影响金黄色葡萄球菌杀白细胞素ED转录表达的培养条件
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摘要
为探讨影响金黄色葡萄球菌杀白细胞素ED(leukocidin ED,LukED)转录表达的最佳培养条件,采用定量反转录-聚合酶链反应(quantitative reverse transcription-polymerase chain reaction,qRT-PCR)检测金黄色葡萄球菌Newman菌株lukE在TSB、BHI、YCP、MHB和LB培养基中不同生长阶段(对数生长早期、中期、晚期和平台期)的表达情况。以表达量较低的培养基为基础,分别加入表达量最高的培养基的不同成分,分析促进lukED表达的物质。结果显示,lukE mRNA水平在YCP培养基中生长至对数晚期时表达最高;YCP组分酵母浸出物、酪蛋白氨基酸及丙酮酸钠均能显著促进lukE转录表达,前两者效果更好。
This study aimed to explore the optimal culture conditions for transcriptional expression of leukocidin ED(lukED)of Staphylococcus aureus(S.aureus).The transcription level of lukEin S.aureus strain Newman was detected at different growth phases(early-exponential,middle-exponential,lateexponential and stationary phases)in TSB,BHI,YCP,MHB and LB media by quantitative reverse transcription-polymerase chain reaction(qRT-PCR). Based on the medium mediating the lowest transcription level of lukE,the different compositions of medium producing the highest mRNA level of lukE was added to discover the substances promoting lukED expression.The findings showed that lukE mRNA level was highest at the late-exponential phase in YCP medium,in which yeast extract,casamino acids and sodium pyruvate could all strikingly increase the expression of lukE,with the first two being better.
This study aimed to explore the optimal culture conditions for transcriptional expression of leukocidin ED(lukED)of Staphylococcus aureus(S.aureus).The transcription level of lukEin S.aureus strain Newman was detected at different growth phases(early-exponential,middle-exponential,lateexponential and stationary phases)in TSB,BHI,YCP,MHB and LB media by quantitative reverse transcription-polymerase chain reaction(qRT-PCR). Based on the medium mediating the lowest transcription level of lukE,the different compositions of medium producing the highest mRNA level of lukE was added to discover the substances promoting lukED expression.The findings showed that lukE mRNA level was highest at the late-exponential phase in YCP medium,in which yeast extract,casamino acids and sodium pyruvate could all strikingly increase the expression of lukE,with the first two being better.
引文
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[4]杨涵,刘庆中.金黄色葡萄球菌杀白细胞素ED的研究进展[J].微生物与感染,2017,12(6):385-390.
[5]Alonzo F 3rd,Torres VJ.The bicomponent pore-forming leucocidins of Staphylococcus aureus[J].Microbiol Mol Biol Rev,2014,78(2):199-230.
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[12]Alonzo F 3rd,Kozhaya L,Rawlings SA,Reyes-Robles T,DuMont AL,Myszka DG,Landau NR,Unutmaz D,Torres VJ.CCR5 is a receptor for Staphylococcus aureus leukotoxin ED[J].Nature,2013,493(7430):51-55.
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[14]Gravet A,Colin DA,Keller D,Girardot R,Monteil H,Prévost G.Characterization of a novel structural member,LukE-LukD,of the bi-component staphylococcal leucotoxins family[J].FEBS Lett,1998,436(2):202-208.
[15]Alonzo F 3rd,Benson MA,Chen J,Novick RP,Shopsin B,Torres VJ.Staphylococcus aureus leucocidin ED contributes to systemic infection by targeting neutrophils and promoting bacterial growth in vivo[J].Mol Microbiol,2012,83(2):423-435.
[16]He C,Xu S,Zhao H,Hu F,Xu X,Jin S,Yang H,Gong F,Liu Q.Leukotoxin and pyrogenic toxin Superantigen gene backgrounds in bloodstream and wound Staphylococcus aureus isolates from eastern region of China[J].BMCInfect Dis,2018,18(1)395.doi:10.1186/s12879-018-3297-0.
[17]Bronner S,Stoessel P,Gravet A,Monteil H,Prevost G.Variable expressions of Staphylococcus aureus bicomponent leucotoxins semiquantified by competitive reverse transcription-PCR[J].Appl Environ Microbiol,2000,66(9):3931-3938.
[18]Balasubramanian D,Ohneck EA,Chapman J,Weiss A,Kim MK,Reyes-Robles T,Zhong J,Shaw LN,Lun DS,Ueberheide B,Shopsin B,Torres VJ.Staphylococcus aureus coordinates leukocidin expression and pathogenesis by sensing metabolic fluxes via RpiRc[J].MBio,2016.doi:10.1128/mBio.00818-16.
[19]Gaupp R,Wirf J,Wonnenberg B,Biegel T,Eisenbeis J,Graham J,Herrmann M,Lee CY,Beisswenger C,Wolz C,Tschernig T,Bischoff M,Somerville GA.RpiRc is a pleiotropic effector of virulence determinant synthesis and attenuates pathogenicity in Staphylococcus aureus[J].Infect Immun,2016,84(7):2031-2041.
[20]Seilie ES,Bubeck Wardenburg J.Staphylococcus aureus pore-forming toxins:The interface of pathogen and host complexity[J].Semin Cell Dev Biol,2017,72:101-116.
[21]Novick RP,Geisinger E.Quorum sensing in staphylococci[J].Annu Rev Genet,2008,42:541-564.
[22]Grunberg-Manago M.Messenger RNA stability and its role in control of gene expression in bacteria and phages[J].Annu Rev Genet,1999,33:193-227.
[23]陈雄,黄煌,胡成远,杜支红,李沛,姚娟,肖冬光,俞学锋.酵母浸出物的营养特性及其在微生物发酵中的应用[J].食品科技,2009,34(12):253-257.
[24]冯梦雪,刘伶俐,钱生财,宋志文.丙酮酸钠对硝化细菌制剂活性的影响[J].河北渔业,2012,19(12):7-10.
[25]Giandomenico AR,Cerniglia GE,Biaglow JE,Stevens CW,Koch CJ.The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide[J].Free Radic Biol Med,1997,23(3):426-434.
[26]Pasquaroli S,Zandri G,Vignaroli C,Vuotto C,Donelli G,Biavasco F.Antibiotic pressure can induce the viable but non-culturable state in Staphylococcus aureus growing in biofilms[J].J Antimicrob Chemother,2013,68(8):1812-1817.
[27]Graves SF,Kobayashi SD,Braughton KR,Diep BA,Chambers HF,Otto M,Deleo FR.Relative contribution of Panton-Valentine leukocidin to PMN plasma membrane permeability and lysis caused by USA300 and USA400culture supernatants[J].Microbes Infect,2010,12(6):446-456.
[2]Vandenesch F,Lina G,Henry T.Staphylococcus aureus hemolysins,bi-component leukocidins,and cytolytic peptides:a redundant arsenal of membrane-damaging virulence factors?[J].Front Cell Infect Microbiol,2012,2:12.doi:10.3389/fcimb.2012.00012.
[3]Otto M.Staphylococcus aureus toxins[J].Curr Opin Microbiol,2014,17:32-37.
[4]杨涵,刘庆中.金黄色葡萄球菌杀白细胞素ED的研究进展[J].微生物与感染,2017,12(6):385-390.
[5]Alonzo F 3rd,Torres VJ.The bicomponent pore-forming leucocidins of Staphylococcus aureus[J].Microbiol Mol Biol Rev,2014,78(2):199-230.
[6]Alonzo F 3rd,Torres VJ.Bacterial survival amidst an immune onslaught:the contribution of the Staphylococcus aureus leukotoxins[J].PLoS Pathog,2013,9(2):e1003143.
[7]Nocadello S,Minasov G,Shuvalova L,Dubrovska I,Sabini E,Bagnoli F,Grandi G,Anderson WF.Crystal structures of the components of the Staphylococcus aureus leukotoxin ED[J].Acta Crystallogr D Struct Biol,2016,72(Pt 1):113-120.
[8]Galy R,Bergeret F,Keller D,Mourey L,Prevost G,Maveyraud L.Crystallization and preliminary crystallographic studies of both components of the staphylococcal LukE-LukD leukotoxin[J].Acta Crystallogr Sect F Struct Biol Cryst Commun,2012,68(Pt 6):663-667.
[9]Bakthavatchalam YD,Nabarro LEB,Ralph R,Veeraraghavan B.Diagnosis and management of PantonValentine leukocidin toxin associated Staphylococcus aureus infection:an update[J].Virulence,2017.doi:10.1080/21505594.2017.1362532.
[10]Spaan AN,Reyes-Robles T,Badiou C,Cochet S,Boguslawski KM,Yoong P,Day CJ,de Haas CJ,van Kessel KP,Vandenesch F,Jennings MP,Le Van Kim C,Colin Y,van Strijp JA,Henry T,Torres VJ.Staphylococcus aureus targets the duffy antigen receptor for chemokines(DARC)to lyse erythrocytes[J].Cell Host Microbe,2015,18(3):363-370.
[11]Reyes-Robles T,Alonzo F 3rd,Kozhaya L,Lacy DB,Unutmaz D,Torres VJ.Staphylococcus aureus leukotoxin ED targets the chemokine receptors CXCR1and CXCR2to kill leukocytes and promote infection[J].Cell Host Microbe,2013,14(4):453-459.
[12]Alonzo F 3rd,Kozhaya L,Rawlings SA,Reyes-Robles T,DuMont AL,Myszka DG,Landau NR,Unutmaz D,Torres VJ.CCR5 is a receptor for Staphylococcus aureus leukotoxin ED[J].Nature,2013,493(7430):51-55.
[13]DuMont AL,Torres VJ.Cell targeting by the Staphylococcus aureus pore-forming toxins:it’s not just about lipids[J].Trends Microbiol,2014,22(1):21-27.
[14]Gravet A,Colin DA,Keller D,Girardot R,Monteil H,Prévost G.Characterization of a novel structural member,LukE-LukD,of the bi-component staphylococcal leucotoxins family[J].FEBS Lett,1998,436(2):202-208.
[15]Alonzo F 3rd,Benson MA,Chen J,Novick RP,Shopsin B,Torres VJ.Staphylococcus aureus leucocidin ED contributes to systemic infection by targeting neutrophils and promoting bacterial growth in vivo[J].Mol Microbiol,2012,83(2):423-435.
[16]He C,Xu S,Zhao H,Hu F,Xu X,Jin S,Yang H,Gong F,Liu Q.Leukotoxin and pyrogenic toxin Superantigen gene backgrounds in bloodstream and wound Staphylococcus aureus isolates from eastern region of China[J].BMCInfect Dis,2018,18(1)395.doi:10.1186/s12879-018-3297-0.
[17]Bronner S,Stoessel P,Gravet A,Monteil H,Prevost G.Variable expressions of Staphylococcus aureus bicomponent leucotoxins semiquantified by competitive reverse transcription-PCR[J].Appl Environ Microbiol,2000,66(9):3931-3938.
[18]Balasubramanian D,Ohneck EA,Chapman J,Weiss A,Kim MK,Reyes-Robles T,Zhong J,Shaw LN,Lun DS,Ueberheide B,Shopsin B,Torres VJ.Staphylococcus aureus coordinates leukocidin expression and pathogenesis by sensing metabolic fluxes via RpiRc[J].MBio,2016.doi:10.1128/mBio.00818-16.
[19]Gaupp R,Wirf J,Wonnenberg B,Biegel T,Eisenbeis J,Graham J,Herrmann M,Lee CY,Beisswenger C,Wolz C,Tschernig T,Bischoff M,Somerville GA.RpiRc is a pleiotropic effector of virulence determinant synthesis and attenuates pathogenicity in Staphylococcus aureus[J].Infect Immun,2016,84(7):2031-2041.
[20]Seilie ES,Bubeck Wardenburg J.Staphylococcus aureus pore-forming toxins:The interface of pathogen and host complexity[J].Semin Cell Dev Biol,2017,72:101-116.
[21]Novick RP,Geisinger E.Quorum sensing in staphylococci[J].Annu Rev Genet,2008,42:541-564.
[22]Grunberg-Manago M.Messenger RNA stability and its role in control of gene expression in bacteria and phages[J].Annu Rev Genet,1999,33:193-227.
[23]陈雄,黄煌,胡成远,杜支红,李沛,姚娟,肖冬光,俞学锋.酵母浸出物的营养特性及其在微生物发酵中的应用[J].食品科技,2009,34(12):253-257.
[24]冯梦雪,刘伶俐,钱生财,宋志文.丙酮酸钠对硝化细菌制剂活性的影响[J].河北渔业,2012,19(12):7-10.
[25]Giandomenico AR,Cerniglia GE,Biaglow JE,Stevens CW,Koch CJ.The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide[J].Free Radic Biol Med,1997,23(3):426-434.
[26]Pasquaroli S,Zandri G,Vignaroli C,Vuotto C,Donelli G,Biavasco F.Antibiotic pressure can induce the viable but non-culturable state in Staphylococcus aureus growing in biofilms[J].J Antimicrob Chemother,2013,68(8):1812-1817.
[27]Graves SF,Kobayashi SD,Braughton KR,Diep BA,Chambers HF,Otto M,Deleo FR.Relative contribution of Panton-Valentine leukocidin to PMN plasma membrane permeability and lysis caused by USA300 and USA400culture supernatants[J].Microbes Infect,2010,12(6):446-456.