Extra copies of the Aspergillus fumigatus squalene epoxidase gene confer resistance to terbinafine:: Genetic approach to studying gene dose-dependent resistance to antifungals in A-fumigatus
Extra copies of the Aspergillus fumigatus squalene epoxidase gene confer resistance to terbinafine:: Genetic approach to studying gene dose-dependent resistance to antifungals in A-fumigatus
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DOI：
10.1128/aac.48.7.2490-2496.2004
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发表时间：
2004-07-01
影响因子：
4.9
通讯作者：
Kontoyiannis, DP
中科院分区：
文献类型：
--
作者：
Liu, W;May, GS;Kontoyiannis, DP
随着侵袭性曲霉病高危患者越来越多地使用抗真菌药，如阿替霉素B、伊曲康唑、伏立康唑、卡泊芬净和特比萘芬（TR B），烟曲霉对这些药物的耐药性最终将出现。由于对A.烟曲霉，很少有研究涉及其对抗真菌药物的抗性机制。我们改造了A.用基于pyrG的A.烟曲霉基因组DNA文库（构建在多拷贝非整合载体pRG 3-AMA 1-NotI中，其也具有用于选择的pyr-4基因）。我们获得了一个pyrG（+）菌株，它生长在含有杀真菌浓度（0.625 μ g/ml）的TRB的培养基中。为了确定TRB抗性是否在该细菌中是质粒依赖性的，我们驱逐质粒，发现伴随的尿嘧啶原养型和TRB抗性的丧失。DNA序列分析表明，与TRB抗性相关的基因为A.烟曲霉角鲨烯环氧酶基因（ERG 1），编码TRB的靶酶。正宗A。从基因组扩增并克隆到pRG 3-AMA 1-NotI中的烟曲霉ERG 1也赋予TRB特异性抗性。这种分子方法有可能增强我们对A.烟曲霉对现代抗真菌剂的抗性。
With the increasing use of antifungals such as amphotericin B, itraconazole, voriconazole, caspofungin, and terbinafine (TRB) in patients at high risk for invasive aspergillosis, resistance of Aspergillus fumigatus to these agents will ultimately emerge. Due to the limited availability of molecular genetics for A. fumigatus, few studies have addressed its mechanisms of resistance to antifungals. We transformed A. fumigatus protoplasts with a pyrG-based A. fumigatus genomic DNA library (constructed in the multicopy nonintegrating vector pRG3-AMA1-NotI, which also has the pyr-4 gene for selection). We obtained one pyrG(+) transformant that grew in medium containing a fungicidal concentration (0.625 mug/ml) of TRB. To determine whether TRB resistance in that transformant was plasmid dependent, we evicted the plasmid and found concomitant loss of uracil prototrophy and TRB resistance. DNA sequence analysis identified the gene responsible for TRB resistance as the A. fumigatus squalene epoxidase gene (ERG1), which encodes the target enzyme of TRB. Authentic A. fumigatus ERG1, amplified from the genome and cloned into pRG3-AMA1-NotI, also conferred TRB-specific resistance. This molecular approach has the potential to enhance our knowledge of the mechanisms of A. fumigatus resistance to modern antifungals.