含有额外拷贝黄曲霉cyp51同源基因的烟曲霉对抗真菌药物的敏感性测定

中国真菌学杂志　 2011, Vol. 6 　Issue (1): 15-21.

含有额外拷贝黄曲霉cyp51同源基因的烟曲霉对抗真菌药物的敏感性测定

刘伟霞, 孙毅, 万喆, 李若瑜, 刘伟

北京大学第一医院皮肤性病科真菌室, 北京大学真菌和真菌病研究中心, 北京, 100034

收稿日期:2010-11-12 出版日期:2011-02-28 发布日期:2011-02-28
通讯作者: 刘伟,E-mail:liuw90@yahoo.com E-mail:liuw90@yahoo.com
作者简介:刘伟霞,女(汉族),硕士研究生在读.E-mail:liuweixia1986@yahoo.cn
基金资助:
国家自然科学基金(30970131);北京市自然科学基金(7102149);国家“十一五艾滋病和病毒性肝炎等重大传染病防治”科技重大专项(2008ZX10004-002)

Antifungal susceptibility of the A.fumigatus transformants containing extra copies of A.flavus cyp51 gene homologues to the common antifungal drugs

LIU Wei-xia, SUN Yi, WAN Zhe, LI Ruo-yu, LIU Wei

Department of Dermatology and Venereology, Peking University First Hospital, and the Research Center for Medical Mycology of Peking University, Beijing 100034, China

Received:2010-11-12 Online:2011-02-28 Published:2011-02-28

摘要/Abstract

摘要： 目的通过构建黄曲霉cyp51同源基因额外拷贝株,研究这些基因对抗真菌药物敏感性的影响。方法通过序列同源性比对,在黄曲霉基因组中找出其cyp51基因的开放读码框(ORF)及其上下游约1 000 bp的基因序列,PCR扩增后利用DNA重组技术将该片段克隆到穿梭质粒pRG3-AMA1-NotI;用重组后的质粒和空质粒转化烟曲霉嘧啶营养缺陷株(pyrG-)AF293.1,并利用美国临床实验室标准化研究所(CLSI)M38-A2中的微量液基稀释法和E-test法测定转化株对两性霉素B(AMB)、伊曲康唑(ITC)、伏立康唑(VRC)、泊沙康唑(POS)和卡泊芬净(CAS)敏感性。结果黄曲霉cyp51同源基因有3个:cyp51A、cyp51B和cyp51C,ORF大小约为1 400～2 000 bp;将其克隆到穿梭质粒pRG3-AMA1-NotI产生重组质粒pRG3-AMA1-CYP51A、pRG3-AMA1-CYP51B和pRG3-AMA1-CYP51C;连同空质粒转化AF293.1后得到阳性转化子rC-YP51A、rCYP51B、rCYP51C和rpRG;微量液基稀释法和E-test法显示,rCYP51A和rCYP51B对VRC和ITC交叉耐药,对AMB和CAS敏感;rCYP51C和rpRG对VRC和ITC中度敏感,而对AMB和CAS敏感。结论额外拷贝黄曲霉cyp51A或cyp51B基因影响烟曲霉对唑类药物的敏感性,对AMB和CAS的敏感性没有影响;额外拷贝黄曲霉cyp51C对测试药物的敏感性没有明显影响。

关键词: 黄曲霉, 唑类, cyp51, 抗真菌药物敏感性

Abstract: Objective To investigate the effect of Aspergillus flavus cyp51 genes on antifungal susceptibility by cloning and constucting the extra copies of Aspergillus flavus cyp51 genes.Methods A.flavus cyp51 gene homologues were identified by tblastn searches in A.flavus genome database.PCR fragments composed of the 5′flanking sequence(approximately 1 000 bp) of cyp51,cyp51ORF,and its 3′flanking sequence(approximately 1 000 bp),were subcloned into shuttle plasmid pRG3-AMA1-NotI to produce recombinant plasmids.These plasmids and empty plasmid pRG3-AMA1-NotI were transformed into A.fumigatus strain AF293.1(pyrG-) respectively to produce transformants.The Clinical Laboratory Standard Institute broth microdilution method M38-A2 and E-test method were used to assay the minimal inhibitory concentrations(MICs) of itraconazole(ITC),voriconazole(VRC),amphotericin B(AMB),and posaconazole(POS),or minimal effect concentration(MEC) of caspofungin(CAS),against these transformants.Results A.flavus genome contains three cyp51 gene homologues,cyp51A,cyp51B and cyp51C,of which the ORF size are 1 400-2 000 bp.When these genes were subcloned into shuttle plasmid pRG3-AMA1-NotI,we get plasmids pRG3-AMA1-CYP51A,pRG3-AMA1-CYP51B and pRG3-AMA1-CYP51C.These plasmids and empty plasmid were transformed into A.fumigatus strain AF293.1(pyrG-) to produce transformants rCYP51A,rCYP51B,rCYP51C and rpRG.The antifungal susceptibility of these A.fumigatus transformants to the antifungal drugs by broth microdilution assaying and E-test method showed that,rCYP51A and rCYP51B were cross-resistant to VRC and ITC,susceptible to both AMB and CAS;rCYP51C and rpRG were intermediate to ITC and VRC,susceptible to both AMB and CAS.Conclusion In A.fumigatus,extra copies of A.flavus′cyp51A gene or cyp51B gene have effect on antifungal susceptibility to azoles,have no effect on AMB and CAS.Extra copy of cyp51C has no obvious effect on all the tested drugs.

Key words: A.flavus, azoles, cyp51, antifungal susceptibility

中图分类号:

R379.6

刘伟霞, 孙毅, 万喆, 李若瑜, 刘伟. 含有额外拷贝黄曲霉cyp51同源基因的烟曲霉对抗真菌药物的敏感性测定[J]. 中国真菌学杂志, 2011, 6(1): 15-21.

LIU Wei-xia, SUN Yi, WAN Zhe, LI Ruo-yu, LIU Wei. Antifungal susceptibility of the A.fumigatus transformants containing extra copies of A.flavus cyp51 gene homologues to the common antifungal drugs[J]. Chinese Journal of Mycology, 2011, 6(1): 15-21.

参考文献

[1] Denning DW.Invasive aspergillosis[J].Clin Infect Dis,1998,26(4):781-803.
[2] Segal BH,Walsh TJ.Current approaches to diagnosis and treatment of invasive aspergillosis[J].Am J Respir Crit Care Med,2006,173(7):707-717.
[3] Krishnan S,Manavathu EK,Chandrasekar PH.Aspergillus flavus:an emerging non-Aspergillus fumigates species of significance[J].Mycoses,2009,52(3):206-222.
[4] Walsh TJ,Anaissie EJ,Denning DW,et al.Treatment of aspergillosis:clinical practice guidelines of the infectious diseases society of America[J].Clin Infect Dis,2008,46(3):327-360.
[5] Thompson CR,Cadena J,Patterson TF.Overview of antifungal agents[J] ,Clin Chest Med,2009,30(2):203-215.
[6] Chamilos G,Kontoyiannis DP.Update.antifungal drug resistance mechanisms ofAspergillus fumigatus[J].Drug Resistance Updates,2005,8(6):344-358.
[7] Hedayati MT,Pasqualotto AC,Warn PA.Aspergillus flanus:human pathogen,allergen and mycotoxin producer[J].Microbiology,2007,153(Pt6):1677-1692
[8] Osherov N,Kontoyiannis DP,Romans A,et al.Resistance to itraconazole in Aspergillus nidulans and Aspergillus fumigates is conferred by extra copies of the A.nidulans P-450 14-alpha-demethylase gene,pdmA[J].J Antimicrob Chemother,2001,48 (1):75-81.
[9] Osherov N,Mathew J,May GS.Polarity-defective mutants of Aspergillus nidulans[J].Fungal Genet Biol,2000,31 (3):181-188.
[10] 彭秀玲,袁汉英,谢毅.等.基因工程实验技术[M].第二版.长沙:湖南科学技术出版社,1998.
[11] McCluskey K.The fungal genetics stock center:from molds to molecules[J].Adv Appl Microbiol,2003,52:245-262.
[12] 刘伟,万品,陈伟,等.对多烯类药物耐药的1株黄曲霉临床株耐药性的初步研究[J].中国真菌学杂志,2007,2(3):133-136.
[13] 卢圣栋.现代分子生物学实验技术[M] ,北京:高等教育出版社,1993.
[14] Liu W,May GS,Lionakis MS,et al.Extra copies of the Aspergillus fumigatussqualene epoxidase gene confer resistance to terbinafiine:genetic approach to studying gene dose-dependent resistance to antifungals in A.fumigatus[J].Antimicrob Agents Chemother,2004,48(7):2490-2496.
[15] Clinical and Laboratory Standards Institute.Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; Approved standard.Document M38-A2[S].CLSI,Wayne,PA,USA,2008.
[16] Kurtz MB,Heath IB,Marrinan J,et al.Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against(1,3) -beta-D-glucan synthase[J].Antimicrob Agents Chemother,1994,38 (7):1480-1489.
[17] Seo K,Akiyoshi H,Ohnishi Y.Alteration of cell wall composition leads to amphotericin B resistance in Aspergillus flavus[J].Microbiol Immunol,1999,43 (11):1017-1025.
[18] Krishnan-Natesan S,Chandrasekar PH,Alangaden GJ,et al.Molecular characterisation of cyp5I A and cyp51 B genes coding for P450 14a-lanosterol demethylases A(CYP51 Ap) and B (CYP51 Bp) from voriconazole-resistant laboratory isolates of Aspergillus lavus[J].Int J Antimicrob Agents,2008,32 (6):519-524.
[19] 乔建军,刘伟,李若瑜,等.额外拷贝ERG6基因对烟曲霉的影响[J].微生物学通报,2007,34(1):191-194
[20] 乔建军,刘伟,李若瑜,等.含额外拷贝ERG3 B基因烟曲霉的表型分析[J] 中国皮肤性病学杂志,2008,22(1);19-22.
[21] Verweij PE,Howard SJ,Melchers WJ,et al.Azole-resistance in Aspergillus:Proposed nomenclature and breakpoints[J].Drug Resist Updat,2009,12(6):141-147.