Bruch Alexander, Lazarova Valentina, Berg Maximilian, Krüger Thomas, Schäuble Sascha, Kelani Abdulrahman A, Mertens Birte, Lehenberger Pamela, Kniemeyer Olaf, Kaiser Stefanie, Panagiotou Gianni, Gsaller Fabio, Blango Matthew G
Junior Research Group RNA Biology of Fungal Infections, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute (Leibniz-HKI), Beutenbergstraße 11A, 07745 Jena, Germany.
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany.
Nucleic Acids Res. 2025 Jan 24;53(3). doi: 10.1093/nar/gkae1205.
Increasing antifungal drug resistance is a major concern associated with human fungal pathogens like Aspergillus fumigatus. Genetic mutation and epimutation mechanisms clearly drive resistance, yet the epitranscriptome remains relatively untested. Here, deletion of the A. fumigatus transfer RNA (tRNA)-modifying isopentenyl transferase ortholog, Mod5, led to altered stress response and unexpected resistance against the antifungal drug 5-fluorocytosine (5-FC). After confirming the canonical isopentenylation activity of Mod5 by liquid chromatography-tandem mass spectrometry and Nano-tRNAseq, we performed simultaneous profiling of transcriptomes and proteomes to reveal a comparable overall response to 5-FC stress; however, a premature activation of cross-pathway control (CPC) genes in the knockout was further increased after antifungal treatment. We identified several orthologues of the Aspergillus nidulans Major Facilitator Superfamily transporter nmeA as specific CPC-client genes in A. fumigatus. Overexpression of Mod5-target tRNATyrGΨA in the Δmod5 strain rescued select phenotypes but failed to reverse 5-FC resistance, whereas deletion of nmeA largely, but incompletely, reverted the resistance phenotype, implying additional relevant exporters. In conclusion, 5-FC resistance in the absence of Mod5 and i6A likely originates from multifaceted transcriptional and translational changes that skew the fungus towards premature CPC-dependent activation of antifungal toxic-intermediate exporter nmeA, offering a potential mechanism reliant on RNA modification to facilitate transient antifungal resistance.
抗真菌药物耐药性的增加是与烟曲霉等人类真菌病原体相关的一个主要问题。基因突变和表观突变机制显然驱动了耐药性，但表观转录组仍相对未得到充分研究。在这里，烟曲霉转移RNA（tRNA）修饰的异戊烯基转移酶直系同源物Mod5的缺失导致应激反应改变以及对抗真菌药物5-氟胞嘧啶（5-FC）产生意外的耐药性。通过液相色谱-串联质谱法和纳米tRNA测序确认了Mod5的典型异戊烯基化活性后，我们同时对转录组和蛋白质组进行了分析，以揭示对5-FC应激的总体反应相当；然而，在抗真菌治疗后，敲除菌株中交叉途径控制（CPC）基因的过早激活进一步增加。我们鉴定出构巢曲霉主要促进剂超家族转运蛋白nmeA的几个直系同源物，它们是烟曲霉中特定的CPC客户基因。在Δmod5菌株中过表达Mod5靶向的tRNATyrGΨA挽救了部分表型，但未能逆转5-FC耐药性，而删除nmeA在很大程度上但不完全恢复了耐药表型，这意味着还有其他相关的转运蛋白。总之，在没有Mod5和i6A的情况下，5-FC耐药性可能源于多方面的转录和翻译变化，这些变化使真菌倾向于过早地依赖CPC激活抗真菌毒性中间产物转运蛋白nmeA，这提供了一种依赖RNA修饰来促进短暂抗真菌耐药性的潜在机制。
