Invasive candidiasis caused by non-albicans species has been on the rise, with Candida glabrata emerging as the second most common etiological agent. Candida glabrata possesses an intrinsically lower susceptibility to azoles and an alarming propensity to rapidly develop high-level azole resistance during treatment. In this study, we have developed an efficient piggyBac (PB) transposon-mediated mutagenesis system in C. glabrata to conduct genome-wide genetic screens and applied it to profile genes that contribute to azole resistance. When challenged with the antifungal drug fluconazole, PB insertion into 270 genes led to significant resistance. A large subset of these genes has a role in the mitochondria, including almost all genes encoding the subunits of the F₁F₀ ATPase complex. We show that deleting ATP3 or ATP22 results in increased azole resistance but does not affect susceptibility to polyenes and echinocandins. The increased azole resistance is due to increased expression of PDR1 that encodes a transcription factor known to promote drug efflux pump expression. Deleting PDR1 in the atp3Δ or atp22Δ mutant resulted in hypersensitivity to fluconazole. Our results shed light on the mechanisms contributing to azole resistance in C. glabrata. This PB transposon-mediated mutagenesis system can significantly facilitate future genome-wide genetic screens.

中文翻译：

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PiggyBac 转座子插入突变体的全基因组分析揭示 F1F0 ATP 酶复合物的丢失导致光滑念珠菌对氟康唑耐药

由非白色念珠菌引起的侵袭性念珠菌病呈上升趋势，其中光滑念珠菌成为第二常见的病原体。光滑念珠菌本质上对唑类药物具有较低的敏感性，并且在治疗过程中具有迅速产生高水平唑类耐药性的惊人倾向。在这项研究中，我们在光滑念珠菌中开发了一种高效的piggyBac（PB）转座子介导的诱变系统，用于进行全基因组遗传筛选，并将其应用于分析有助于唑类抗性的基因。当受到抗真菌药物氟康唑的挑战时，PB插入 270 个基因导致了显着的耐药性。这些基因的很大一部分在线粒体中发挥作用，包括几乎所有编码 F ₁ F ₀ ATP 酶复合物亚基的基因。我们发现，删除ATP3或ATP22会导致唑类耐药性增加，但不会影响对多烯和棘白菌素的敏感性。唑类耐药性增加是由于PDR1表达增加所致，PDR1 编码一种已知可促进药物外排泵表达的转录因子。删除atp3 Δ 或atp22 Δ 突变体中的PDR1会导致对氟康唑过敏。我们的结果揭示了光滑念珠菌中唑类抗性的机制。这种PB转座子介导的诱变系统可以显着促进未来的全基因组遗传筛选。