Broad antifungal resistance mediated by RNAi-dependent epimutation in the basal human fungal pathogen Mucor circinelloides
Broad antifungal resistance mediated by RNAi-dependent epimutation in the basal human fungal pathogen Mucor circinelloides
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DOI：
10.1371/journal.pgen.1007957
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发表时间：
2019-02-01
期刊：
影响因子：
4.5
通讯作者：
Heitman, Joseph
中科院分区：
文献类型：
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作者：
Chang, Zanetta;Billmyre, R. Blake;Heitman, Joseph
毛霉菌病新出现的致命真菌感染很难治疗，部分原因是致病菌表现出广泛的临床抗真菌耐药性。然而，这些感染中耐药性的机制仍然知之甚少。我们之前的工作表明，毛霉菌病的一种主要病原体卷枝毛霉（Mucor circinelloides）可以通过一种称为表观突变的新型瞬时 RNA 干扰依赖性机制对抗真菌药物 FK506 和雷帕霉素产生耐药性。表观突变使药物靶基因沉默，并通过药物暴露进行选择；无需药物传代后，靶基因重新表达并且敏感性恢复。该沉默过程涉及通过核心 RNAi 途径蛋白生成针对靶基因的小 RNA (sRNA)。为了进一步阐明表突变在毛霉广泛抗真菌耐药性中的作用，分离了对另一种抗真菌药物 5-氟乳清酸 (5-FOA) 产生耐药性的表突变体。我们鉴定了对 5-FOA 表现出抗性的表突变菌株，但 PyrF 或 PyrG 没有突变，PyrF 或 PyrG 是可将 5-FOA 转化为活性毒性形式的酶。使用sRNA杂交以及sRNA文库分析，我们证明这些表观突变体含有针对pyrF或pyrG的sRNA，并进一步表明该sRNA在恢复药物敏感性后丢失。我们得出的结论是，表观突变是一种能够靶向多个基因的机制，使毛霉能够对多种抗真菌药物产生耐药性。阐明 RNAi 在表突变中的作用可以更全面地了解毛霉菌病。此外，它还提高了我们对真菌发病机制和应激适应的理解，包括耐药性的进化。作者摘要 新出现的感染毛霉菌病导致高死亡率，部分原因是主要致病真菌，包括卷枝毛霉菌，对大多数临床可用的抗真菌药物具有抗药性。我们之前发现了一种基于 RNA 干扰的耐药机制，即表观突变，通过该机制，M. circinelloides 通过改变内源 RNA 表达，对抗真菌剂 FK506 产生短暂的耐药性。我们通过分离两个基因的表突变来进一步表征这种新机制，这两个基因赋予对另一种抗真菌剂 5-氟乳清酸的抗性。因此，我们证明表观突变可以通过靶向多种基因来诱导对多种抗真菌药物的耐药性。这些结果表明，表观突变发挥着广泛的作用，使真菌能够对环境压力做出快速、可逆的反应，包括药物暴露、控制抗真菌药物耐药性和 RNA 表达。随着抗真菌药物耐药性的出现，更深入地了解其致病机制对于改善治疗至关重要。
Mucormycosisan emergent, deadly fungal infectionis difficult to treat, in part because the causative species demonstrate broad clinical antifungal resistance. However, the mechanisms underlying drug resistance in these infections remain poorly understood. Our previous work demonstrated that one major agent of mucormycosis, Mucor circinelloides, can develop resistance to the antifungal agents FK506 and rapamycin through a novel, transient RNA interference-dependent mechanism known as epimutation. Epimutations silence the drug target gene and are selected by drug exposure; the target gene is re-expressed and sensitivity is restored following passage without drug. This silencing process involves generation of small RNA (sRNA) against the target gene via core RNAi pathway proteins. To further elucidate the role of epimutation in the broad antifungal resistance of Mucor, epimutants were isolated that confer resistance to another antifungal agent, 5-fluoroorotic acid (5-FOA). We identified epimutant strains that exhibit resistance to 5-FOA without mutations in PyrF or PyrG, enzymes which convert 5-FOA into the active toxic form. Using sRNA hybridization as well as sRNA library analysis, we demonstrate that these epimutants harbor sRNA against either pyrF or pyrG, and further show that this sRNA is lost after reversion to drug sensitivity. We conclude that epimutation is a mechanism capable of targeting multiple genes, enabling Mucor to develop resistance to a variety of antifungal agents. Elucidation of the role of RNAi in epimutation affords a fuller understanding of mucormycosis. Furthermore, it improves our understanding of fungal pathogenesis and adaptation to stresses, including the evolution of drug resistance.Author summary The emerging infection mucormycosis causes high mortality in part because the major causative fungi, including Mucor circinelloides, are resistant to most clinically available antifungal drugs. We previously discovered an RNA interference-based resistance mechanism, epimutation, through which M. circinelloides develops transient resistance to the antifungal agent FK506 by altering endogenous RNA expression. We further characterize this novel mechanism by isolating epimutations in two genes that confer resistance to another antifungal agent, 5-fluoroorotic acid. Thus, we demonstrate epimutation can induce resistance to multiple antifungals by targeting a variety of genes. These results reveal epimutation plays a broad role enabling rapid and reversible fungal responses to environmental stresses, including drug exposure, and controlling antifungal drug resistance and RNA expression. As resistance to antifungals emerges, a deeper understanding of the causative mechanisms is crucial for improving treatment.