白念珠菌MRR2基因错义突变C1409A与氟康唑耐药的相关性

doi:10.3969/j.issn.1673-8640.2016.09.003

检验医学 ›› 2016, Vol. 31 ›› Issue (9): 744-749.DOI: 10.3969/j.issn.1673-8640.2016.09.003

• 深部真菌的耐药性及耐药机制专题 • 上一篇下一篇

白念珠菌MRR2基因错义突变C1409A与氟康唑耐药的相关性

王影¹, 张嵘¹, 刘锦燕², 史册¹, 李文静¹, 赵悦¹, 项明洁²

1. 上海交通大学医学院附属瑞金医院检验科,上海 200020
2. 上海交通大学医学院附属瑞金医院卢湾分院放免检验科,上海 200020

收稿日期:2016-03-14 出版日期:2016-09-30 发布日期:2016-10-11
作者简介:null
作者简介：王影,女,1989年生,学士,主要从事真菌耐药机制及流行病学研究。张嵘,女,1967年生,学士,主管技师,主要从事临床检验诊断学工作。张嵘与王影对本研究具有同等贡献,并列为第一作者。
基金资助:
黄浦区卫生局项目（2012-HGG-44）;上海市自然科学基金项目（15ZR1426900）

Correlation between C1409A missense mutation in MRR2 gene and fluconazole resistance of Candida albicans

WANG Ying¹, ZHANG Rong¹, LIU Jinyan², SHI Ce¹, LI Wenjing¹, ZHAO Yue¹, XIANG Mingjie²

1. Department of Clinical Laboratory,Ruijin Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200020,China
2. Department of Radioimmunology and Laboratory Medicine,Luwan Branch,Ruijin Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200020,China

Received:2016-03-14 Online:2016-09-30 Published:2016-10-11

摘要/Abstract

摘要：

目的研究锌簇转录因子——多药耐药调节因子2（Mrr2）编码基因MRR2错义突变C1409A与白念珠菌氟康唑耐药的相关性。方法利用白念珠菌工程菌株SN152构建MRR2基因敲除菌株,通过一步法克隆及定点突变技术构建MRR2基因C1409A突变型表达质粒,再用高效醋酸锂转染法将质粒片段转染入MRR2基因敲除菌株,异位表达于ADE2位点以构建MRR2基因定点突变菌株。通过体外药物敏感性试验及实时荧光定量聚合酶链反应（PCR）分析错义突变C1409A与氟康唑耐药的关系。结果氟康唑对MRR2基因C1409A定点突变的白念珠菌的最低抑菌浓度（MIC）较对工程菌株SN152的MIC增加4倍,CDR1表达上调约3倍。结论MRR2基因错义突变C1409A可上调白念珠菌CDR1表达并介导白念珠菌对氟康唑的耐药。

关键词: 白念珠菌, 氟康唑, 耐药, MRR2基因, 错义突变

Abstract:

Objective To study the correlation between C1409A missense mutation in MRR2 gene [the gene encoding zinc cluster transcription factor-multidrug resistance regulator 2（Mrr2）] and fluconazole resistance of Candida albicans. Methods The isolates of Candida albicans containing C1409A missense mutation in MRR2 gene were generated by one-step cloning technology and site-directed mutagenesis based on MRR2Δ/Δ mutant,which was recombined from Candida albicans engineering isolate SN152. The mutated MRR2 gene was expressed at ADE2 locus using classical lithium acetate transfection method. Fluconazole susceptibility test and real-time fluorescence quantitation polymerase chain reaction （PCR） were performed to analyze the correlation between C1409A missense mutation in MRR2 gene and fluconazole resistance. Results The recombinant isolates contributed to an almost 3-time increase for CDR1 expression and a 4-time increase for minimal inhibitory concentration（MIC） in fluconazole resistance compared with SN152. Conclusions The C1409A missense mutation in MRR2 gene contributes to fluconazole resistance of Candida albicans with upregulating CDR1 expression.

Key words: Candida albicans, Fluconazole, Drug resistance, MRR2 gene, Missense mutation

中图分类号:

R446.5

王影, 张嵘, 刘锦燕, 史册, 李文静, 赵悦, 项明洁. 白念珠菌MRR2基因错义突变C1409A与氟康唑耐药的相关性[J]. 检验医学, 2016, 31(9): 744-749.

WANG Ying, ZHANG Rong, LIU Jinyan, SHI Ce, LI Wenjing, ZHAO Yue, XIANG Mingjie. Correlation between C1409A missense mutation in MRR2 gene and fluconazole resistance of Candida albicans[J]. Laboratory Medicine, 2016, 31(9): 744-749.

图/表 5

参考文献 19

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引物名称	引物序列（5'~3'）
MRR2-F	AGGTAATCATTGTCAATGACCAAACGTG- ATCGTACA
MRR2-R	CAATGGCACTACAGCGGGTTTACGAGAT- TTTGAGGA
CP-F	GCTGTAGTGCCATTGCATTGTTCACCACA- AATGTTTCT
CP-R	TGACAATGATTACCTATGGTAGCGATGCA- CGGT
VP-F	ATCCACTGTGCTCCGAAAAC
VP-R	TGCCTTGGGTGGCTATTTTA
MP-F	CCGAAGCTAGTAATGTGTTCTACAACGAT- TTGGTCATT
MP-R	TTGTAGAACACATTACTAGCTTCGGAATC- AATGGTGGC
UP-F	TTGGACGCAGCCAAATCAC
UP-R	TGAGTTCAAATGGCACACCAA
CDR1-F	TTTAGCCAGAACTTTCACTCATGATT
CDR1-R	TATTTATTTCTTCATGTTCATATGGATTGA

引物名称	引物序列（5'~3'）
MRR2-F	AGGTAATCATTGTCAATGACCAAACGTG- ATCGTACA
MRR2-R	CAATGGCACTACAGCGGGTTTACGAGAT- TTTGAGGA
CP-F	GCTGTAGTGCCATTGCATTGTTCACCACA- AATGTTTCT
CP-R	TGACAATGATTACCTATGGTAGCGATGCA- CGGT
VP-F	ATCCACTGTGCTCCGAAAAC
VP-R	TGCCTTGGGTGGCTATTTTA
MP-F	CCGAAGCTAGTAATGTGTTCTACAACGAT- TTGGTCATT
MP-R	TTGTAGAACACATTACTAGCTTCGGAATC- AATGGTGGC
UP-F	TTGGACGCAGCCAAATCAC
UP-R	TGAGTTCAAATGGCACACCAA
CDR1-F	TTTAGCCAGAACTTTCACTCATGATT
CDR1-R	TATTTATTTCTTCATGTTCATATGGATTGA

菌株	基因型	备注
SN152	ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434his1::hisG/his1::hisGleu2/leu2 arg4/arg4	参考文献[14]
MRR2Δ/Δ	SN152^*mrr2:: his1/ mrr2:: C.m leu2	参考文献[13]
S0	MRR2Δ/Δ^*ADE2/ade2:: ADH1p-PAP	本研究
S1	MRR2Δ/Δ^*ADE2/ade2:: ADH1p-MRR2^SN152-PAP	本研究
SM	MRR2Δ/Δ^*ADE2/ade2:: ADH1p-MRR2^C1409A-PAP	本研究

菌株	基因型	备注
SN152	ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434his1::hisG/his1::hisGleu2/leu2 arg4/arg4	参考文献[14]
MRR2Δ/Δ	SN152^*mrr2:: his1/ mrr2:: C.m leu2	参考文献[13]
S0	MRR2Δ/Δ^*ADE2/ade2:: ADH1p-PAP	本研究
S1	MRR2Δ/Δ^*ADE2/ade2:: ADH1p-MRR2^SN152-PAP	本研究
SM	MRR2Δ/Δ^*ADE2/ade2:: ADH1p-MRR2^C1409A-PAP	本研究

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白念珠菌MRR2基因错义突变C1409A与氟康唑耐药的相关性

Correlation between C1409A missense mutation in MRR2 gene and fluconazole resistance of Candida albicans

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