群体感应调控子AphA对河弧菌Ⅵ型分泌系统VflT6SS2的调控研究  

Regulation mechanism of the quorum sensing regulator AphA on the typeⅥsecretion system VflT6SS2 in Vibrio fluvialis

作　　者：程倩 韩雨 黄元铭[1] 冀赛森 李杰[1] 刁保卫[1] 梁未丽[1] Cheng Qian;Han Yu;Huang Yuanming;Ji Saisen;Li Jie;Diao Baowei;Liang Weili(National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]传染病溯源预警与智能决策全国重点实验室,中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《中华流行病学杂志》2024年第4期566-573,共8页Chinese Journal of Epidemiology

基　　金：国家重点研发计划(2023YFC2604400,2021YFC2300302);国家自然科学基金(81772242)。

摘　　要：目的研究河弧菌群体感应调控子AphA对Ⅵ型分泌系统VflT6SS2功能活性的调控机制。方法采用Western Blot检测河弧菌野生株、aphA缺失株(ΔaphA)和aphA回补株中VflT6SS2标志性组件溶血素共调节蛋白(Hcp)的表达和分泌。采用实时荧光定量PCR和启动子-lux融合冷光系统检测野生株和ΔaphA中VflT6SS2核心基因簇和附属基因簇代表基因tssB2、hcp(tssD2)和vgrG(tssI2)以及群体感应调控子HapR的mRNA相对表达量和启动子活性。采用定点突变实验结合启动子活性测定确定AphA在tssD2b启动子区的调控结合位点;采用电泳迁移率位移测定(EMSA)确定AphA与hapR启动子的结合。结果ΔaphA中tssB2、hcp(tssD2)、vgrG(tssI2)和hapR的mRNA相对表达量及Hcp蛋白的表达分泌明显高于野生株。VflT6SS2核心基因簇、tssD2a、tssI2a和hapR的启动子活性在ΔaphA中均高于野生株,而tssD2b启动子活性则低于野生株。tssD2a和tssD2b的启动子序列分析显示-335 bp~-229 bp区差异较大,在tssD2b中该区域存在2个潜在AphA结合位点,将其中的保守位点ATG替换为CGA,tssD2b启动子活性明显降低。EMSA结果显示,AphA与hapR启动子直接结合。结论AphA在转录水平直接抑制hapR的表达,间接参与对VflT6SS2核心基因簇和附属基因簇的调控。AphA对tssD2a和tssD2b呈现相反的调控模式,AphA可直接与tssD2b启动子区(-335 bp~-229 bp)结合正向调控tssD2b的表达。Objective To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of typeⅥsecretion system VflT6SS2 in Vibrio fluvialis.Methods Western Blot analysis was used to detect the relative expression and secretion of VflT6SS2 signature component hemolysin-coregulated protein(Hcp)in wild type(WT),ΔaphA,and corresponding complementary strains.Quantitative reverse transcription PCR and luminescence activity assay of the promoter-lux fusion system was used to measure the mRNA expression levels and promoter activity of the VflT6SS2 core and accessory gene-cluster representative genes tssB2,hcp(tssD2)and vgrG(tssI2),and the quorum sensing regulator HapR in WT andΔaphA strains.A point mutation experiment combined with a luminescence activity assay was used to verify the regulatory binding site of AphA in the tssD2b promoter region.Electrophoretic mobility shift assay(EMSA)was used to determine AphA binding to the hapR promoter.Results The mRNA expression levels of tssB2,hcp(tssD2),vgrG(tssI2),and hapR as well as the protein expression and secretion levels of Hcp inΔaphA strain,were significantly higher than those in the WT strain.The promoter activities of the VflT6SS2 core cluster,tssD2a,tssI2a,and hapR were higher inΔaphA strain than in the WT strain,while the promoter activity of tssD2b showed the opposite trend.The promoter sequence analysis of tssD2a and tssD2b found significant differences in the region from-335 bp to-229 bp,and two potential AphA binding sites on tssD2b.The promoter activity of tssD2b decreased significantly after the point mutation of the two potential AphA binding sites.EMSA results showed that AphA binds directly to the promoter region of hapR.Conclusions AphA indirectly inhibits the regulation of the VflT6SS2 core and accessory gene clusters at the promoter level by directly repressing the expression of hapR.AphA showed opposite regulation patterns for tssD2a and tssD2b,and AphA could positively regulate the expression of tssD2b by directly binding t

关 键 词：河弧菌 群体感应调控子 Ⅵ型分泌系统 转录调控 

分 类 号：R378.3[医药卫生—病原生物学]