摘要
目的研究河弧菌85003 VI型分泌系统VflT6SS2核心基因簇上游paar基因(AL536_RS29530)编码蛋白的结构特征、功能及其对河弧菌VflT6SS2功能的影响。方法利用自杀质粒介导的同源重组技术构建paar基因缺失株,PCR扩增并克隆paar基因于pSRKTc表达载体获得回补质粒,导入相应缺失株得到回补株。Western Blot(WB)检测缺失株和回补株中T6SS效应蛋白Hcp的表达和分泌,细菌杀菌实验检测菌株杀菌毒力变化。荧光定量反转录聚合酶链式反应检测tssB2和hcp mRNA表达水平。启动子-Lux报告系统的冷光表型检测确定VflT6SS2核心基因簇启动子及hcp启动子在野生株和缺失株背景下的活性差异。结果成功构建河弧菌85003的paar基因的精确缺失株和相应的回补株;WB检测缺失株中Hcp的表达和分泌较野生株明显降低,同时其对大肠埃希菌的菌间杀伤能力也降低,导入相应回补质粒可使缺失株Hcp的表达、分泌和杀菌表型恢复到野生株水平;hcp和tssB2的mRNA水平检测显示缺失株低于野生株,差异有显著性,但VflT6SS2核心基因簇的启动子活性和hcp启动子活性在野生株及缺失株中无明显差异,提示paar基因可能在转录后水平调控VflT6SS2。结论河弧菌中paar(AL536_RS29530)基因编码产物是河弧菌VflT6SS2的组成部分,为其功能所需,缺失该基因导致VflT6SS2的分泌和杀菌能力缺陷,但其具体的调控机制有待进一步研究。
Objective This study aimed to investigate the structure and function of paar(AL536_RS29530), an upstream gene of VflT6 SS2 core cluster and its effect on the function of VflT6 SS2 in Vibrio fluvialis 85003. Methods The deletion mutant of paar was constructed through homologous recombination mediated by suicide plasmid. The complementation plasmid constructed by cloning the coding sequence of paar by PCR into plasmid pSRKTc was mobilized into the deletion mutant by conjugation. Western Blot analysis was used to detect the relative expression and secretion of the Hcp effector of the derivative strains. Bacterial killing assay was used to measure the change of antibacterial virulence in derivative strains, in comparison with wild type(WT) strain. The relative expression of tssB2 and hcp mRNA was detected by quantitative reverse transcription PCR. Luminescence activity of promoter reporting system was used to test the promoter activities of VflT6 SS2 core cluster and hcp-vgrG clusters in the wild type and the deletion mutant. Results The accurate deletion of paar coding sequence and its complemented strain were successfully constructed. The expression and secretion of Hcp and the antibacterial virulence severely decreased in the deletion mutant, but restored when its trans-complemented plasmid was introduced.Significantly, deletion of paar decreased the mRNA levels of tssB2 and hcp, while promoter activities of VflT6 SS2 core cluster and hcp-vgrG clusters showed no significant difference between the wild type and the deletion mutant. It suggested that paar might regulate VflT6 SS2 at posttranscriptional level. Conclusion paar(AL536_RS29530) is an integral part of VflT6 SS2 in V. fluvialis, which contributes to the function of VflT6 SS2. Deletion of paar decreased expression and antibacterial virulence of VflT6 SS2. Further study on its specific regulation mechanism is needed.
作者
刘笑舒
张安冉
黄元铭
刘平
韩雨
阚飙
梁未丽
Liu Xiaoshu;Zhang Anran;Huang Yuanming;Liu Ping;Han Yu;Kan Biao;Liang Weili(National Institute for Communicable Disease Prevention and Control,Chinese Center for Disease Control and Prevention,Beijing 102206,China)
出处
《疾病监测》
CAS
2020年第4期294-300,共7页
Disease Surveillance
基金
国家自然科学基金(No.81772242)。
关键词
河弧菌
Ⅵ型分泌系统
PAAR
HCP
转录调控
Vibrio fluvialis
TypeⅥsecretion system
Proline-Alanine-Alanine-Arginine
Haemolysin co-regulated protein
Transcriptional regulation
