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

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。

ObjectiveTo 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. MethodsThe 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. ResultsThe 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. ConclusionsThe C1409A missense mutation in MRR2 gene contributes to fluconazole resistance of Candida albicans with upregulating CDR1 expression.