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：
10.1093/jac/42.2.241
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发表时间：
1998-02-01
影响因子：
4.9
通讯作者：
Bille, J
中科院分区：
文献类型：
--
作者：
Sanglard, D;Ischer, F;Bille, J
酵母细胞色素P-450羊毛甾醇14α-脱甲基酶参与了麦角甾醇生物合成的重要步骤，由于细胞色素P51A1是唑类抗真菌药物的靶标，该酶容易发生改变，导致对这些药物的耐药性，其中，该酶对这些药物的亲和力降低是可能的。我们在一组来自艾滋病患者的白色念珠菌中发现，多药外排转运蛋白在白色念珠菌对唑类抗真菌药物的耐药性中起着重要的作用，但不排除其他因素的参与(D.Sanglard，K.Kuchler，F.Ischer，J.-L，Pagani，M.Monod，和J.Bille，Antimmicrob，Agents Chemther，39：2378-2386,1995)，因此，我们更详细地分析了CYP51A1对唑类抗真菌药物的亲和力的变化，设计了一种由患者连续临床分离的白念珠菌CYP51A1基因在酿酒酵母中的功能表达组成的策略，该选择与对唑衍生物氟康唑、酮康唑和伊曲康唑的敏感性测试相结合，使得能够检测不同克隆的CYP51A1基因的突变，其产物对唑衍生物的亲和力可能受到影响，这一选择使得能够检测到与临床分离的白色念珠菌对唑类耐药相关的5种不同的突变：第129位甘氨酸替换为丙氨酸(G129A)、Y132H、S405F、G464S和R467K。在一株唑类耐药酵母菌的CYP51A1基因中，发现S405F突变为单一氨基酸替换，同时发现其他突变，即R467K突变G464S，S405F突变Y132H，G129A突变G464S，R467K突变G464S和Y132H。通过对野生型CYP51A1基因进行定点突变，以评估这些突变对唑衍生物耐药性的影响，除G129A外，每个突变都对靶酶与特定的唑衍生物的亲和力有可测量的影响，我们推测这些特定的突变可能与临床分离株的多药外排转运体的作用相结合，导致对唑衍生物的不同模式和逐步增加的耐药性。
The cytochrome P-450 lanosterol 14 alpha-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 of C. 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 in Saccharomyces 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 cloned CYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives, This selection enabled the detection of five different mutations in the cloned CYP51A1 genes 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 CYP51A1 gene 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.