肺炎链球菌血红素结合脂蛋白PiuA的表达、纯化和表征
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摘要
【背景】肺炎链球菌是社区获得性肺炎最常见的致病菌之一,它也会引起脑膜炎、鼻窦炎、中耳炎、菌血症等一系列疾病,对人类(特别是儿童、老人、免疫缺陷患者)健康造成重大威胁。铁是肺炎链球菌生存和感染所必需的元素之一,其中血红素转运系统PiuABCD是肺炎链球菌最重要的铁转运系统。【目的】克隆、表达和纯化肺炎链球菌血红素转运系统脂蛋白PiuA,并在体外表征PiuA蛋白的血红素结合特性。【方法】将肺炎链球菌D39菌株中的piuA(spd_1652)基因连接到载体pBAD-HisA上,在大肠杆菌Top10菌株中进行异源表达,然后运用Ni-NTA亲和层析纯化PiuA-His蛋白,并用肠激酶切掉His标签获得不含标签的PiuA蛋白,最后运用圆二色谱、紫外光谱和荧光光谱表征PiuA蛋白的血红素结合特性。【结果】构建了pBAD/HisA-PiuA重组表达载体,获得了纯度大于95%的PiuA蛋白,圆二色谱显示PiuA蛋白与Hemin结合后,其二级结构不发生改变;紫外光谱结果显示PiuA蛋白具有血红素结合能力;荧光光谱结果显示apo-PiuA蛋白与Hemin结合常数K=3.4×10~5 L/mol。【结论】肺炎链球菌血红素转运系统脂蛋白PiuA能够特异地结合血红素,为肺炎链球菌的生存和感染提供必需的铁源,PiuA蛋白的体外表征结果为针对PiuABCD血红素转运系统设计抗菌药物奠定了基础。
[Background] Streptococcus pneumoniae is one of the most common pathogens of community-acquired pneumonia, and causes a variety of other serious diseases including bacterial meningitis, sinusitis, otitis media and bacteremia, and poses health threats to humans, especially among children and the elderly, or individuals with immature/compromised immune systems. Iron is essential for Streptococcal survival and infection, and haem ABC uptake system PiuABCD is the most iron ABC uptake system for S. pneumoniae. [Objective] Clone, express and purify hemin-binding lipoprotein PiuA belonging to haem ABC uptake system PiuABCD, and characterize its hemin-binding properties in vitro. [Methods] The piuA(spd_1652) gene from S. pneumoniae D39 strain was ligated into pBAD-HisA vector, and expressed in E. coli Top 10 strain, then the PiuA-His fusion protein was purified using Ni-NTA affinity chromatography, and the His tag free PiuA protein was obtained by cutting off the His tag with enterokinase. Finally, the hemin-binding properties of PiuA protein were characterized by circular dichroism, UV spectroscopy and fluorescence spectroscopy. [Results] We successfully constructed recombinant vector pBAD/HisA-PiuA, and acquired the PiuA protein with the purity higher than 95%. CD spectra showed that hemin binding did not induce any significant structural change in PiuA, UV spectroscopy revealed that PiuA protein can specifically bind hemin, and fluorescence spectroscopy demonstrated that the binding constant K between the PiuA and hemin is 3.4×10~5 L/mol. [Conclusion] Hemin-binding lipoprotein PiuA can specifically bind to hemin and provide the iron for Streptococcal survival and infection, these characterization results of PiuA protein provide basis data for design of antibacterial drug targeted for haem ABC uptake system PiuABCD.
[Background] Streptococcus pneumoniae is one of the most common pathogens of community-acquired pneumonia, and causes a variety of other serious diseases including bacterial meningitis, sinusitis, otitis media and bacteremia, and poses health threats to humans, especially among children and the elderly, or individuals with immature/compromised immune systems. Iron is essential for Streptococcal survival and infection, and haem ABC uptake system PiuABCD is the most iron ABC uptake system for S. pneumoniae. [Objective] Clone, express and purify hemin-binding lipoprotein PiuA belonging to haem ABC uptake system PiuABCD, and characterize its hemin-binding properties in vitro. [Methods] The piuA(spd_1652) gene from S. pneumoniae D39 strain was ligated into pBAD-HisA vector, and expressed in E. coli Top 10 strain, then the PiuA-His fusion protein was purified using Ni-NTA affinity chromatography, and the His tag free PiuA protein was obtained by cutting off the His tag with enterokinase. Finally, the hemin-binding properties of PiuA protein were characterized by circular dichroism, UV spectroscopy and fluorescence spectroscopy. [Results] We successfully constructed recombinant vector pBAD/HisA-PiuA, and acquired the PiuA protein with the purity higher than 95%. CD spectra showed that hemin binding did not induce any significant structural change in PiuA, UV spectroscopy revealed that PiuA protein can specifically bind hemin, and fluorescence spectroscopy demonstrated that the binding constant K between the PiuA and hemin is 3.4×10~5 L/mol. [Conclusion] Hemin-binding lipoprotein PiuA can specifically bind to hemin and provide the iron for Streptococcal survival and infection, these characterization results of PiuA protein provide basis data for design of antibacterial drug targeted for haem ABC uptake system PiuABCD.
引文
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[26]Haraguchi H.Metallomics as integrated biometal science[J].Journal of Analytical Atomic Spectrometry,2004,19(1):5-14
[27]Shi W,Chance MR.Metallomics and metalloproteomics[J].Cellular and Molecular Life Sciences,2008,65(19):3040-3048
[28]Ding XL,Gao HQ.Applications and experimental methods of circular dichroism spectroscopy[J].Experimental Technology and Management,2008,25(10):48-52(in Chinese)丁晓岚,高红旗.圆二色光谱技术应用和实验方法[J].实验技术与管理,2008,25(10):48-52
[29]Wierzchowski KL.Photochemistry and UV spectroscopy of proteins and nucleic acids[J].Postepy Biochemii,2015,61(3):242-252
[30]Yin YX,Xiang BQ,Tong L.The application of studying fluorescence spectroscopy on protein[J].Experimental Technology and Management,2010,27(2):33-36,40(in Chinese)尹燕霞,向本琼,佟丽.荧光光谱法在蛋白质研究中的应用[J].实验技术与管理,2010,27(2):33-36,40
[2]Kadioglu A,Weiser JN,Paton JC,et al.The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease[J].Nature Reviews Microbiology,2008,6(4):288-301
[3]WHO.Pneumococcal disease[EB/OL].http://www.who.int/ith/vaccines/pneumococcal/en/
[4]Kim GL,Seon SH,Rhee DK.Pneumonia and Streptococcus pneumoniae vaccine[J].Archives of Pharmacal Research,2017,40(8):885-893
[5]Yesilkaya H,Andisi VF,Andrew PW,et al.Streptococcus pneumoniae and reactive oxygen species:an unusual approach to living with radicals[J].Trends in Microbiology,2013,21(4):187-195
[6]Turner AG,Ong CIY,Walker MJ,et al.Transition metal homeostasis in Streptococcus pyogenes and Streptococcus pneumoniae[J].Advances in Microbial Physiology,2017,70:123-191
[7]Ge RG,Sun XS.Iron acquisition and regulation systems in Streptococcus species[J].Metallomics,2014,6(5):996-1003
[8]Cassat JE,Skaar EP.Iron in infection and immunity[J].Cell Host&Microbe,2013,13(5):509-519
[9]Nguyen MT,Gotz F.Lipoproteins of gram-positive bacteria:key players in the immune response and virulence[J].Microbiology and Molecular Biology Reviews,2016,80(3):891-903
[10]Kohler S,VoβF,Gómez Mejia A,et al.Pneumococcal lipoproteins involved in bacterial fitness,virulence,and immune evasion[J].FEBS Letters,2016,590(21):3820-3839
[11]Counago RM,McDevitt CA,Ween MP,et al.Prokaryotic substrate-binding proteins as targets for antimicrobial therapies[J].Curr Drug Targets,2012,13(11):1400-1410
[12]Yang XY,Sun B,Zhang L,et al.Chemical interference with iron transport systems to suppress bacterial growth of Streptococcus pneumoniae[J].PLoS One,2014,9(8):e105953
[13]Kim HK,DeDent A,Cheng AG,et al.IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge[J].Vaccine,2010,28(38):6382-6392
[14]Ballouche M,Cornelis P,Baysse C.Iron metabolism:a promising target for antibacterial strategies[J].Recent Patents on Anti-Infective Drug Discovery,2009,4(3):190-205
[15]Kaneko Y,Thoendel M,Olakanmi O,et al.The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibiofilm activity[J].The Journal of Clinical Investigation,2007,117(4):877-888
[16]Yang XY,He K,Du G,et al.Integrated translatomics with proteomics to identify novel iron-transporting proteins in Streptococcus pneumoniae[J].Frontiers in Microbiology,2016,7(1):78
[17]Brown JS,Gilliland SM,Ruiz-Albert J,et al.Characterization of pit,a Streptococcus pneumoniae iron uptake ABC transporter[J].Infection and Immunity,2002,70(8):4389-4398
[18]Brown JS,Gilliland SM,Holden DW.A Streptococcus pneumoniae pathogenicity island encoding an ABC transporter involved in iron uptake and virulence[J].Molecular Microbiology,2001,40(3):572-585
[19]Jomaa M,Terry S,Hale C,et al.Immunization with the iron uptake ABC transporter proteins PiaA and PiuA prevents respiratory infection with Streptococcus pneumoniae[J].Vaccine,2006,24(24):5133-5139
[20]Jomaa M,Yuste J,Paton JC,et al.Antibodies to the iron uptake ABC transporter lipoproteins PiaA and PiuA promote opsonophagocytosis of Streptococcus pneumoniae[J].Infection and Immunity,2005,73(10):6852-6859
[21]Brown JS,Ogunniyi AD,Woodrow MC,et al.Immunization with components of two iron uptake ABC transporters protects mice against systemic Streptococcus pneumoniae infection[J].Infection and Immunity,2001,69(11):6702-6706
[22]Chen YX,Zhou CZ,Shi WW.Streptococcus pneumonia PiuAprotein fragment and preparation method thereof.China,CN102807611(B)[P].2015-01-07.https://worldwide.espacenet.com/publicationDetails/biblio?CC=CN&NR=102807611B&KC=B&FT=D&ND=4&date=20150107&DB=&locale=en_EP
[23]Schaible UE,Kaufmann SH.Iron and microbial infection[J].Nature Reviews Microbiology,2004,2(12):946-953
[24]Andrews SC,Robinson AK,Rodríguez-Qui?ones F.Bacterial iron homeostasis[J].FEMS Microbiology Reviews,2003,27(2/3):215-237
[25]Wilks A,Burkhard KA.Heme and virulence:how bacterial pathogens regulate,transport and utilize heme[J].Natural Product Reports,2007,24(3):511-522
[26]Haraguchi H.Metallomics as integrated biometal science[J].Journal of Analytical Atomic Spectrometry,2004,19(1):5-14
[27]Shi W,Chance MR.Metallomics and metalloproteomics[J].Cellular and Molecular Life Sciences,2008,65(19):3040-3048
[28]Ding XL,Gao HQ.Applications and experimental methods of circular dichroism spectroscopy[J].Experimental Technology and Management,2008,25(10):48-52(in Chinese)丁晓岚,高红旗.圆二色光谱技术应用和实验方法[J].实验技术与管理,2008,25(10):48-52
[29]Wierzchowski KL.Photochemistry and UV spectroscopy of proteins and nucleic acids[J].Postepy Biochemii,2015,61(3):242-252
[30]Yin YX,Xiang BQ,Tong L.The application of studying fluorescence spectroscopy on protein[J].Experimental Technology and Management,2010,27(2):33-36,40(in Chinese)尹燕霞,向本琼,佟丽.荧光光谱法在蛋白质研究中的应用[J].实验技术与管理,2010,27(2):33-36,40