Multiple resistance mechanisms among Aspergillus fumigatus mutants with high-level resistance to itraconazole
Multiple resistance mechanisms among Aspergillus fumigatus mutants with high-level resistance to itraconazole
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DOI：
10.1128/aac.47.5.1719-1726.2003
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发表时间：
2003-05-01
影响因子：
4.9
通讯作者：
Perlin, DS
中科院分区：
文献类型：
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作者：
Nascimento, AM;Goldman, GH;Perlin, DS
在体外（以紫外线照射为初步步骤）筛选了对伊曲康唑（itraconazole， RIT）高度耐药的烟曲霉（Aspergillus fumigatus）突变体，以研究这种临床重要病原体的耐药机制。发现8个RIT突变体在唑靶基因CYP51A的Gly54 （G54E， -K或-R）处发生突变。针对多药耐药（MDR）泵高度保守区域设计引物，利用逆转录酶PCR扩增反应鉴定烟曲霉多药耐药（MDR）外排泵的新基因。两个基因AfuMDR3和AfuMDR4在许多RIT突变体中表现出显著的表达水平变化，并得到了更详细的表征。对AfuMDR3编码的氨基酸序列分析显示，AfuMDR4与主要的促进剂转运蛋白超家族高度相似，而AfuMDR4是atp结合盒超家族的典型成员。采用分子信标探针实时荧光定量PCR检测AfuMDR3和AfuMDR4的表达水平。大多数RIT突变体在暴露于伊曲康唑后表现出两种基因的组成性高水平表达或诱导表达。我们的研究结果表明，烟曲霉一种或两种新发现的药物外排泵基因的过表达和/或药物靶点突变的选择与伊曲康唑的高水平耐药性有关，并且是临床伊曲康唑耐药性出现的机制考虑因素。
A collection of Aspergillus fumigatus mutants highly resistant to itraconazole (RIT) at 100 mug ml(-1) were selected in vitro (following UV irradiation as a preliminary step) to investigate mechanisms of drug resistance in this clinically important pathogen. Eight of the RIT mutants were found to have a mutation at Gly54 (G54E, -K, or -R) in the azole target gene CYP51A. Primers designed for highly conserved regions of multidrug resistance (MDR) pumps were used in reverse transcriptase PCR amplification reactions to identify novel genes encoding potential MDR efflux pumps in A. fumigatus. Two genes, AfuMDR3 and AfuMDR4, showed prominent changes in expression levels in many RIT mutants and were characterized in more detail. Analysis of the deduced amino acid sequence encoded by AfuMDR3 revealed high similarity to major facilitator superfamily transporters, while AfuMDR4 was a typical member of the ATP-binding cassette superfamily. Real-time quantitative PCR with molecular beacon probes was used to assess expression levels of AfuMDR3 and AfuMDR4. Most RIT mutants showed either constitutive high-level expression of both genes or induction of expression upon exposure to itraconazole. Our results suggest that overexpression of one or both of these newly identified drug efflux pump genes of A. fumigatus and/or selection of drug target site mutations are linked to high-level itraconazole resistance and are mechanistic considerations for the emergence of clinical resistance to itraconazole.