Identification of Essential Genes and Fluconazole Susceptibility Genes in Candida glabrata by Profiling Hermes Transposon Insertions.
Identification of Essential Genes and Fluconazole Susceptibility Genes in Candida glabrata by Profiling Hermes Transposon Insertions.
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DOI：
10.1534/g3.120.401595
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发表时间：
2020-10-05
期刊：
影响因子：
--
通讯作者：
Cunningham KW
中科院分区：
文献类型：
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作者：
Gale AN;Sakhawala RM;Levitan A;Sharan R;Berman J;Timp W;Cunningham KW
在出芽酵母中，光秃假丝酵母菌和其他中aseomyces分支的成员已经发展出独立于白色念珠菌和金黄色念珠菌的毒力特征。为了开始探索C. glabrata毒力及其抗真菌先天抗性的遗传基础，我们从质粒中启动了Hermes转座子，并在整个基因组中测序了超过50万个不同的半随机插入。通过机器学习，我们确定了1278个蛋白质编码基因（占总数的25%），这些基因不能耐受转座子插入，并且可能是体外适能性所必需的。有趣的是，参与mRNA剪接的基因在C. glabrata中不像在S. cerevisiae中那样重要，而参与着丝粒功能和染色体分离的基因则相反。当一组插入突变体被一线抗真菌药物氟康唑攻击时，插入的几个已知抗性基因（如PDR1、CDR1、PDR16、PDR17、UPC2A、DAP1、STV1）和15个附加基因（包括KGD1、KGD2、YHR045W）对氟康唑过敏。另外200个基因的插入对氟康唑产生了显著的抗性，其中三分之二的基因在线粒体中起作用，可能下调Pdr1的表达或功能。KGD2和IDH2敲除突变体在线粒体中消耗和产生α -酮戊二酸，通过依赖于Pdr1的过程表现出对氟康唑的抗性增加和减少。这些发现建立了转座子插入谱在人类这一重要病原体的前瞻性遗传研究中的效用。
Within the budding yeasts, the opportunistic pathogen Candida glabrata and other members of the Nakaseomyces clade have developed virulence traits independently from C. albicans and C. auris. To begin exploring the genetic basis of C. glabrata virulence and its innate resistance to antifungals, we launched the Hermes transposon from a plasmid and sequenced more than 500,000 different semi-random insertions throughout the genome. With machine learning, we identified 1278 protein-encoding genes (25% of total) that could not tolerate transposon insertions and are likely essential for C. glabrata fitness in vitro. Interestingly, genes involved in mRNA splicing were less likely to be essential in C. glabrata than their orthologs in S. cerevisiae, whereas the opposite is true for genes involved in kinetochore function and chromosome segregation. When a pool of insertion mutants was challenged with the first-line antifungal fluconazole, insertions in several known resistance genes (e.g., PDR1, CDR1, PDR16, PDR17, UPC2A, DAP1, STV1) and 15 additional genes (including KGD1, KGD2, YHR045W) became hypersensitive to fluconazole. Insertions in 200 other genes conferred significant resistance to fluconazole, two-thirds of which function in mitochondria and likely down-regulate Pdr1 expression or function. Knockout mutants of KGD2 and IDH2, which consume and generate alpha-ketoglutarate in mitochondria, exhibited increased and decreased resistance to fluconazole through a process that depended on Pdr1. These findings establish the utility of transposon insertion profiling in forward genetic investigations of this important pathogen of humans.