转录因子Eha直接调控迟缓爱德华菌的靶基因
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摘要
目的 Eha是一种重要转录调控因子,它可以影响迟缓爱德华氏菌(Et)的胞内存活,本实验探究Eha直接调控的靶基因如何抵御巨噬细胞杀灭细菌的分子机制。方法构建pGEX-4T-ehaflag重组质粒,电击导入eha基因缺失株的ET-13菌,得到Cehaflag ET13重组菌;用Western blot检测重组菌Eha-Flag融合蛋白的表达,并用细菌胞内存活实验检测细菌EhaFlag融合蛋白的活性;我们釆用染色质免疫共沉淀(CHIP)技术,用抗Flag标签抗体对靶基因沉淀Eha-DNA片段,除去结合的蛋白并纯化DNA片段;以RNA-Sequencing的差异表达基因设计引物,以CHIP得到的DNA样品为模板,进行qRT-PCR; PCR扩增靶基因的启动子区域,构建了pBAD-P_靶lac Z重组质粒,电击分别导入ET-13野生株和eha基因缺失株,比较它们β-半乳糖苷酶活性的差异;SDS-PAGE电泳比较野生株和缺失株外膜蛋白、厌氧C4二羧酸转运蛋白DcuA1和鞭毛钩蛋白FlgK表达的差异,并用细菌胞内存活实验比较野生株和缺失株的差异。结果 Western blot表明,Cehaflag ET13重组菌能够表达Eha-Flag融合蛋白;细菌胞内存活实验表明,该融合蛋白完全可以恢复缺失株在胞内降低的生存能力,Flag融合标签不影响Eha蛋白的功能;通过qPCR鉴定CHIP,富集到Eha的结合靶点,最终筛选出10个Eha直接结合的基因;通过野生株和缺失株中β-半乳糖苷酶活性的差异,证明eha基因对5个靶基因启动子有直接调控作用;通过检测野生株和缺失株表型的差异,证明eha基因对靶蛋白DcuA1,TnaA和FlgK的表达和活性有调控作用。结论 Eha可以通过直接调控这些靶基因,使Et菌在巨噬细胞内的存活受到影响。
Eha is a virulence regulator of Edwardsiella tarda replicating within RAW264.7 macrophages. Our studies try to demonstrate Eha to regulate the target genes to survive against killing bacteria by macrophages. We constructed pGEX-4 T-ehaflag, introduced the combination vector into eha mutant strain by electric shock and obtained Cehaflag ET13 strain. The expression and the activity of Eha-Flag fusion protein were inspected by Western blot and intracellular survival experiment, respectively. The results suggested that Eha-Flag fusion protein could express and restore the survival ability within macrophages in the Cehaflag ET13 strain. We used monoclonal anti-Flag antibody to precipitate EhaFlag-DNA complex, removed the binding protein and purified the DNA fragment with CHIP. We designed primers according to the DEGs of RNA-sequencing and identified the genes combined with Eha enriched by CHIP in qPCR. Finally, we got the ten genes combined immediately with Eha. We amplified the promoter regions of the genes from ET-13 genome, constructed the promoter recombinant vector of pBAD-P_(target)LacZ, and introduced the combination vector into the eha mutant and the wild strain by electric shock. We compared the β-gal activity of pBAD-P_(target)LacZ-Δeha strains with the one of pBAD-P_(target) LacZ-ET13 strains. The results indicated that the eha gene regulated immediately the promoters of five target genes. We compared the expression of the transporter protein DcuA1 of anaerobic C4 dicarboxylic acid and the flgellar hook protein FlgK in the eha mutant strain with the ones in the wild strain by SDS-PAGE. We compared the survival ability of Et13-pACYC184, Et13-tnaA, △eha-tnaA and △eha-pACYC184 strain within macrophages. The results suggest that the eha gene is able to regulate the expression and activity of TnaA, FlgK and DcuA1 proteins. Therefore, Eha can regulate directly these target genes to affect the survival ability of Edwardsiella tarda within macrophage.
Eha is a virulence regulator of Edwardsiella tarda replicating within RAW264.7 macrophages. Our studies try to demonstrate Eha to regulate the target genes to survive against killing bacteria by macrophages. We constructed pGEX-4 T-ehaflag, introduced the combination vector into eha mutant strain by electric shock and obtained Cehaflag ET13 strain. The expression and the activity of Eha-Flag fusion protein were inspected by Western blot and intracellular survival experiment, respectively. The results suggested that Eha-Flag fusion protein could express and restore the survival ability within macrophages in the Cehaflag ET13 strain. We used monoclonal anti-Flag antibody to precipitate EhaFlag-DNA complex, removed the binding protein and purified the DNA fragment with CHIP. We designed primers according to the DEGs of RNA-sequencing and identified the genes combined with Eha enriched by CHIP in qPCR. Finally, we got the ten genes combined immediately with Eha. We amplified the promoter regions of the genes from ET-13 genome, constructed the promoter recombinant vector of pBAD-P_(target)LacZ, and introduced the combination vector into the eha mutant and the wild strain by electric shock. We compared the β-gal activity of pBAD-P_(target)LacZ-Δeha strains with the one of pBAD-P_(target) LacZ-ET13 strains. The results indicated that the eha gene regulated immediately the promoters of five target genes. We compared the expression of the transporter protein DcuA1 of anaerobic C4 dicarboxylic acid and the flgellar hook protein FlgK in the eha mutant strain with the ones in the wild strain by SDS-PAGE. We compared the survival ability of Et13-pACYC184, Et13-tnaA, △eha-tnaA and △eha-pACYC184 strain within macrophages. The results suggest that the eha gene is able to regulate the expression and activity of TnaA, FlgK and DcuA1 proteins. Therefore, Eha can regulate directly these target genes to affect the survival ability of Edwardsiella tarda within macrophage.
引文
[1] 陈爱平,江育林,钱冬,等.迟缓爱德华氏菌病 [J].中国水产,2011,7:49-50.
[2] Gao D,Cheng J,Zheng E,et al.Eha,a transcriptional regulator of hemolytic activity of Edwardsiella tarda[J].FEMS Microbiol Lett,2014,353 (2):132-140.DOI:10.1111/1574-6968.12420.
[3] 刘念,李玉红,郑恩金,等.基于RNA-seq 分析Eha调控迟缓爱德华菌抵抗酸化的作用[J].中国人兽共患病学报,2017,33(7):25-33.DOI:10.3969/j.issn.1002-2694
[4] 孟祥荣,苏山春,邓仲良,等.建立基于RNA免疫共沉淀技术的鼠疫耶尔森菌Hfq蛋白相关sRNA的体内验证方法[J].生物技术通讯,2013,24(5):631-635.DOI:10.3969/j.issn.1009-0002.2013.05.008
[5] Gao D,Li Y,Zheng E,et al.Eha,a regulator of Edwardsiella tarda,required for resistance to oxidative stress in macrophages[J].FEMS Microbiol Lett.2016:363(20).DOI:10.1093 /femsle/ fnw192
[6] Pitchandi P,Hopper W,Rao R,et al.Comprehensive database of Chorismate synthase enzyme from shikimate pathway in pathogenic bacteria[J].BMC Pharmacol Toxicol,2013,14:29.DOI:10.1186/2050-6511-14-29
[7] Wosten MM,van de Lest CH,van Dijk L,et al.Function and regulation of the C4-dicarboxylate transporters in Campylobacter jejuni[J].Front Microbiol,2017,8:174.DOI:10.3389/fmicb.2017.00174
[8] Goeser L,Fan TJ,Tchaptchet S,et al.Small heat-shock proteins,IbpAB,protect non-pathogenic Escherichia coli from killing by macrophage-derived reactive oxygen species[J].PLoS One,2015,10(3):e0120249.DOI:10.1371/journal.pone.0120249
[9] Yeh HY,Serrano KV,Acosta AS,et al.Production of recombinant Salmonella flagellar protein,FlgK,and its uses in detection of anti-Salmonella antibodies in chickens by automated capillary immunoassay[J].J Microbiologic Methods,2016,122:27-32.DOI:10.1016/j.mimet.2016.01.007
[10] Han Y,Yang CL,Yang Q,et al.Mutation of tryptophanase gene tnaA in Edwardsiella tarda reduces lipopolysaccharide production,antibiotic resistance and virulence[J].Environ Microbiol Rep,2011,3(5):603-612.DOI:10.1111/j.1758-2229.2011.00269.x
[2] Gao D,Cheng J,Zheng E,et al.Eha,a transcriptional regulator of hemolytic activity of Edwardsiella tarda[J].FEMS Microbiol Lett,2014,353 (2):132-140.DOI:10.1111/1574-6968.12420.
[3] 刘念,李玉红,郑恩金,等.基于RNA-seq 分析Eha调控迟缓爱德华菌抵抗酸化的作用[J].中国人兽共患病学报,2017,33(7):25-33.DOI:10.3969/j.issn.1002-2694
[4] 孟祥荣,苏山春,邓仲良,等.建立基于RNA免疫共沉淀技术的鼠疫耶尔森菌Hfq蛋白相关sRNA的体内验证方法[J].生物技术通讯,2013,24(5):631-635.DOI:10.3969/j.issn.1009-0002.2013.05.008
[5] Gao D,Li Y,Zheng E,et al.Eha,a regulator of Edwardsiella tarda,required for resistance to oxidative stress in macrophages[J].FEMS Microbiol Lett.2016:363(20).DOI:10.1093 /femsle/ fnw192
[6] Pitchandi P,Hopper W,Rao R,et al.Comprehensive database of Chorismate synthase enzyme from shikimate pathway in pathogenic bacteria[J].BMC Pharmacol Toxicol,2013,14:29.DOI:10.1186/2050-6511-14-29
[7] Wosten MM,van de Lest CH,van Dijk L,et al.Function and regulation of the C4-dicarboxylate transporters in Campylobacter jejuni[J].Front Microbiol,2017,8:174.DOI:10.3389/fmicb.2017.00174
[8] Goeser L,Fan TJ,Tchaptchet S,et al.Small heat-shock proteins,IbpAB,protect non-pathogenic Escherichia coli from killing by macrophage-derived reactive oxygen species[J].PLoS One,2015,10(3):e0120249.DOI:10.1371/journal.pone.0120249
[9] Yeh HY,Serrano KV,Acosta AS,et al.Production of recombinant Salmonella flagellar protein,FlgK,and its uses in detection of anti-Salmonella antibodies in chickens by automated capillary immunoassay[J].J Microbiologic Methods,2016,122:27-32.DOI:10.1016/j.mimet.2016.01.007
[10] Han Y,Yang CL,Yang Q,et al.Mutation of tryptophanase gene tnaA in Edwardsiella tarda reduces lipopolysaccharide production,antibiotic resistance and virulence[J].Environ Microbiol Rep,2011,3(5):603-612.DOI:10.1111/j.1758-2229.2011.00269.x