双组分系统EnvZ/OmpR促进副溶血弧菌抵抗碱胁迫的作用机制

The two-component system EnvZ/OmpR mediates alkaline stress tolerance of Vibrio parahaemolyticus

摘要:

【目的】探究双组分系统(two-component system，TCS)EnvZ/OmpR对副溶血弧菌(Vibrio parahaemolyticus，VP)抵抗碱胁迫的作用机制。【方法】用SMART在线工具(https://smart.embl.de/)鉴定出副溶血弧菌基因组中的双组分系统EnvZ/OmpR，再利用同源重组技术将envZ和ompR基因分别进行缺失，构建相应回补株，比较各菌株的生长曲线来检测相应基因对细菌适应高渗透胁迫和碱胁迫的作用，并结合qRT-PCR及荧光检测系统，筛选参与EnvZ/OmpR抵抗碱胁迫的下游靶基因，鉴定该双组分系统对下游基因的调控机制。【结果】在副溶血弧菌基因组中鉴定出vp0155/vp0154编码EnvZ/OmpR双组分系统同源蛋白。△ompR菌株在高渗透胁迫和碱胁迫中的生长能力明显弱于野生株，而回补株C△ompR、△envZ和C△envZ菌株生长能力与野生株类似。在△ompR菌株中，孔道蛋白基因vp1218、vp0493、vpa1745、vpa0085与vpa1308的转录水平均明显低于野生株，并且发现这些孔道蛋白基因缺失株(△vpa1308除外)在碱性环境中生长能力均明显弱于野生株。OmpR蛋白可直接抑制调控因子AphB基因转录，而△aphB菌株在碱胁迫中的生长能力明显强于野生株。此外，AphB蛋白可直接抑制孔道蛋白基因vp0493和vpa0085转录。【结论】双组分系统EnvZ/OmpR促进副溶血弧菌抵抗碱胁迫，其中OmpR蛋白可通过抑制调控因子AphB的表达，以促进部分孔道蛋白的表达，从而增强副溶血弧菌抵抗碱胁迫的能力。

Abstract:

[Objective] Vibrio parahaemolyticus is a significant foodborne pathogen that causes economic and public health problems worldwide. In this study, we explored the roles of the two-component system (TCS) EnvZ/OmpR in mediating the alkaline stress tolerance of V.parahaemolyticus.[Methods] We identified the two-component system EnvZ/OmpR in the V.parahaemolyticus chromosomes with SMART (https://smart.embl.de/). Then, we constructed the envZ-deleted strain, the ompR-deleted strain, as well as the complemented strains. On this basis, we compared the alkaline stress tolerance of the wild-type strain and the mutants, and the mechanisms whereby the EnvZ/OmpR regulated the alkaline stress tolerance of V.parahaemolyticus were explored by qRT-PCR and bioluminescence reporter assay. [Results] Homology analysis of the VP0155 and VP0154 proteins revealed that their amino acid sequences shared a high identity with the two-component system EnvZ/OmpR. ompR deletion decreased the tolerance of V.parahaemolyticus to osmotic stress and alkaline stress, while the growth status of C△ompR, △envZ, and C△envZ was similar to that of the wild type. The transcription levels of porin genes vp1218, vp0493, vpa1745, vpa0085, and vpa1308 in △ompR were lower than those in the wild type, and the deletion strains of the porin genes (except for △vpa1308) showed low growth ability in the alkaline-shock environment as compared with the wild strains. In addition, OmpR inhibited the expression of AphB and △aphB was significantly more tolerant to alkaline stress than the wild type. Moreover, AphB can directly repress the transcription of vp0493 and vpa0085. [Conclusion] The two-component system EnvZ/OmpR mediates alkaline stress tolerance of V. parahaemolyticus, which depends on the transcriptional factor AphB to regulate the expression of the porins.