Nascimento Adriana M, Goldman Gustavo H, Park Steven, Marras Salvatore A E, Delmas Guillaume, Oza Uma, Lolans Karen, Dudley Michael N, Mann Paul A, Perlin David S
Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil.
Antimicrob Agents Chemother. 2003 May;47(5):1719-26. doi: 10.1128/AAC.47.5.1719-1726.2003.
A collection of Aspergillus fumigatus mutants highly resistant to itraconazole (RIT) at 100 micro g 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.
为了研究这种临床上重要病原体的耐药机制，在体外（以紫外线照射作为初步步骤）筛选出了对100μg/ml伊曲康唑（RIT）具有高度抗性的烟曲霉突变体库。发现8个RIT突变体在唑类靶基因CYP51A的Gly54（G54E、-K或-R）处发生了突变。设计用于多药耐药（MDR）泵高度保守区域的引物用于逆转录PCR扩增反应，以鉴定编码烟曲霉潜在MDR外排泵的新基因。两个基因，AfuMDR3和AfuMDR4，在许多RIT突变体中的表达水平有显著变化，并进行了更详细的表征。对AfuMDR3编码的推导氨基酸序列分析显示与主要转运体超家族转运蛋白高度相似，而AfuMDR4是ATP结合盒超家族的典型成员。使用分子信标探针进行实时定量PCR来评估AfuMDR3和AfuMDR4的表达水平。大多数RIT突变体显示这两个基因组成型高表达或在暴露于伊曲康唑后诱导表达。我们的结果表明，烟曲霉这两个新鉴定的药物外排泵基因中的一个或两个的过表达和/或药物靶位点突变的选择与高水平伊曲康唑抗性有关，并且是伊曲康唑临床耐药性出现的机制性考虑因素。
