嗜水气单胞菌AraC家族转录调控因子AetY的功能
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摘要
为研究嗜水气单胞菌中国流行菌株NJ-35的AraC家族转录调控因子AetY(U876_01475)的功能,本实验采用同源重组技术构建了该基因的缺失株ΔaetY及互补株CΔaetY,并对野生株、缺失株及互补株生物被膜形成能力、自凝集活性、对恩诺沙星的耐药性和对斑马鱼的致病力进行检测。结果显示,与野生株相比,ΔaetY生物被膜形成能力、自凝集活性、对恩诺沙星的最小抑菌浓度(MIC)均显著降低,对斑马鱼致病力明显降低,半数致死量(LD_(50))增加400余倍。CΔaetY各项指标均恢复至野生株水平。本研究首次针对嗜水气单胞菌AraC家族成员进行了基因功能分析,为进一步探究该菌的致病机理提供参考。
In order to investigate the function of AraC-family transcriptional factor AetY(U876_01475), we constructed the aetY deletion mutant strain ΔaetY using homologous recombination with Aeromonas hydrophila Chinese epidemic strain NJ-35 as a parental strain. The corresponding gene-complemented strain CΔaetY was also constructed. Biofilm formation ability, auto-aggregation activity, drug resistance to enrofloxacin and pathogenicity were detected in NJ-35, ΔaetY and CΔaetY strains. The inactivation of aetY caused a significant decrease in biofilm formation, auto-aggregation and MIC value for enrofloxacin. The virulence of ΔaetY to Danio rerio was significantly reduced, and the LD_(50) value had an increase of more than 400 times. The above characteristics of CΔaetY were restored to the same level of the wide type strain. This study for the first time investigated the function of AraC-family transcriptional factor AetY in A. hydrophila, which will provide a basis for further exploration of AetY role in pathogenic mechanism.
In order to investigate the function of AraC-family transcriptional factor AetY(U876_01475), we constructed the aetY deletion mutant strain ΔaetY using homologous recombination with Aeromonas hydrophila Chinese epidemic strain NJ-35 as a parental strain. The corresponding gene-complemented strain CΔaetY was also constructed. Biofilm formation ability, auto-aggregation activity, drug resistance to enrofloxacin and pathogenicity were detected in NJ-35, ΔaetY and CΔaetY strains. The inactivation of aetY caused a significant decrease in biofilm formation, auto-aggregation and MIC value for enrofloxacin. The virulence of ΔaetY to Danio rerio was significantly reduced, and the LD_(50) value had an increase of more than 400 times. The above characteristics of CΔaetY were restored to the same level of the wide type strain. This study for the first time investigated the function of AraC-family transcriptional factor AetY in A. hydrophila, which will provide a basis for further exploration of AetY role in pathogenic mechanism.
引文
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[22]Zalucki Y M,Dhulipala V,Shafer W M.Dueling regulatory properties of a transcriptional activator(Mtr A)and repressor(Mtr R)that control efflux pump gene expression in Neisseria gonorrhoeae[J].m Bio,2012,3(6):e00446-12.
[23]Jiménez-Castellanos J C,Wan Ahmad Kamil W N I,Cheung C H P,et al.Comparative effects of overproducing the Ara C-type transcriptional regulators Mar A,Sox S,Rar A and Ram A on antimicrobial drug susceptibility in Klebsiella pneumoniae[J].Journal of Antimicrobial Chemotherapy,2016,71(7):1820-1825.
[24]Duval V,Lister I M.Mar A,Sox S and Rob of Escherichia coli-Global regulators of multidrug resistance,virulence and stress response[J].International Journal of Biotechnology for Wellness Industries,2013,2(3):101-124.
[2]Pang M D,Jiang J W,Xie X,et al.Novel insights into the pathogenicity of epidemic Aeromonas hydrophila ST251 clones from comparative genomics[J].Scientific Reports,2015,5:9833.
[3]胡萌,潘子豪,陆承平,等.嗜水气单胞菌流行菌株的生物学特性[J].中国兽医科学,2013,43(5):441-445.Hu M,Pan Z H,Lu C P,et al.Biological characterization of epidemic Aeromonas hydrophila strains[J].Chinese Veterinary Science,2013,43(5):441-445(in Chinese).
[4]任良云.基于转录因子结合位点的特性对转录因子分类的新方法[D].济南:山东大学,2012.Ren L Y.A novel method for classification of transcription factors based on properties of transcription factor binding sites[D].Ji’nan:Shandong University,2012(in Chinese).
[5]Tobes R,Ramos J L.Ara C-Xyl S database:a family of positive transcriptional regulators in bacteria[J].Nucleic Acids Research,2002,30(1):318-321.
[6]庞茂达.嗜水气单胞菌流行株基因组特征及毒力相关基因研究[D].南京:南京农业大学,2015.Pang M D.Genomic characteristics and virulence-associated genes analysis of Aeromonas hydrophila[D].Nanjing:Nanjing Agricultural University,2015(in Chinese).
[7]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2?￠￠CT method[J].Methods,2001,25(4):402-408.
[8]Stepanovi?S,Vukovi?D,Daki?I,et al.A modified microtiter-plate test for quantification of Staphylococcal biofilm formation[J].Journal of Microbiological Methods,2000,40(2):175-179.
[9]吴亚锋,王旭远,庞茂达,等.水族箱气单胞菌的鉴定及致病特性分析[J].水产学报,2015,39(4):573-579.Wu Y F,Wang X Y,Pang M D,et al.Identification and pathogenic characteristics of Aeromonas aquariorum,a new member of the genus Aeromonas[J].Journal of Fisheries of China,2015,39(4):573-579(in Chinese).
[10]Frota C C,Papavinasasundaram K G,Davis E O,et al.The Ara C family transcriptional regulator Rv1931c plays a role in the virulence of Mycobacterium tuberculosis[J].Infection and Immunity,2004,72(9):5483-5486.
[11]Rowe S E,Campbell C,Lowry C,et al.Ara C-type regulator Rbf controls the Staphylococcus epidermidis biofilm phenotype by negatively regulating the ica ADBC repressor Sar R[J].Journal of Bacteriology,2016,198(21):2914-2924.
[12]Li T M,He L,Song Y,et al.Ara C-type regulator Rsp adapts Staphylococcus aureus gene expression to acute infection[J].Infection and Immunity,2016,84(3):723-734.
[13]Krukonis E S,Yu R R,Dirita V J.The Vibrio cholerae Tox R/Tcp P/Tox T virulence cascade:distinct roles for two membrane-localized transcriptional activators on a single promoter[J].Molecular Microbiology,2000,38(1):67-84.
[14]Withey J H,Di Rita V J.Activation of both acf A and acf D transcription by Vibrio cholerae Tox T requires binding to two centrally located DNA sites in an inverted repeat conformation[J].Molecular Microbiology,2005,56(4):1062-1077.
[15]Lowden M J,Skorupski K,Pellegrini M,et al.Structure of Vibrio cholerae Tox T reveals a mechanism for fatty acid regulation of virulence genes[J].Proceedings of the National Academy of Sciences of the United States of America,2010,107(7):2860-2865.
[16]Hsiao A,Liu Z,Joelsson A,et al.Vibrio cholerae vir-ulence regulator-coordinated evasion of host immunity[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(39):14542-14547.
[17]Schwiesow L,Lam H,Dersch P,et al.Yersinia Type III secretion system master regulator Lcr F[J].Journal of Bacteriology,2016,198(4):604-614.
[18]Emanuele A A,Garcia G A.Mechanism of action and initial,in vitro SAR of an inhibitor of the Shigella flexneri virulence regulator Vir F[J].PLo S One,2015,10(9):e0137410.
[19]Zhao Y H,Shaw J G.Cross-talk between the Aeromonas hydrophila Type III secretion system and lateral flagella system[J].Frontiers in Microbiology,2016,7:1434.
[20]崔佳佳.淡水鱼源嗜水气单胞菌耐药机制初探[D].上海:上海海洋大学,2016.Cui J J.Study on drug resistance mechanism of Aeromonas hydrophila isolated from freshwater fish species[D].Shanghai:Shanghai Ocean University,2016(in Chinese).
[21]方一风,潘晓艺,蔺凌云,等.嗜水气单胞菌对喹诺酮类药物耐药的分子机制[J].微生物学报,2014,54(2):174-182.Fang Y F,Pan X Y,Lin L Y,et al.Molecular mechanisms of quinolone resistance in Aeromonas hydrophilia[J].Acta Microbiologica Sinica,2014,54(2):174-182(in Chinese).
[22]Zalucki Y M,Dhulipala V,Shafer W M.Dueling regulatory properties of a transcriptional activator(Mtr A)and repressor(Mtr R)that control efflux pump gene expression in Neisseria gonorrhoeae[J].m Bio,2012,3(6):e00446-12.
[23]Jiménez-Castellanos J C,Wan Ahmad Kamil W N I,Cheung C H P,et al.Comparative effects of overproducing the Ara C-type transcriptional regulators Mar A,Sox S,Rar A and Ram A on antimicrobial drug susceptibility in Klebsiella pneumoniae[J].Journal of Antimicrobial Chemotherapy,2016,71(7):1820-1825.
[24]Duval V,Lister I M.Mar A,Sox S and Rob of Escherichia coli-Global regulators of multidrug resistance,virulence and stress response[J].International Journal of Biotechnology for Wellness Industries,2013,2(3):101-124.