VPA1500基因缺失对副溶血弧菌生物学特性和致病性的影响

Study on biological characteristics and pathogenicity of VPA1500 gene knock-out mutant of Vibrio parahaemolyticus

型分泌系统(T6SS)是细菌的一种毒力因子分泌系统,通过分泌蛋白参与调控细菌的环境适应性和毒力。副溶血弧菌具有两个T6SS系统(T6SS1和T6SS2)。【目的】前期通过差异蛋白质组学技术筛选到副溶血弧菌T6SS1相关的分泌蛋白,本文选择其中的VPA1500为研究对象,研究其基因缺失对副溶血弧菌的生物学特性及致病性的影响。【方法】利用同源重组技术构建缺失株ΔVPA1500和互补株CΔVPA1500;分析各菌株生长特性、在体外的细菌竞争能力、运动性、细菌鞭毛相关基因的转录水平及生物被膜形成能力的差异,比较各菌株对细胞毒性、小鼠毒力、动物组织载菌量以及组织病理学变化的影响。【结果】与野生株相比,VPA1500基因缺失后不影响细菌的生长能力、生物被膜形成能力和群集运动,然而ΔVPA1500的浮泳运动能力显著下降;进一步通过透射电镜观察和实时定量PCR检测发现,VPA1500缺失影响副溶血弧菌鞭毛的形成;细菌竞争实验显示缺失VPA1500基因降低了副溶血弧菌野生株体外对大肠杆菌的杀伤能力;ΔVPA1500对细胞毒性、小鼠毒力以及在动物组织的定殖能力均显著低于野生株,互补株毒力基本恢复至野生株水平;组织病理学结果进一步表明,缺失VPA1500基因能够降低副溶血弧菌对小鼠组织的损伤。【结论】VPA1500参与副溶血弧菌的体外细菌竞争能力、浮游运动能力和致病性。

The type Ⅵ secretion system(T6 SS) is widespread in bacterial pathogens and used to deliver virulence effector proteins into target cells. Vibrio parahaemolyticus also harbours T6 SS gene clusters and possesses two T6 SSs,T6 SS1 and T6 SS2. [Objective] In previous work, we identified several potential T6 SS effectors by using comparative proteomics. In this study, VPA1500 was chosen to explore the roles on biological characteristics and pathogenicity of Vibrio parahaemolyticus. [Methods] The deletion mutant ΔVPA1500 and complementary strain CΔVPA1500 were constructed by using homologous recombination technology. Growth characteristics, anti-bacterial activity in vivo,motility, the transcription level of flagella-related genes, and biofilm formation ability were analyzed in the wild-type strain(WT), ΔVPA1500 and CΔVPA1500. Furthermore, cytotoxicity to host cell, lethality rate in mice, bacterial colonization, and histopathological changes were also analyzed between WT and ΔVPA1500. [Results] Compared with WT, there were no significant changes in growth characteristics, swarming ability, and biofilm formation of ΔVPA1500(P>0.05), while swimming ability was significantly decreased(P<0.05). Transmission electron micrographs showed that VPA1500 deletion affected the formation of bacterial flagella in V. parahaemolyticus. qPCR results also showed that the VPA1500 gene significantly inhibited the transcription level of some flagella-related genes in WT. Bacterial competition experiments showed that the deletion of VPA1500 reduced the anti-bacterial activity of Vibrio parahaemolyticus against E. coli in vitro. However, ΔVPA1500 showed significantly weaker cytotoxicity to Hela cells than WT. In addition, ΔVPA1500 exhibited attenuated virulence in mice that showed lower a lethality rate than that of the wild-type strain. Moreover, VPA1500 deletion affected the colonization in heart, liver, spleen, and kidney of mice, whereas the complementation strain restored the virulence to resemble that of WT. Histopathological analyses further demonstrated that detection of VPA1500 could reduce the damage of Vibrio parahaemolyticus to tissues in mice. [Conclusion] The VPA1500 plays an important role in swimming motility, pathogenicity, and anti-bacterial activity in V. parahaemolyticus.