Reconstitution of high-level micafungin resistance detected in a clinical isolate of Candida glabrata identifies functional homozygosity in glucan synthase gene expression
Reconstitution of high-level micafungin resistance detected in a clinical isolate of Candida glabrata identifies functional homozygosity in glucan synthase gene expression
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DOI：
10.1093/jac/dks112
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发表时间：
2012-07-01
影响因子：
5.2
通讯作者：
Monk, Brian C.
中科院分区：
文献类型：
--
作者：
Niimi, Kyoko;Woods, Matthew A.;Monk, Brian C.
光滑念珠菌获得高水平棘白菌素耐药性的机制进行了研究。构建FKS突变体的目的是：确定临床上显著的米卡芬净耐药是否需要FKS 1中的热点突变和FKS 2中的提前终止密码子，如在临床分离株中观察到的那样;选择敏感性降低的变体并定位FKS基因中的突变;并评估FKS 1和FKS 2的作用。比较了突变体之间的药物敏感性、基因表达和葡聚糖合酶活性。通过选择获得的突变通过来自所选变体的FKS基因的DNA序列分析来鉴定。构建了单个FKS缺失株，并检测了其表型。仅在FKS 1中引入热点突变可使易感性中度降低，而仅在FKS 2中引入提前终止密码子对易感性没有影响，而与临床分离株相当的易感性严重降低则需要这两种突变。敏感菌株暴露于米卡芬净产生了敏感性中度降低的变体，其在FKS 1中具有热点突变。进一步暴露于米卡芬净产生了易感性严重降低的变异体，这些变异体在FKS 2中获得了各种单突变。fks 1和fks 2突变体的表型表明，这两个FKS基因是功能冗余的，而FKS 1和FKS 2的缺失赋予合成致死性。glabrata，临床上显著降低米卡芬净敏感性需要FKS 1和FKS 2的单核苷酸变化，两个基因编码-1，3-葡聚糖合酶催化亚基。
A mechanism for the acquisition of high-level echinocandin resistance in Candida glabrata was investigated. FKS mutants were constructed to: determine whether clinically significant micafungin resistance requires a hot-spot mutation in FKS1 and a premature stop codon in FKS2, as was observed in a clinical isolate; select for variants with reduced susceptibility and locate mutations in FKS genes; and assess the roles of FKS1 and FKS2.A panel of FKS mutants was constructed using micafungin-susceptible parents by site-directed mutagenesis. Drug susceptibility, gene expression and glucan synthase activities were compared between mutants. Mutations acquired by selection were identified by DNA sequence analysis of FKS genes from selected variants. Single FKS deletants were constructed and their phenotypes examined.Introduction of the hot-spot mutation in FKS1 alone conferred an intermediate reduction in susceptibility, and the premature stop codon in FKS2 alone had no effect on susceptibility, while severely reduced susceptibility equivalent to that of the clinical isolate required both mutations. Exposure of susceptible strains to micafungin yielded variants with an intermediate reduction in susceptibility that possessed a hot-spot mutation in FKS1. Further exposure to micafungin yielded variants with severely reduced susceptibility that acquired various single mutations in FKS2. The phenotypes of fks1 and fks2 mutants indicate that the two FKS genes are functionally redundant, while deletion of both FKS1 and FKS2 conferred synthetic lethality.In the laboratory mutants of C. glabrata, clinically significant reduced susceptibility to micafungin required single nucleotide changes in both FKS1 and FKS2, and both genes encoded -1,3-glucan synthase catalytic subunits.