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浙江大学学报(医学版)  2013, Vol. 42 Issue (2): 156-163    DOI: 10.3785/j.issn.1008-9292.2013.02.005
专题报道     
感染过程中钩端螺旋体外膜蛋白抗原表达水平变化及其调控机制
郑林立,葛玉梅,胡玮琳,严杰
浙江大学医学院病原生物学系,浙江 杭州 310058
Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism
ZHENG Lin-Li, GE Yu-Mei, HU Wei-Lin, YAN Jie
Department of Medical Microbiology and Parasitology,Zhejiang University School of Medicine,Hangzhou,Zhejiang 310058,China
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摘要:

目的:了解感染人巨噬细胞过程中问号钩端螺旋体(简称钩体)黄疸出血群赖型赖株主要外膜蛋白(OMP)抗原表达水平变化及其OmpR相关基因调控机制。
方法:采用生物信息学技术预测问号钩体赖株OmpR及其组氨酸激酶(HK)基因和结构功能域。采用实时荧光定量RT-PCR检测钩体感染人THP-1巨噬细胞前后钩体主要OMP编码基因mRNA水平的变化。采用HK胞外区多肽抗血清封闭试验及氯氰碘柳胺阻断试验,检测OmpR及其HK对感染过程中问号钩体OMP编码基因mRNA水平变化的影响。
结果:生物信息学分析结果显示,LB015和LB333可能是问号钩体赖株OmpR编码基因,LB014可能是其HK编码基因。问号钩体赖株感染THP-1细胞后,lipL21、lipL32、lipL41基因mRNA水平迅速并持续下降(P<0.01),groEL、mce、loa22、ligB基因mRNA水平瞬时迅速升高(P<0.01)。氯氰碘柳胺或HK抗血清处理可使感染THP-1细胞而显著下降的问号钩体lipL21和lipL48基因mRNA水平明显回升(P<0.01),氯氰碘柳胺处理可使感染THP-1细胞而显著升高的groEL、mce、loa22、ligB基因mRNA水平明显下降(P<0.01)。
结论:问号钩体赖株感染人巨噬细胞后主要OMP抗原表达水平发生明显变化。问号钩体赖株染色体中存在一组编码OmpR及其HK的基因,其主要功能可能是下调感染过程中部分OMP抗原表达水平。

关键词: 问号钩端螺旋体细菌外膜蛋白质类基因表达逆转录聚合酶链反应感染外膜蛋白表达OmpR氯氰碘柳胺    
Abstract:
Objective: To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism.
Methods: OmpR encoding genes and OmpR-related histidine kinase(HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique.mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR.Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays.
Results: The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete,while LB014 might act as a OmpR-related HK-encoding gene.After the spirochete infecting THP-1 cells,mRNA levels of leptospiral lipL21,lipL32 and lipL41 genes were rapidly and persistently down-regulated(P<0.01),whereas mRNA levels of leptospiral groEL,mce,loa22 and ligB genes were rapidly but transiently up-regulated(P<0.01).The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes(P<0.01).Moreover,closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL,mce,loa22 and ligB genes(P<0.01).
Conclusions: Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages.There is a group of OmpR- and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.
Key words: Leptospira interrogans    Bacterial outer membrane proteins    Gene expression    Reverse transcriptase polymerase chain reaction    Infection    Outer membrane proteins    Expression    OmpR    Closantel
收稿日期: 2012-12-28 出版日期: 2013-03-25
:  R 337.5  
基金资助:

国家自然科学基金资助项目(81261160321,81171534).

通讯作者: 严杰(1956-),男,医学博士,教授,博导,主要从事病原菌致病与耐药机制及相关新型药物与疫苗研究     E-mail: med_bp@zju.edu.cn
作者简介: 郑林立(1987-),男,硕士研究生,主要从事病原微生物感染与免疫的研究.
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引用本文:

郑林立, 葛玉梅, 胡玮琳, 严杰. 感染过程中钩端螺旋体外膜蛋白抗原表达水平变化及其调控机制[J]. 浙江大学学报(医学版), 2013, 42(2): 156-163.

ZHENG Lin-Li, GE Yu-Mei, HU Wei-Lin, YAN Jie. Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism. Journal of ZheJiang University(Medical Science), 2013, 42(2): 156-163.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2013.02.005        http://www.zjujournals.com/med/CN/Y2013/V42/I2/156


[1]MCBRIDE A J,ATHANAZIO D A,REIS M G,et al.Leptospirosis
[J].Curr Opin Infect Dis,2005,18(5):376-386.

[2]YAN Jie,DAI Baoming,YU Enshu(严杰,戴保民,于恩庶).Leptospirology(钩端螺旋体病学)
[M].Third Edition,北京:The People′s Healthy Publication House,China 2006.(in Chinese)

[3]YAN Jie(严杰).Advances in research of pathogenic mechanism and novel genetic engineering vaccines of Leptospira interrogans and bacterial drug resistance
[J].Journal of Zhejiang University: Medical Science(浙江大学学报:医学版),2008,37(6):537-543.(in Chinese)

[4]HAAKE D A,CHAMPION C I,MARFINCH C,et al.Molecular cloning and sequence analysis of the gene encoding OmpL1,a transmembrane outer membrane protein of pathogenic Leptospira spp
[J].J Bateriol,1993,175(13):4225-4234.

[5]HAAKE D A,CHAO G,ZUERNER R L,et al.The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection
[J].Infect Immun,2000,68(4):2276-2285.

[6]CULLEN P A,HAAKE D A,BULACH D M,et al.LipL21 is a novel surface-exposed lipoprotein of pathogenic Leptospira species
[J].Infect Immun,2003,71(5):2414-2421.

[7]LUO Dongjiao,QIU Xiaofeng,WANG Jiang,et al(罗冬娇,邱晓枫,王江,等).Prokaryotic expression of trigeminy artificial fusion gene of Leptospira interrogans and the immunogenicity of its products
[J].Journal of Zhejiang University: Medical Science(浙江大学学报:医学版),2009,37(6):599-604.(in Chinese)

[8]QIU Xiaofeng,XU Hanfei,GUO Zongqi,et al(邱晓枫,徐韩飞,郭宗琪,等).Establishment and application of ELISAs based on rLipL32/1-LipL21-OmpL1/2 fusion antigen of Leptospira interrogans
[J].Journal of Zhejiang University: Medical Science(浙江大学学报:医学版),2009,37(6):592-598.(in Chinese)

[9]XUE F,DONG H Y,WU J Y,et al.Transcriptional responses of Leptospira interrogans to host innate immunity: significant changes in metabolism,oxygen tolerance,and outer membrane
[J].PLoS Negl Trop Dis,2010,4(10): e857.

[10]CASINO P,RUBIO V,MARINA A.The mechanism of signal transduction by two-component systems
[J].Curr Opin Struc Biol,2010,20(6):763-771.

[11]KENNEY L J.Structure / function relationships in OmpR and other winged-helix transcription factors
[J].Curr Opin Microbiol,2002,5(2):135-141.

[12]REN S X,FU G,JIANG X G,et al.Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing
[J].Nature,2003,422(6934):888-893.

[13]DeCOURSEY T E,KIM S Y,SILVER M R,et al.Ion channel expression in PMA-differentiated human THP-1 macrophages
[J].J Membr Biol,1996,152(2):141-157.

[14]SUN A H,WANG Y,DU P,et al.A sensitive and specific IgM-ELISA for the serological diagnosis of human leptospirosis using a rLipL32/1-LipL21-OmpL1/2 fusion protein
[J].Biomed Environ Sci,2011,24(3):291-299.

[15]SCHREIER S,TRIAMPO W,DOUNGCHAWEE G,et al.Leptospirosis research: fast,easy and reliable enumeration of mobile leptospires
[J].Biol Res,2009,42(1):5-12.

[16]JIN D D,OJCIUS D M,SUN D,et al.Leptospira interrogans induces apoptosis in macrophages via caspase-8- and caspase-3-dependent pathways
[J].Infect Immun,2009,77(2):799-809.

[17]SAMBROOK J,FRITSCH E F,MANIATIS T.Molecular Cloning: A Laboratory Manual
[M].2nd edition,New York: Cold Sping Harbor Labooratory Press,1989,1.21-1.52,2.60-2.80,7.3-7.35,9.14-9.22.

[18]ZHANG L,ZHANG C L,OJCIUS D M,et al.Identification of Mce as a critical outer membrane protein of pathogenic Leptospira species responsible for RGD motif-dependent adherence and host-cell invasion
[J].Mol Microbiol,2012,83(5):1006-1023.

[19]WANG H,WU Y F,OJCIUS D M,et al.Leptospiral hemolysins induce proinflammatory cytokines through Toll-like receptor 2- and 4-mediated JNK and NF-κB signaling pathways
[J].PLoS One,2012,7(8):e42266.

[20]KUMAGAI Y,CHENG Z,LIN M,et al.Biochemical activities of three pairs of Ehrlichia chaffeensis two-component regulatory system proteins involved in inhibition of lysosomal fusion
[J].Infect Immun,2006,74(9):5014-5022.

[21]MURRAY G L,ELLIS K M,LO M,et al.Leptospira interrogans requires a functional heme oxygenase to scavenge iron from hemoglobin.
[J].Microbes Infect,2008,10(7):791-797.

[22]FINLAY B B,MCFADDEN G.Anti-immunology: evasion of the host immune system by bacterial and viral pathogens
[J].Cell,2006,124(4):767-782.

[23]SUN Pingping,LIN Xv′ai,LI Liwei(孙萍萍,林旭瑷,李立伟,等),et al.Diversity of outer membrane protein expression of Leptospira interrogans during infection of human THP-1 monocytes
[J].Chinese Journal of Microbiology and Immunology(中华微生物学与免疫学杂志),2012,32(3):224-231.(in Chinese)

[24]GRIFANTINI R,BARTOLINI E,MUZZI A,et al.Previously unrecognized vaccine candidates against group B meningococcus identified by DNA microarrays
[J].Nat Biotechnol,2002,20(9):914-921.

[25]ZHANG C L,WANG H,YAN J.Leptospirosis prevalence in Chinese populations in the last two decades
[J].Microbes Infect,2012,14(4):317-323.

[26]DAVIS J M,HAAKE D A,RAMAKRISHNAN L.Leptospira interrogans stably infects zebrafish embryos,altering phagocyte behavior and homing to specific tissues
[J].PLoS Negl Trop Dis,2009,3(6):e463.

[27]THEODOROU E C,THEODOROU M C,KYRIAKIDIS D A.Inhibition of the signal transduction through the AtoSC system by histidine kinase inhibitors in Escherichia coli
[J].Cell Signal,2011,23(8):1327-1337.

[28]STEPHENSON K,YAMAGUCHI Y,HOCH J A.The mechanism of action of inhibitors of bacterial two-component signal transduction systems
[J].J Biol Chem,2000,275(49):38900-38904.
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