Transcription factor, Eha directly regulates the target genes of Edwardsiella tarda
TIAN Chang1, LIU Nian1, ZHENG En-jin1, GAO Da-qing1, LU Cheng-ping2
1.School of Medicine, Southeast University, Nanjing 210009,China; 2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Abstract：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-4T-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-PtargetLacZ, and introduced the combination vector into the eha mutant and the wild strain by electric shock. We compared the β-gal activity of pBAD-PtargetLacZ-Δeha strains with the one of pBAD-PtargetLacZ-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.
[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
2019, Vol. 35 
