背景:创伤弧菌NCIMB2137,革兰氏阴性,从患病的鳗鱼中分离出金属蛋白酶阴性的河口菌株。最近已报道称为VvsA的45kDa胰凝乳蛋白酶样碱性丝氨酸蛋白酶是负责该菌株的发病机理的主要毒力因子之一。vvsA基因和下游基因vvsB,其函数仍然未知构成指定为vvsAB的操作数。
目的:本研究考察了VvsB对VvsA功能的贡献。
方法:在本研究中,VvsB使用快速翻译系统(RTS系统)单独表达,然后分析其在调节VvsA的丝氨酸蛋白酶活性中的作用。
结果:在将纯化的VvsB添加到创伤弧菌的培养上清后,VvsA的蛋白水解活性增加。然而,使用大肠杆菌系统的蛋白质表达的尝试揭示了值得注意的观察,即与细胞质部分内的vvsAB基因相比,来自vvsA基因的蛋白质表达表现出更高的蛋白酶活性。这些发现表明了VvsB和VvsA之间复杂的相互作用,其中VvsB可能与细菌内的VvsA相互作用并抑制蛋白水解活性。在细菌环境之外,VvsB似乎刺激非活性VvsA的激活。
结论:研究结果表明,创伤弧菌通过VvsB的作用调节VvsA的活性,细胞内和细胞外,以确保其生存。
BACKGROUND: Vibrio vulnificus NCIMB2137, a Gram-negative, metalloprotease negative estuarine strain was isolated from a diseased eel. A 45 kDa chymotrypsin-like alkaline serine protease known as VvsA has been recently reported as one of the major virulence factor responsible for the pathogenesis of this strain. The vvsA gene along with a downstream gene vvsB, whose function is still unknown constitute an operon designated as vvsAB.
OBJECTIVE: This study examines the contribution of VvsB to the functionality of VvsA.
METHODS: In this study, VvsB was individually expressed using Rapid Translation System (RTS system), followed by an analysis of its role in regulating the serine protease activity of VvsA.
RESULTS: The proteolytic activity of VvsA increased upon the addition of purified VvsB to the culture supernatant of V. vulnificus. However, the attempts of protein expression using an E. coli system revealed a noteworthy observation that protein expression from the vvsA gene exhibited higher protease activity compared to that from the vvsAB gene within the cytoplasmic fraction. These findings suggest an intricate interplay between VvsB and VvsA, where VvsB potentially interacts with VvsA inside the bacterium and suppress the proteolytic activity. While outside the bacterial milieu, VvsB appears to stimulate the activation of inactive VvsA.
CONCLUSIONS: The findings suggest that Vibrio vulnificus regulates VvsA activity through the action of VvsB, both intracellularly and extracellularly, to ensure its survival.