Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Canada.
Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Canada.
PLoS Genet. 2023 Oct 19;19(10):e1011002. doi: 10.1371/journal.pgen.1011002. eCollection 2023 Oct.
Pathogenic fungi are a cause of growing concern. Developing an efficient and safe antifungal is challenging because of the similar biological properties of fungal and host cells. Consequently, there is an urgent need to better understand the mechanisms underlying antifungal resistance to prolong the efficacy of current molecules. A major step in this direction would be to be able to predict or even prevent the acquisition of resistance. We leverage the power of experimental evolution to quantify the diversity of paths to resistance to the antifungal 5-fluorocytosine (5-FC), commercially known as flucytosine. We generated hundreds of independent 5-FC resistant mutants derived from two genetic backgrounds from wild isolates of Saccharomyces cerevisiae. Through automated pin-spotting, whole-genome and amplicon sequencing, we identified the most likely causes of resistance for most strains. Approximately a third of all resistant mutants evolved resistance through a pleiotropic drug response, a potentially novel mechanism in response to 5-FC, marked by cross-resistance to fluconazole. These cross-resistant mutants are characterized by a loss of respiration and a strong tradeoff in drug-free media. For the majority of the remaining two thirds, resistance was acquired through loss-of-function mutations in FUR1, which encodes an important enzyme in the metabolism of 5-FC. We describe conditions in which mutations affecting this particular step of the metabolic pathway are favored over known resistance mutations affecting a step upstream, such as the well-known target cytosine deaminase encoded by FCY1. This observation suggests that ecological interactions may dictate the identity of resistance hotspots.
致病真菌引起的关注度日益增加。由于真菌和宿主细胞具有相似的生物学特性，开发高效且安全的抗真菌药物具有挑战性。因此，迫切需要更好地了解抗真菌耐药性的机制，以延长现有分子的疗效。朝这个方向迈出的重要一步是能够预测甚至预防耐药性的产生。我们利用实验进化的力量来量化对抗真菌药物 5-氟胞嘧啶（5-FC）的耐药性的多种途径，5-FC 的商品名为氟胞嘧啶。我们从野生酵母属酿酒酵母的两个遗传背景中生成了数百个独立的 5-FC 耐药突变体。通过自动化针点印迹、全基因组和扩增子测序，我们确定了大多数菌株最有可能的耐药原因。大约三分之一的耐药突变体通过多效药物反应进化出耐药性，这是一种对 5-FC 的潜在新机制，表现为对氟康唑的交叉耐药性。这些交叉耐药突变体的特征是呼吸丧失和无药物培养基中的强烈权衡。对于其余三分之二的大部分，耐药性是通过编码 5-FC 代谢中重要酶的 FUR1 基因的功能丧失突变获得的。我们描述了在哪些条件下，影响代谢途径这一特定步骤的突变会比影响上游步骤的已知耐药突变更有利，例如已知的靶标胞嘧啶脱氨酶，其由 FCY1 编码。这一观察结果表明，生态相互作用可能决定耐药热点的特征。
