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

doi:10.3969/j.issn.1673-8640.2016.09.003

Abstract

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

CLC Number:

R446.5

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.

Figures/Tables 5

References 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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