Amino Acid Substitutions in the Cytochrome P-450 Lanosterol 14α-Demethylase (CYP51A1) from Azole-Resistant Candida albicans Clinical Isolates Contribute to Resistance to Azole Antifungal Agents
Amino Acid Substitutions in the Cytochrome P-450 Lanosterol 14α-Demethylase (CYP51A1) from Azole-Resistant Candida albicans Clinical Isolates Contribute to Resistance to Azole Antifungal Agents
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DOI：
10.1128/aac.42.2.241
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发表时间：
1998-02
影响因子：
4.9
通讯作者：
D. Sanglard;F. Ischer;L. Koymans;J. Billé
中科院分区：
文献类型：
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作者：
D. Sanglard;F. Ischer;L. Koymans;J. Billé
摘要酵母细胞色素P-450羊毛甾醇14α-脱甲基酶（CYP 51 A1）是麦角甾醇生物合成的重要环节。由于CYP 51 A1是唑类抗真菌药物的靶点，因此该酶可能易于发生改变，导致对这些药物的耐药性。其中，CYP 51 A1对这些药物的亲和力可能降低。我们发现，在一组从艾滋病患者分离的白色念珠菌中，多药外排转运蛋白在念珠菌耐药中起着重要作用。白念珠菌对唑类抗真菌药物敏感，但不排除其他因素的参与（D。桑格拉德湾Kuchler，F. Ischer，J. L.帕加尼，M. Monod和J. Bille，Antimicrob.探员Chemother 39：2378-2386，1995）。因此，我们更详细地分析了CYP 51 A1对唑类抗真菌药物亲和力的变化。提出了在酿酒酵母中进行功能性表达的策略。白念珠菌CYP 51 A1基因序列设计。这种选择，这是再加上一个测试的敏感性唑类衍生物氟康唑，酮康唑，伊曲康唑，使不同的clonedCYP 51 A1基因突变的检测，其产品是潜在的影响，在他们的亲和力唑类衍生物。这种选择使得能够在克隆的CYP 51 A1基因中检测到与临床C.白色念珠菌分离株。这些突变如下：用丙氨酸（G129 A）、Y132 H、S405 F、G464 S和R467 K替换129位的甘氨酸。虽然S405 F突变是作为来自唑抗性酵母的CYP 51 A1基因中的单个氨基酸取代而发现的，但在单个CYP 51 A1基因中同时发现了其他突变，即，R467 K与G464 S、S405 F与Y132 H、G129 A与G464 S以及R467 K与G464 S和Y132 H。对野生型CYP 51 A1基因进行定点突变，以评估这些突变对唑类衍生物耐药性的影响。除G129 A外，每个单一突变对靶酶对特定唑类衍生物的亲和力都有可测量的影响。我们推测，这些特定的突变可能与临床分离株中多药外排转运蛋白的作用联合收割机结合，导致对唑类衍生物的耐药的不同模式和逐步增加。
ABSTRACT The cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance ofC. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378–2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression inSaccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different clonedCYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.