霍乱弧菌ToxS蛋白间的互作增强ToxR蛋白诱导毒力基因的表达
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摘要
【目的】阐明霍乱弧菌ToxR蛋白功能调控的分子机制。【方法】利用巯基捕获(thiol-trapping)的方法分析DsbA蛋白对ToxR周质空间结构域半胱氨酸残基的氧化作用;采用定点突变的方法构建ToxR半胱氨酸突变株(ToxR_(C236/293S));利用荧光素酶基因作为报告基因分析ToxR野生型(ToxR_(wt))和半胱氨酸突变体(ToxR_(C236/293S))诱导下游基因表达的活性;通过细菌双杂交系统分析ToxR_(wt)和ToxR_(C236/293S)蛋白之间、ToxR与ToxS之间以及ToxS之间的相互作用。【结果】ToxR周质空间结构域半胱氨酸残基确实可以被DsbA蛋白氧化,且当ToxR与ToxS共表达时,ToxR诱导ctxAB转录表达的活性显著增强,且在dsbA基因缺失突变株中ToxR诱导ctxAB转录表达的活性更高;成功构建株霍乱弧菌ToxR半胱氨酸突变株(ToxR_(C236/293S)),在没有ToxS存在的条件下,ToxR_(C236/293S)诱导毒力基因表达的活性与ToxRwt相当;细菌双杂交系统分析发现当ToxR与ToxS共转录表达时,ToxS极大增强ToxR蛋白之间的互作;在dsbA基因缺失突变株中,ToxS之间的相互作用显著增强。【结论】ToxR蛋白本身的氧还状态对其诱导毒力基因表达的活性没有影响;ToxS通过增强ToxR形成二聚体的能力从而增强其诱导毒力基因的表达,而DsbA对ToxS蛋白之间的相互作用具有抑制作用,DsbA通过影响ToxS的蛋白互作从而影响ToxR蛋白的功能。本文为进一步阐明霍乱弧菌毒力基因表达调控的分子机制提供重要的理论依据。
[Objective] In this study, we demonstrated the mechanism of ToxR activating virulence gene expression by modifying its protein function in Vibrio cholerae. [Methods] ToxR redox status assay with or without DsbA was analyzed by thiol-trapping assay. ToxR cysteine mutant ToxR_(C263/293S) was obtained by site-directed mutagenesis.Escherichia coli strains containing ctxAB promoter luxCDABE transcriptional fusion and a plasmid-coded ToxR expressed under the control of arabinose were used to analyze the transcriptional level. ToxR and ToxS proteins interaction was detected by the bacterial two-hybrid system. [Results] The two cysteines in the periplasmic domain of ToxR could be oxidized by DsbA. When ToxR and ToxS were co-transcribed under the control of the same promoter, ToxR activated ctxAB expression to a higher level in E. coli dsbA knock out mutant. However, when ToxR was expressed alone without ToxS, the redox status of ToxR had no effect on its activation of ctxAB expression in E. coli. Bacterial two-hybrid system assay showed that ToxS greatly enhanced ToxR homodimerization regardless of the redox status of ToxR, and DsbA strongly repressed ToxS-ToxS interaction.[Conclusion] The redox status of ToxR has no effect on its activating virulence gene expression and ToxS enhances ToxR activity by promoting ToxR homodimerization. DsbA indirectly affects ToxR activating virulence genes expression by repressing ToxS homodimerization.
[Objective] In this study, we demonstrated the mechanism of ToxR activating virulence gene expression by modifying its protein function in Vibrio cholerae. [Methods] ToxR redox status assay with or without DsbA was analyzed by thiol-trapping assay. ToxR cysteine mutant ToxR_(C263/293S) was obtained by site-directed mutagenesis.Escherichia coli strains containing ctxAB promoter luxCDABE transcriptional fusion and a plasmid-coded ToxR expressed under the control of arabinose were used to analyze the transcriptional level. ToxR and ToxS proteins interaction was detected by the bacterial two-hybrid system. [Results] The two cysteines in the periplasmic domain of ToxR could be oxidized by DsbA. When ToxR and ToxS were co-transcribed under the control of the same promoter, ToxR activated ctxAB expression to a higher level in E. coli dsbA knock out mutant. However, when ToxR was expressed alone without ToxS, the redox status of ToxR had no effect on its activation of ctxAB expression in E. coli. Bacterial two-hybrid system assay showed that ToxS greatly enhanced ToxR homodimerization regardless of the redox status of ToxR, and DsbA strongly repressed ToxS-ToxS interaction.[Conclusion] The redox status of ToxR has no effect on its activating virulence gene expression and ToxS enhances ToxR activity by promoting ToxR homodimerization. DsbA indirectly affects ToxR activating virulence genes expression by repressing ToxS homodimerization.
引文
[1]Davies HG,Bowman C,Luby SP.Cholera-management and prevention.Journal of Infection,2017,74(Suppl 1):S66-S73.
[2]Hall RH.Curbing cholera.Science Translational Medicine,2018,10(445):eaat9483.
[3]Krukonis ES,Di Rita VJ.From motility to virulence:sensing and responding to environmental signals in Vibrio cholerae.Current Opinion in Microbiology,2013,6(2):186-190.
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[5]Hung DT,Mekalanos JJ.Bile acids induce cholera toxin expression in Vibrio cholerae in a Tox T-independent manner.Proceedings of the National Academy of Sciences of the United States of America,2005,102(8):3028-3033.
[6]Taylor JL,de Silva RS,Kovacikova G,Lin W,Taylor RK,Skorupski K,Kull FJ.The crystal structure of Aph B,a virulence gene activator from Vibrio cholerae,reveals residues that influence its response to oxygen and p H.Molecular Microbiology,2012,83(3):457-470.
[7]Mazumdar S,Bhattacharyya S,Ghosh S,Majumdar S,Ganguly NK.The role of a heat shock protein from V.cholerae 0139 in the gut immune response.Molecular and Cellular Biochemistry,2007,297(1/2):9-19.
[8]Spagnuolo AM,Dirita V,Kirschner D.A model for Vibrio cholerae colonization of the human intestine.Journal of Theoretical Biology,2011,289:247-258.
[9]Morgan SJ,Felek S,Gadwal S,Koropatkin NM,Perry JW,Bryson AB,Krukonis ES.The two faces of Tox R:activator of omp U,co-regulator of tox T in Vibrio cholerae.Molecular Microbiology,2011,81(1):113-128.
[10]Kazi MI,Conrado AR,Mey AR,Payne SM,Davies BW.Toxr antagonizes H-NS regulation of horizontally acquired genes to drive host colonization.PLo S Pathogens,2016,12(4):e1005570.
[11]Di Rita VJ,Mekalanos JJ.Periplasmic interaction between two membrane regulatory proteins,Tox R and Tox S,results in signal transduction and transcriptional activation.Cell,1991,64(1):29-37.
[12]Midgett CR,Almagro-Moreno S,Pellegrini M,Taylor RK,Skorupski K,Kull FJ.Bile salts and alkaline p H reciprocally modulate the interaction between the periplasmic domains of Vibrio cholerae Tox R and Tox S.Molecular Microbiology,2017,105(2):258-272.
[13]Almagro-Moreno S,Root MZ,Taylor RK.Role of Tox S in the proteolytic cascade of virulence regulator Tox R in Vibrio cholerae.Molecular Microbiology,2015,98(5):963-976.
[14]Peterson KM.Expression of Vibrio cholerae virulence genes in response to environmental signals.Current Issues in Intestinal Microbiology,2002,3(2):29-38.
[15]Xue YY,Tu F,Shi MT,Wu CQ,Ren GP,Wang XJ,Fang WH,Song HH,Yang MH.Redox pathway sensing bile salts activates virulence gene expression in Vibrio cholerae.Molecular Microbiology,2016,102(5):909-924.
[16]Yang MH,Liu Z,Hughes C,Stern AM,Wang H,Zhong ZT,Kan B,Fenical W,Zhu J.Bile salt-induced intermolecular disulfide bond formation activates Vibrio cholerae virulence.Proceedings of the National Academy of Sciences of the United States of America,2013,110(6):2348-2353.
[17]Heras B,Shouldice SR,Totsika M,Scanlon MJ,Schembri MA,Martin JL.DSB proteins and bacterial pathogenicity.Nature Reviews Microbiology,2009,7(3):215-225.
[18]Ren GP,Champion MM,Huntley JF.Identification of disulfide bond isomerase substrates reveals bacterial virulence factors.Molecular Microbiology,2014,94(4):926-944.
[19]Karimova G,Pidoux J,Ullmann A,Ladant D.A bacterial two-hybrid system based on a reconstituted signal transduction pathway.Proceedings of the National Academy of Sciences of the United States of America,1998,95(10):5752-5756.
[20]Lippa AM,Goulian M.Perturbation of the oxidizing environment of the periplasm stimulates the Pho Q/Pho Psystem in Escherichia coli.Journal of Bacteriology,2012,194(6):1457-1463.
[21]Miller J.Experiments in Molecular Genetics.Cold Spring Harbor,N.Y.:Cold Spring Harbor Laboratory Press,1972.
[22]Herrington DA,Hall RH,Losonsky G,Mekalanos JJ,Taylor RK,Levine MM.Toxin,toxin-coregulated pili,and the tox Rregulon are essential for Vibrio cholerae pathogenesis in humans.The Journal of Experimental Medicine,1988,168(4):1487-1492.
[23]Miller VL,Taylor RK,Mekalanos JJ.Cholera toxin transcriptional activator tox R is a transmembrane DNAbinding protein.Cell,1987,48(2):271-279.
[24]Goss TJ,Seaborn CP,Gray MD,Krukonis ES.Identification of the Tcp P-binding site in the tox T promoter of Vibrio cholerae and the role of Tox R in Tcp P-mediated activation.Infection and Immunity,2010,78(10):4122-4133.
[25]Champion GA,Neely MN,Brennan MA,Di Rita VJ.A branch in the Tox R regulatory cascade of Vibrio cholerae revealed by characterization of tox T mutant strains.Molecular Microbiology,1997,23(2):323-331.
[26]Wang QM,Ma Y,Liu LJ,Zhu J,Liu Z.Biofilm development and environmental determinants in Vibrio cholerae.Chinese Journal of Biotechnology,2017,33(9):1533-1546.(in Chinese)王全民,马遥,刘丽钧,朱军,刘智.霍乱弧菌生物被膜发育与环境调控.生物工程学报,2017,33(9):1533-1546.
[2]Hall RH.Curbing cholera.Science Translational Medicine,2018,10(445):eaat9483.
[3]Krukonis ES,Di Rita VJ.From motility to virulence:sensing and responding to environmental signals in Vibrio cholerae.Current Opinion in Microbiology,2013,6(2):186-190.
[4]Matson JS,Withey JH,Di Rita VJ.Regulatory networks controlling Vibrio cholerae virulence gene expression.Infection and Immunity,2007,75(12):5542-5549.
[5]Hung DT,Mekalanos JJ.Bile acids induce cholera toxin expression in Vibrio cholerae in a Tox T-independent manner.Proceedings of the National Academy of Sciences of the United States of America,2005,102(8):3028-3033.
[6]Taylor JL,de Silva RS,Kovacikova G,Lin W,Taylor RK,Skorupski K,Kull FJ.The crystal structure of Aph B,a virulence gene activator from Vibrio cholerae,reveals residues that influence its response to oxygen and p H.Molecular Microbiology,2012,83(3):457-470.
[7]Mazumdar S,Bhattacharyya S,Ghosh S,Majumdar S,Ganguly NK.The role of a heat shock protein from V.cholerae 0139 in the gut immune response.Molecular and Cellular Biochemistry,2007,297(1/2):9-19.
[8]Spagnuolo AM,Dirita V,Kirschner D.A model for Vibrio cholerae colonization of the human intestine.Journal of Theoretical Biology,2011,289:247-258.
[9]Morgan SJ,Felek S,Gadwal S,Koropatkin NM,Perry JW,Bryson AB,Krukonis ES.The two faces of Tox R:activator of omp U,co-regulator of tox T in Vibrio cholerae.Molecular Microbiology,2011,81(1):113-128.
[10]Kazi MI,Conrado AR,Mey AR,Payne SM,Davies BW.Toxr antagonizes H-NS regulation of horizontally acquired genes to drive host colonization.PLo S Pathogens,2016,12(4):e1005570.
[11]Di Rita VJ,Mekalanos JJ.Periplasmic interaction between two membrane regulatory proteins,Tox R and Tox S,results in signal transduction and transcriptional activation.Cell,1991,64(1):29-37.
[12]Midgett CR,Almagro-Moreno S,Pellegrini M,Taylor RK,Skorupski K,Kull FJ.Bile salts and alkaline p H reciprocally modulate the interaction between the periplasmic domains of Vibrio cholerae Tox R and Tox S.Molecular Microbiology,2017,105(2):258-272.
[13]Almagro-Moreno S,Root MZ,Taylor RK.Role of Tox S in the proteolytic cascade of virulence regulator Tox R in Vibrio cholerae.Molecular Microbiology,2015,98(5):963-976.
[14]Peterson KM.Expression of Vibrio cholerae virulence genes in response to environmental signals.Current Issues in Intestinal Microbiology,2002,3(2):29-38.
[15]Xue YY,Tu F,Shi MT,Wu CQ,Ren GP,Wang XJ,Fang WH,Song HH,Yang MH.Redox pathway sensing bile salts activates virulence gene expression in Vibrio cholerae.Molecular Microbiology,2016,102(5):909-924.
[16]Yang MH,Liu Z,Hughes C,Stern AM,Wang H,Zhong ZT,Kan B,Fenical W,Zhu J.Bile salt-induced intermolecular disulfide bond formation activates Vibrio cholerae virulence.Proceedings of the National Academy of Sciences of the United States of America,2013,110(6):2348-2353.
[17]Heras B,Shouldice SR,Totsika M,Scanlon MJ,Schembri MA,Martin JL.DSB proteins and bacterial pathogenicity.Nature Reviews Microbiology,2009,7(3):215-225.
[18]Ren GP,Champion MM,Huntley JF.Identification of disulfide bond isomerase substrates reveals bacterial virulence factors.Molecular Microbiology,2014,94(4):926-944.
[19]Karimova G,Pidoux J,Ullmann A,Ladant D.A bacterial two-hybrid system based on a reconstituted signal transduction pathway.Proceedings of the National Academy of Sciences of the United States of America,1998,95(10):5752-5756.
[20]Lippa AM,Goulian M.Perturbation of the oxidizing environment of the periplasm stimulates the Pho Q/Pho Psystem in Escherichia coli.Journal of Bacteriology,2012,194(6):1457-1463.
[21]Miller J.Experiments in Molecular Genetics.Cold Spring Harbor,N.Y.:Cold Spring Harbor Laboratory Press,1972.
[22]Herrington DA,Hall RH,Losonsky G,Mekalanos JJ,Taylor RK,Levine MM.Toxin,toxin-coregulated pili,and the tox Rregulon are essential for Vibrio cholerae pathogenesis in humans.The Journal of Experimental Medicine,1988,168(4):1487-1492.
[23]Miller VL,Taylor RK,Mekalanos JJ.Cholera toxin transcriptional activator tox R is a transmembrane DNAbinding protein.Cell,1987,48(2):271-279.
[24]Goss TJ,Seaborn CP,Gray MD,Krukonis ES.Identification of the Tcp P-binding site in the tox T promoter of Vibrio cholerae and the role of Tox R in Tcp P-mediated activation.Infection and Immunity,2010,78(10):4122-4133.
[25]Champion GA,Neely MN,Brennan MA,Di Rita VJ.A branch in the Tox R regulatory cascade of Vibrio cholerae revealed by characterization of tox T mutant strains.Molecular Microbiology,1997,23(2):323-331.
[26]Wang QM,Ma Y,Liu LJ,Zhu J,Liu Z.Biofilm development and environmental determinants in Vibrio cholerae.Chinese Journal of Biotechnology,2017,33(9):1533-1546.(in Chinese)王全民,马遥,刘丽钧,朱军,刘智.霍乱弧菌生物被膜发育与环境调控.生物工程学报,2017,33(9):1533-1546.
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