Genomewide screening for genes associated with gliotoxin resistance and sensitivity in Saccharomyces cerevisiae
Genomewide screening for genes associated with gliotoxin resistance and sensitivity in Saccharomyces cerevisiae
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DOI：
10.1128/aac.01393-07
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发表时间：
2008-04-01
影响因子：
4.9
通讯作者：
Kontoyiannis, Dimitrios P.
中科院分区：
文献类型：
--
作者：
Chamilos, Georgios;Lewis, Russell E.;Kontoyiannis, Dimitrios P.
胶霉毒素 (GT) 是一种次生真菌代谢产物，具有多效性免疫抑制特性，与曲霉菌毒力有关。然而，GT细胞毒性的机制及其在真核细胞中的分子靶点尚未得到充分表征。我们筛选了酿酒酵母单基因缺失突变体的单倍体文库（EUROSCARF 中的 4,787 个菌株），以鉴定与 GT 抗性增加 (GT-IR) 和敏感性增加 (GT-IS) 相关的非必需基因。野生型亲本菌株 BY4741 对 GT 的敏感性最初通过使用不同培养基的微量肉汤稀释方法进行评估。 GT-IR 和 GT-IS 被定义为 MIC 分别增加四倍和减少四倍，并且这还通过琼脂酵母提取物-蛋白胨-葡萄糖平板上的药敏测试得到证实。在研究 GT-IR 和 GT-IS 突变体对常规抗真菌剂、放线菌酮和 H2O2 的敏感性的研究中，进一步测试了与野生型菌株相比表现出正常生长的 GT-IR 和 GT-IS 突变体的特异性。 GT-IR 与参与一般代谢（OP11、SNF1、IFA38）、线粒体功能（RTG2）、DNA 损伤修复（RAD18）和囊泡运输（APL2）的基因以及功能未知的基因（YGL235W、YOR345C、YLR456W、YGL072C）的破坏相关。编码转硫作用 (CYS3)、线粒体功能 (MEF2) 和未知功能 (YKL037W) 的三个基因的破坏导致了 GT-IS。在所有突变体中均观察到 GT-IR 和 GT-IS 的特异性。重要的是，GT-IR (6/10) 和 GT-IS (2/3) 涉及的大多数基因 (69%) 具有人类同源物。我们鉴定了与 GT-IR 或 GT-IS 特别相关的新酵母基因。由于大多数这些基因在进化上是保守的，对其功能的进一步表征可以提高我们对人类 GT 细胞毒性机制的理解。
Gliotoxin (GT) is a secondary fungal metabolite with pleiotropic immunosuppressive properties that have been implicated in Aspergillus virulence. However, the mechanisms of GT cytotoxicity and its molecular targets in eukaryotic cells have not been fully characterized. We screened a haploid library of Saccharomyces cerevisiae single-gene deletion mutants (4,787 strains in EUROSCARF) to identify nonessential genes associated with GT increased resistance (GT-IR) and increased sensitivity (GT-IS). The susceptibility of the wild-type parental strain BY4741 to GT was initially assessed by broth microdilution methods using different media. GT-IR and GT-IS were defined as a fourfold increase and decrease, respectively, in MIC, and this was additionally confirmed by susceptibility testing on agar yeast extract-peptone-glucose plates. The specificity of GT-IR and GT-IS mutants exhibiting normal growth compared with the wild-type strain was further tested in studies of their susceptibility to conventional antifungal agents, cycloheximide, and H2O2. GT-IR was associated with the disruption of genes acting in general metabolism (OP11, SNF1, IFA38), mitochondrial function (RTG2), DNA damage repair (RAD18), and vesicular transport (APL2) and genes of unknown function (YGL235W, YOR345C, YLR456W, YGL072C). The disruption of three genes encoding transsulfuration (CYS3), mitochondrial function (MEF2), and an unknown function (YKL037W) led to GT-IS. Specificity for GT-IR and GT-IS was observed in all mutants. Importantly, the majority (69%) of genes implicated in GT-IR (6/10) and GT-IS (2/3) have human homologs. We identified novel Saccharomyces genes specifically implicated in GT-IR or GT-IS. Because most of these genes are evolutionarily conserved, further characterization of their function could improve our understanding of GT cytotoxicity mechanisms in humans.