副溶血性弧菌CalR调控vopB2的分子机制
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摘要
【背景】副溶血性弧菌是一种非常重要的食源性致病菌,CalR蛋白是一种全局转录调节因子。III型分泌系统2 (Type 3 secretion systems 2 T3SS2)是副溶血性弧菌主要的毒力因子,vopB2是T3SS2中的一个关键效应蛋白。【目的】研究副溶血弧菌CalR对vopB2的转录调控机制。【方法】利用引物延伸实验鉴定vopB2及vtrA的转录起始位点,并根据产物的丰度判断CalR对靶基因的调控关系;采用实时定量RT-PCR研究靶基因mRNA在WT和ΔcalR中转录丰度,验证CalR对靶基因的转录调控关系,进一步利用LacZ实验通过比较β-半乳糖苷酶活性的差异来判定CalR对靶基因的调控关系;利用凝胶阻滞实验分析His-CalR对靶基因启动子区是否具有直接的结合作用。【结果】vopB2有两个转录起始位点A(-130和-28)且其活性受CalR的直接抑制;引物延伸和LacZ结果表明CalR对vtrA的转录并无调控作用。【结论】CalR直接抑制vopB2的转录,该抑制作用与vtrA无关联。
[Background] Vibrio parahaemolyticus is a seafood-borne pathogen. CalR is a global transcriptional regulator in V. parahaemolyticus. Type 3 secretion systems 2(T3 SS2) is one of the major virulence determinants of V. parahaemolyticus. VopB2 is a key effector protein of T3 SS2. [Objective] To study the transcriptional regulation of vopB2 by CalR in Vibrio parahaemolyticus. [Methods] Primer extension assay was employed to detect the transcription start sites and the amount of primer extension porroducts of target genes in ΔcalR and WT. Quantitative RT-PCR was then carried out to calculate the transcriptional variation of target genes between ΔcalR and WT. LacZ assay was used to verify regulation relationship by measuring the β-galactosidase activities in cellular extracts using a β-Galactosidase Enzyme Assay System(Promega). Finally the electrophoretic mobility shift assay(EMSA) was applied to analyze the DNA-binding activity of His-CalR to target promoters in vitro. [Results] We detected one transcription start site for vopB2, which was located at 130 bp and 28 bp upstream of vopB2 and its transcribed activity was under the negative control of the CalR. We also found that His-CalR was bind to the promoter region of vopB2 directly. Besides, our data showed that CalR had no regulatory effect on vtrA transcription, a known regulator for vopB2. [Conclusion] V. parahaemolyticus CalR represses the transcription of vopB2 directly, which is not associated to vtrA.
[Background] Vibrio parahaemolyticus is a seafood-borne pathogen. CalR is a global transcriptional regulator in V. parahaemolyticus. Type 3 secretion systems 2(T3 SS2) is one of the major virulence determinants of V. parahaemolyticus. VopB2 is a key effector protein of T3 SS2. [Objective] To study the transcriptional regulation of vopB2 by CalR in Vibrio parahaemolyticus. [Methods] Primer extension assay was employed to detect the transcription start sites and the amount of primer extension porroducts of target genes in ΔcalR and WT. Quantitative RT-PCR was then carried out to calculate the transcriptional variation of target genes between ΔcalR and WT. LacZ assay was used to verify regulation relationship by measuring the β-galactosidase activities in cellular extracts using a β-Galactosidase Enzyme Assay System(Promega). Finally the electrophoretic mobility shift assay(EMSA) was applied to analyze the DNA-binding activity of His-CalR to target promoters in vitro. [Results] We detected one transcription start site for vopB2, which was located at 130 bp and 28 bp upstream of vopB2 and its transcribed activity was under the negative control of the CalR. We also found that His-CalR was bind to the promoter region of vopB2 directly. Besides, our data showed that CalR had no regulatory effect on vtrA transcription, a known regulator for vopB2. [Conclusion] V. parahaemolyticus CalR represses the transcription of vopB2 directly, which is not associated to vtrA.
引文
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[12]Hou SN,Meng YY,Zhu WJ,et al.Construction of the calR null mutant and the complemented mutant strains in Vibrio parahaemolyticus[J].Military Medical Sciences,2016,40(5):374-378(in Chinese)侯书宁,孟彦言,朱文君,等.副溶血弧菌calR的基因敲除株与回补株构建[J].军事医学,2016,40(5):374-378
[13]Ono T,Park KS,Ueta M,et al.Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1[J].Infection and Immunity,2006,74(2):1032-1042
[14]Burdette DL,Yarbrough ML,Orth K.Not without cause:Vibrio parahaemolyticus induces acute autophagy and cell death[J].Autophagy,2009,5(1):100-102
[15]Zhou XH,Konkel ME,Call DR.Type III secretion system 1 of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines[J].Microbiology,2009,155:837-851
[16]Wang JJ,Sun WS,Jin MT,et al.Fate of Vibrio parahaemolyticus on shrimp after acidic electrolyzed water treatment[J].International Journal of Food Microbiology,2014,179:50-56
[2]Makino K,Oshima K,Kurokawa K,et al.Genome sequence of Vibrio parahaemolyticus:a pathogenic mechanism distinct from that of V cholerae[J].Lancet,2003,361(9359):743-749
[3]Park KS,Ono T,Rokuda M,et al.Functional characterization of two type III secretion systems of Vibrio parahaemolyticus[J].Infection and Immunity,2004,72(11):6659-6665
[4]Kodama T,Hiyoshi H,Gotoh K,et al.Identification of two translocon proteins of Vibrio parahaemolyticus type III secretion system 2[J].Infection and Immunity,2008,76(9):4282-4289
[5]Kodama T,Gotoh K,Hiyoshi H,et al.Two regulators of Vibrio parahaemolyticus play important roles in enterotoxicity by controlling the expression of genes in the Vp-PAI region[J].PLoSOne,2010,5(1):e8678
[6]Okada R,Matsuda S,Iida T.Vibrio parahaemolyticus VtrA is a membrane-bound regulator and is activated via oligomerization[J].PLoS One,2017,12(11):e0187846
[7]Cai Q,Zhang YQ.Structure,function and regulation of the thermostable direct hemolysin(TDH)in pandemic Vibrio parahaemolyticus[J].Microbial Pathogenesis,2018,123:242-245
[8]Osei-Adjei G,Gao H,Zhang Y,et al.Regulatory actions of ToxRand CalR on their own genes and type III secretion system 1 in Vibrio parahaemolyticus[J].Oncotarget,2017,8(39):65809-65822
[9]Zhang YQ,Hu LF,Osei-Adjei G,et al.Autoregulation of ToxRand its regulatory actions on major virulence gene loci in Vibrio parahaemolyticus[J].Frontiers in Cellular and Infection Microbiology,2018,8:291
[10]Zhang LY,Osei-Adjei G,Zhang Y,et al.CalR is required for the expression of T6SS2 and the adhesion of Vibrio parahaemolyticus to HeLa cells[J].Archives of Microbiology,2017,199(6):931-938
[11]Zhang YQ,Zhang Y,Gao H,et al.Vibrio parahaemolyticus CalRdown regulates the thermostable direct hemolysin(TDH)gene transcription and thereby inhibits hemolytic activity[J].Gene,2017,613:39-44
[12]Hou SN,Meng YY,Zhu WJ,et al.Construction of the calR null mutant and the complemented mutant strains in Vibrio parahaemolyticus[J].Military Medical Sciences,2016,40(5):374-378(in Chinese)侯书宁,孟彦言,朱文君,等.副溶血弧菌calR的基因敲除株与回补株构建[J].军事医学,2016,40(5):374-378
[13]Ono T,Park KS,Ueta M,et al.Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1[J].Infection and Immunity,2006,74(2):1032-1042
[14]Burdette DL,Yarbrough ML,Orth K.Not without cause:Vibrio parahaemolyticus induces acute autophagy and cell death[J].Autophagy,2009,5(1):100-102
[15]Zhou XH,Konkel ME,Call DR.Type III secretion system 1 of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines[J].Microbiology,2009,155:837-851
[16]Wang JJ,Sun WS,Jin MT,et al.Fate of Vibrio parahaemolyticus on shrimp after acidic electrolyzed water treatment[J].International Journal of Food Microbiology,2014,179:50-56