds.2016.03.004. Epub 2016 Apr 18.
BACKGROUND/PURPOSE: The most potent virulence factors of the periodontal pathogen are gingipains, three cysteine proteases (RgpA, RgpB, and Kgp) that bind and cleave a wide range of host proteins. Considerable proof indicates that RgpA contributes to the entire virulence of the organism and increases the risk of periodontal disease by disrupting the host immune defense and destroying the host tissue. However, the functional significance of this proteinase is incompletely understood. It is important to analyze the effect of arginine-specific gingipain A gene () on selected virulence and physiological properties of .
Electroporation and homologous recombination were used to construct an mutant of ATCC33277. The mutant was verified by polymerase chain reaction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cell structures of the mutant were examined by transmission electron microscopy and homotypic biofilm formation was examined by confocal laser scanning microscopy.
Gene analysis revealed that the gene was deleted and replaced by a drug resistance gene marker. The defect of the gene resulted in a complete loss of RgpA proteinase, a reduction of out membrane vesicles and hemagglutination, and an increase in homotypic biofilm formation.
Our data indicate that an gene deficient strain of is successfully isolated. RgpA may have a variety of physiological and pathological roles in .
背景/目的：牙周病原体最强大的毒力因子是牙龈蛋白酶，即三种半胱氨酸蛋白酶（RgpA、RgpB和Kgp），它们能结合并切割多种宿主蛋白。大量证据表明，RgpA通过破坏宿主免疫防御和破坏宿主组织，对该生物体的整体毒力有贡献，并增加了患牙周病的风险。然而，这种蛋白酶的功能意义尚未完全了解。分析精氨酸特异性牙龈蛋白酶A基因（）对所选毒力和生理特性的影响很重要。
采用电穿孔和同源重组构建牙龈卟啉单胞菌ATCC33277的突变体。通过聚合酶链反应和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳对突变体进行验证。通过透射电子显微镜检查突变体的细胞结构，并通过共聚焦激光扫描显微镜检查同型生物膜的形成。
基因分析显示该基因被缺失并被耐药基因标记取代。该基因的缺陷导致RgpA蛋白酶完全丧失、外膜囊泡减少和血凝反应降低，以及同型生物膜形成增加。
我们的数据表明成功分离出牙龈卟啉单胞菌的基因缺陷菌株。RgpA可能在牙龈卟啉单胞菌中具有多种生理和病理作用。
