Contributions of yap1 mutation and subsequent atrF upregulation to voriconazole resistance in Aspergillus flavus [PublishAheadOfPrint]

Aspergillus flavus is the second most significant pathogen that causes invasive aspergillosis; however, its emergence risks and mechanisms of voriconazole (VRC) resistance have not yet been elucidated in detail. Here, we demonstrate that repeated exposure of A. flavus to sub-inhibitory concentrations of VRC in vitro causes the emergence of a VRC-resistant mutant with a novel resistance mechanism. The VRC-resistant mutant shows a MIC of 16 μg/mL for VRC, and of 0.5 μg/mL for itraconazole (ITC). Whole-genome sequencing analysis showed that the mutant possesses a point mutation in yap1, which encodes a bZIP transcription factor working as the master regulator of oxidative stress response, but no mutations in the cyp51 genes. This point mutation in yap1 caused substitution of Leu558 to Trp (Yap1^Leu558Trp) in the putative nuclear export sequence in the carboxy-terminal cysteine-rich domain of Yap1. This Yap1^Leu558Trp substitution was confirmed as being responsible for the VRC-resistant phenotype, but not for ITC, by the revertant to Yap1^wild-type with homologous gene replacement. Furthermore, Yap1^Leu558Trp caused marked upregulation of the atrF ATP-binding cassette transporter, and the deletion of atrF restored susceptibility to VRC in A. flavus. These findings provide new insights into VRC-resistance mechanisms via a transcriptional factor mutation that is independent of the cyp51 gene mutation in A. flavus.

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