Objectives: Aspergillus flavus is the second most common pathogen causing invasive aspergillosis after A. fumigatus. In the tropical and sub-tropical regions of Asia, sino-orbital or cerebral aspergillosis due to invasive Aspergillus sinusitis are emerging diseases and A. flavus is the predominant pathogen in these infections. In invasive aspergillosis, long-term treatment with azole drugs may cause the emergence of azole-resistant A. fumigatus and acquired azole-resistant mechanisms in A. fumigatus have been well studied. On the other hand, most of azole-resistant risks and mechanisms in A. flavus remain to be elucidated. Here, we investigate the risk of acquiring azole-resistance in A. flavus due to long-term exposure to azole drugs in vitro, and the mechanism of azole-resistance in an isolated azole-resistant mutant.
Methods: Serial passage cultures (7 days per culture) of A. flavus ATCC204304 conidia were performed under exposure to itraconazole or voriconazole in RPMI-agar medium. The MIC was determined by the broth microdilution method according to the CLSI M38-A2 standard. Whole genome sequencing analysis was performed on the Illumina MiSeq platform and CLC Genomics Workbench. Homologous gene replacement studies were performed with A. flavus A1421. Gene expression levels were determined using RT-qPCR.
Results: A mutant with reduced susceptibility to voriconazole with MIC of 16 μg/mL was isolated from the fifth passage culture of A. flavus ATCC204304 in the presence of voriconazole whereas the MIC of itraconazole was 0.5 μg/mL. This voriconazole-resistant mutant did not have any mutation in the cyp51A gene and its promoter region. Interestingly, we found a mutation in yap1, encoding a bZIP transcription factor well-known as the master regulator of oxidative stress responses, which caused Leu558Trp substitution in the carboxy-terminal cysteine-rich domain of Yap1. This mutation was confirmed to be responsible for the voriconazole-resistant phenotype but not for itraconazole by homologous gene replacement studies. Furthermore, we evaluated transcriptional levels of genes which have putative Yap1 response elements in promoter regions, and the expression levels of atrF, dsba, gst and cyp51A in the yap1 mutant were 62-, 27-, 15- and 4.7-fold higher compared to the parental strain, respectively. Notably, we focused on the marked upregulation of the atrF, encoding an ABC efflux transporter, and then the deletion of atrF in the yap1 mutant almost restored the susceptibility to voriconazole.
Conclusions: To the best of our knowledge, this is the first report about a point mutation in yap1 and subsequent marked atrF-upregulation causing voriconazole-resistance in A. flavus. These findings provide new insights into azole-resistant mechanisms independent of the cyp51A gene mutation in A. flavus.

A Novel Mechanism of Voriconazole-Resistance in Aspergillus flavus