双组分系统rcsC基因影响禽致病性大肠杆菌的致病性及相关生物学特性
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摘要
【目的】双组分系统Rcs感受外界环境变化,并调控细菌的适应性及生存等。本文探讨Rcs双组分系统传感器激酶RcsC对禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)相关生物学特性及致病性的影响。【方法】采用Red同源重组的方法构建rcsC基因缺失株,并利用互补质粒构建互补株,然后比较野生株、基因缺失株与互补株的生长特性、运动性、生物被膜、凝集沉淀能力、致病力及毒力基因转录水平的差异。【结果】rcsC基因缺失不影响APEC的生长速度,然而,缺失RcsC导致APEC的运动能力升高、生物被膜形成能力降低和凝集能力增强。凝集试验结果显示rcsC基因有助于APEC的凝集沉降。细胞黏附入侵结果表明,rcsC在APEC侵袭DF-1细胞过程中发挥作用,而对黏附能力无影响。动物感染试验结果表明rcsC基因缺失能显著降低APEC的毒力。荧光定量PCR检测结果表明,rcsC基因缺失株中ompA、aatA、fyuA和luxS基因的转录水平均显著降低,而fimC和tsh基因的转录水平显著升高。【结论】RcsC参与调控APEC的运动性、生物被膜形成、凝集沉降和致病力。
[Objective] The two-component system(TCS) Rcs is involved in the regulation of bacterial adaptation to external environment and survival. The aim of this study is to elucidate effects of sensor kinase RcsC on associated biological characteristics and virulence of avian pathogenic Escherichia coli(APEC). [Methods] We constructed the rcsC gene mutant and complementary strain of APEC by the Red recombination system and complementation plasmid. Then we compared the growth curve, motility, biofilm formation, agglutination, pathogenicity and virulence gene transcription levels of mutant, wild-type and complementary strains. [Results] rcsC gene deletion had no influence on bacterial growth. However, inactivation of rcsC gene resulted in enhanced motility, decreased biofilm formation and increased agglutination. Bacterial adherence and invasion assays showed that RcsC contribute to the invasion of APEC to DF-1 cells, whereas, it did not affect the adhesion capacity. Moreover, rcsC gene mutant strain exhibited attenuated virulence in ducks. Quantitative real-time PCR analysis demonstrated that the transcription levels of virulence genes ompA, aatA, fyuA and luxS were significantly decreased in mutant strain. Whereas, the fimC and tsh transcription levels were significantly upregulated. [Conclusion] These data indicated that RcsC play roles in regulation of bacterial motility, biofilm formation, agglutination and virulence of APEC.
[Objective] The two-component system(TCS) Rcs is involved in the regulation of bacterial adaptation to external environment and survival. The aim of this study is to elucidate effects of sensor kinase RcsC on associated biological characteristics and virulence of avian pathogenic Escherichia coli(APEC). [Methods] We constructed the rcsC gene mutant and complementary strain of APEC by the Red recombination system and complementation plasmid. Then we compared the growth curve, motility, biofilm formation, agglutination, pathogenicity and virulence gene transcription levels of mutant, wild-type and complementary strains. [Results] rcsC gene deletion had no influence on bacterial growth. However, inactivation of rcsC gene resulted in enhanced motility, decreased biofilm formation and increased agglutination. Bacterial adherence and invasion assays showed that RcsC contribute to the invasion of APEC to DF-1 cells, whereas, it did not affect the adhesion capacity. Moreover, rcsC gene mutant strain exhibited attenuated virulence in ducks. Quantitative real-time PCR analysis demonstrated that the transcription levels of virulence genes ompA, aatA, fyuA and luxS were significantly decreased in mutant strain. Whereas, the fimC and tsh transcription levels were significantly upregulated. [Conclusion] These data indicated that RcsC play roles in regulation of bacterial motility, biofilm formation, agglutination and virulence of APEC.
引文
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[11]Wang SH,Dai JJ,Meng QM,Han XG,Han Y,Zhao YC,Yang DH,Ding C,Yu SQ.DotU expression is highly induced during in vivo infection and responsible for virulence and Hcp1 secretion in avian pathogenic Escherichia coli.Frontiers in Microbiology,2014,5:588.
[12]Datsenko KA,Wanner BL.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCRproducts.Proceedings of the National Academy of Sciences of the United States of America,2000,97(12):6640-6645.
[13]Wang SH,Xia YJ,Dai JJ,Shi ZY,Kou YH,Li HQ,Bao YL,Lu CP.Novel roles for autotransporter adhesin AatA of avian pathogenic Escherichia coli:colonization during infection and cell aggregation.FEMS Immunology and Medical Microbiology,2011,63(3):328-338.
[14]Wang D,Wang SH,Xu F,Meng QM,Liu X,Xu X,Yang DH,Han XG,Ding C,Zhang HR,Yu SQ.Effects of typeⅥsecretion system 2 core component ClpB on the biological characteristics and virulence of avian pathogenic Escherichia coli.Chinese Veterinary Science,2016,46(7):834-840.(in Chinese)王栋,王少辉,徐凤,孟庆美,刘新,许漩,杨登辉,韩先干,丁铲,张焕容,于圣青.Ⅵ型分泌系统2核心组分ClpB对禽致病性大肠杆菌生物学特性及致病性的影响.中国兽医科学,2016,46(7):834-840.
[15]Wang SH,Niu CL,Shi ZY,Xia YJ,Yaqoob M,Dai JJ,Lu CP.Effects of ibeA deletion on virulence and biofilm formation of avian pathogenic Escherichia coli.Infection and Immunity,2011,79(1):279-287.
[16]Han XG,Bai H,Liu L,Dong HL,Liu R,Song J,Ding C,Qi KZ,Liu HW,Yu SQ.The luxS gene functions in the pathogenesis of avian pathogenic Escherichia coli.Microbial Pathogenesis,2013,55:21-27.
[17]Schmittgen TD,Livak KJ.Analyzing real-time PCR data by the comparative CT method.Nature Protocols,2008,3(6):1101-1108.
[18]Ishihama A.Prokaryotic genome regulation:multifactor promoters,multitarget regulators and hierarchic networks.FEMS Microbiology Reviews,2010,34(5):628-645.
[19]Gao QQ,Ye ZQ,Wang XB,Mu XH,Gao S,Liu XF.RstA is required for the virulence of an avian pathogenic Escherichia coli O2 strain E058.Infection,Genetics and Evolution,2015,29:180-188.
[20]Li CX,Zhang YX,Qi KZ,Han XG,Tu J,Xue T,Zhou XH.The role of phoP/Q two-component system in virulence of avian pathogenic Escherichia coli.Acta Veterinaria et Zootechnica Sinica,2016,47(1):157-164.(in Chinese)李春晓,张宇曦,祁克宗,韩先干,涂健,薛挺,周秀红.phoP/Q双组分系统对禽致病性大肠杆菌的毒力调控作用.畜牧兽医学报,2016,47(1):157-164.
[21]Francez-Charlot A,Laugel B,van Gemert A,Dubarry N,Wiorowski F,Castanié-Cornet MP,Gutierrez C,Cam K.RcsCDB His-Asp phosphorelay system negatively regulates the flhDC operon in Escherichia coli.Molecular Microbiology,2003,49(3):823-832.
[22]Wang QF,Zhao YF,McClelland M,Harshey RM.The RcsCDB signaling system and swarming motility in Salmonella enterica serovar typhimurium:dual regulation of flagellar and SPI-2 virulence genes.Journal of Bacteriology,2007,189(23):8447-8457.
[23]Ferrières L,Clarke DJ.The RcsC sensor kinase is required for normal biofilm formation in Escherichia coli K-12 and controls the expression of a regulon in response to growth on a solid surface.Molecular Microbiology,2003,50(5):1665-1682.
[24]Prigent-Combaret C,Prensier G,Le Thi TT,Vidal O,Lejeune P,Dorel C.Developmental pathway for biofilm formation in curli-producing Escherichia coli strains:role of flagella,curli and colanic acid.Environmental Microbiology,2000,2(4):450-464.
[25]Oropeza R,Salgado-Bravo R,Calva E.Deletion analysis of RcsC reveals a novel signalling pathway controlling poly-N-acetylglucosamine synthesis and biofilm formation in Escherichia coli.Microbiology,2015,161(4):903-913.
[26]Finlay BB,Falkow S.Common themes in microbial pathogenicity revisited.Microbiology and Molecular Biology Reviews,1997,61(2):136-169.
[27]Mouslim C,Delgado M,Groisman EA.Activation of the RcsC/YojN/RcsB phosphorelay system attenuates Salmonella virulence.Molecular Microbiology,2004,54(2):386-395.
[28]Tobe T,Ando H,Ishikawa H,Abe H,Tashiro K,Hayashi T,Kuhara S,Sugimoto N.Dual regulatory pathways integrating the RcsC-RcsD-RcsB signalling system control enterohaemorrhagic Escherichia coli pathogenicity.Molecular Microbiology,2005,58(1):320-333.
[29]Gotoh Y,Eguchi Y,Watanabe T,Okamoto S,Doi A,Utsumi R.Two-component signal transduction as potential drug targets in pathogenic bacteria.Current Opinion in Microbiology,2010,13(2):232-239.
[2]Bergeron CR,Prussing C,Boerlin P,Daignault D,Dutil L,Reid-Smith RJ,Zhanel GG,Manges AR.Chicken as reservoir for extraintestinal pathogenic Escherichia coli in humans,Canada.Emerging Infectious Diseases,2012,18(3):415-421.
[3]Tivendale KA,Logue CM,Kariyawasam S,Jordan D,Hussein A,Li GW,Wannemuehler Y,Nolan LK.Avian-pathogenic Escherichia coli strains are similar to neonatal meningitis E.coli strains and are able to cause meningitis in the rat model of human disease.Infection and Immunity,2010,78(8):3412-3419.
[4]Mora A,Viso S,López C,Alonso MP,García-Garrote F,Dabhi G,Mamani R,Herrera A,Marzoa J,Blanco M,Blanco JE,Moulin-Schouleur M,Schouler C,Blanco J.Poultry as reservoir for extraintestinal pathogenic Escherichia coli O45:K1:H7-B2-ST95 in humans.Veterinary Microbiology,2013,167(3/4):506-512.
[5]Stock AM,Robinson VL,Goudreau PN.Two-component signal transduction.Annual Review of Biochemistry,2000,69(1):183-215.
[6]Koretke KK,Lupas AN,Warren PV,Rosenberg M,Brown JR.Evolution of two-component signal transduction.Molecular Biology and Evolution,2000,17(12):1956-1970.
[7]Hinchliffe SJ,Howard SL,Huang YH,Clarke DJ,Wren BW.The importance of the Rcs phosphorelay in the survival and pathogenesis of the enteropathogenic yersiniae.Microbiology,2008,154(4):1117-1131.
[8]Majdalani N,Gottesman S.The Rcs phosphorelay:a complex signal transduction system.Annual Review of Biochemistry,2005,59:379-405.
[9]Erickson KD,Detweiler CS.The Rcs phosphorelay system is specific to enteric pathogens/commensals and activates ydeI,a gene important for persistent Salmonella infection of mice.Molecular Microbiology,2006,62(3):883-894.
[10]Huang YH,Ferrières L,Clarke DJ.The role of the Rcs phosphorelay in Enterobacteriaceae.Research in Microbiology,2006,157(3):206-212.
[11]Wang SH,Dai JJ,Meng QM,Han XG,Han Y,Zhao YC,Yang DH,Ding C,Yu SQ.DotU expression is highly induced during in vivo infection and responsible for virulence and Hcp1 secretion in avian pathogenic Escherichia coli.Frontiers in Microbiology,2014,5:588.
[12]Datsenko KA,Wanner BL.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCRproducts.Proceedings of the National Academy of Sciences of the United States of America,2000,97(12):6640-6645.
[13]Wang SH,Xia YJ,Dai JJ,Shi ZY,Kou YH,Li HQ,Bao YL,Lu CP.Novel roles for autotransporter adhesin AatA of avian pathogenic Escherichia coli:colonization during infection and cell aggregation.FEMS Immunology and Medical Microbiology,2011,63(3):328-338.
[14]Wang D,Wang SH,Xu F,Meng QM,Liu X,Xu X,Yang DH,Han XG,Ding C,Zhang HR,Yu SQ.Effects of typeⅥsecretion system 2 core component ClpB on the biological characteristics and virulence of avian pathogenic Escherichia coli.Chinese Veterinary Science,2016,46(7):834-840.(in Chinese)王栋,王少辉,徐凤,孟庆美,刘新,许漩,杨登辉,韩先干,丁铲,张焕容,于圣青.Ⅵ型分泌系统2核心组分ClpB对禽致病性大肠杆菌生物学特性及致病性的影响.中国兽医科学,2016,46(7):834-840.
[15]Wang SH,Niu CL,Shi ZY,Xia YJ,Yaqoob M,Dai JJ,Lu CP.Effects of ibeA deletion on virulence and biofilm formation of avian pathogenic Escherichia coli.Infection and Immunity,2011,79(1):279-287.
[16]Han XG,Bai H,Liu L,Dong HL,Liu R,Song J,Ding C,Qi KZ,Liu HW,Yu SQ.The luxS gene functions in the pathogenesis of avian pathogenic Escherichia coli.Microbial Pathogenesis,2013,55:21-27.
[17]Schmittgen TD,Livak KJ.Analyzing real-time PCR data by the comparative CT method.Nature Protocols,2008,3(6):1101-1108.
[18]Ishihama A.Prokaryotic genome regulation:multifactor promoters,multitarget regulators and hierarchic networks.FEMS Microbiology Reviews,2010,34(5):628-645.
[19]Gao QQ,Ye ZQ,Wang XB,Mu XH,Gao S,Liu XF.RstA is required for the virulence of an avian pathogenic Escherichia coli O2 strain E058.Infection,Genetics and Evolution,2015,29:180-188.
[20]Li CX,Zhang YX,Qi KZ,Han XG,Tu J,Xue T,Zhou XH.The role of phoP/Q two-component system in virulence of avian pathogenic Escherichia coli.Acta Veterinaria et Zootechnica Sinica,2016,47(1):157-164.(in Chinese)李春晓,张宇曦,祁克宗,韩先干,涂健,薛挺,周秀红.phoP/Q双组分系统对禽致病性大肠杆菌的毒力调控作用.畜牧兽医学报,2016,47(1):157-164.
[21]Francez-Charlot A,Laugel B,van Gemert A,Dubarry N,Wiorowski F,Castanié-Cornet MP,Gutierrez C,Cam K.RcsCDB His-Asp phosphorelay system negatively regulates the flhDC operon in Escherichia coli.Molecular Microbiology,2003,49(3):823-832.
[22]Wang QF,Zhao YF,McClelland M,Harshey RM.The RcsCDB signaling system and swarming motility in Salmonella enterica serovar typhimurium:dual regulation of flagellar and SPI-2 virulence genes.Journal of Bacteriology,2007,189(23):8447-8457.
[23]Ferrières L,Clarke DJ.The RcsC sensor kinase is required for normal biofilm formation in Escherichia coli K-12 and controls the expression of a regulon in response to growth on a solid surface.Molecular Microbiology,2003,50(5):1665-1682.
[24]Prigent-Combaret C,Prensier G,Le Thi TT,Vidal O,Lejeune P,Dorel C.Developmental pathway for biofilm formation in curli-producing Escherichia coli strains:role of flagella,curli and colanic acid.Environmental Microbiology,2000,2(4):450-464.
[25]Oropeza R,Salgado-Bravo R,Calva E.Deletion analysis of RcsC reveals a novel signalling pathway controlling poly-N-acetylglucosamine synthesis and biofilm formation in Escherichia coli.Microbiology,2015,161(4):903-913.
[26]Finlay BB,Falkow S.Common themes in microbial pathogenicity revisited.Microbiology and Molecular Biology Reviews,1997,61(2):136-169.
[27]Mouslim C,Delgado M,Groisman EA.Activation of the RcsC/YojN/RcsB phosphorelay system attenuates Salmonella virulence.Molecular Microbiology,2004,54(2):386-395.
[28]Tobe T,Ando H,Ishikawa H,Abe H,Tashiro K,Hayashi T,Kuhara S,Sugimoto N.Dual regulatory pathways integrating the RcsC-RcsD-RcsB signalling system control enterohaemorrhagic Escherichia coli pathogenicity.Molecular Microbiology,2005,58(1):320-333.
[29]Gotoh Y,Eguchi Y,Watanabe T,Okamoto S,Doi A,Utsumi R.Two-component signal transduction as potential drug targets in pathogenic bacteria.Current Opinion in Microbiology,2010,13(2):232-239.