Aneuploidy formation in the filamentous fungus Aspergillus flavus in response to azole stress

Authors: BARDA, OMER - Volcani Center (ARO); SADHASIVAM, SUDHARSAN - Volcani Center (ARO); GONG, DI - Volcani Center (ARO); DORON-FAIGENBOIM, ADI - Volcani Center (ARO); ZAKIN, VARDA - Volcani Center (ARO); Drott, Milton; SIONOV, EDWARD - Volcani Center (ARO)

Submitted to: Microbiology Spectrum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/6/2023
Publication Date: 6/26/2023
Citation: Barda, O., Sadhasivam, S., Gong, D., Doron-Faigenboim, A., Zakin, V., Drott, M.T., Sionov, E. 2023. Aneuploidy formation in the filamentous fungus Aspergillus flavus in response to azole stress. Microbiology Spectrum. 11(4). Article e04339-22. https://doi.org/10.1128/spectrum.04339-22.
DOI: https://doi.org/10.1128/spectrum.04339-22

Interpretive Summary: Fungal pathogens cause human disease and threaten global food security by contaminating crops with toxins. Azole antifungal drugs are among the most effective compounds used to control fungi in both clinical and agricultural settings. However, a rise in azole-resistant fungi threatens our main line of defense against these devastating pathogens. Aspergillus flavus is a filamentous fungus that is a leading cause of invasive aspergillosis in humans and contaminates most major crops with the notorious carcinogen, aflatoxin. We show that A. flavus can rapidly evolve azole resistance through the transient duplication of specific genomic regions (i.e., aneuploidy). Our discovery of aneuploidy-mediated resistance in a filamentous fungus represents a paradigm shift as this type of resistance was previously thought to occur only in yeast-like organisms. Knowledge of aneuploidy in filamentous fungi is important to explain fungal pathogenesis and may help develop new strategies to mitigate the emergence of fungicide resistance.

Technical Abstract: Azole antifungal drugs are among the most popular and effective chemical compounds used to control fungal infections of agricultural crops and people. While many azole resistance mechanisms are widespread across fungi, aneuploidy-mediated resistance has previously been reported only in fungi with yeast-like growth patterns. Aspergillus flavus, a filamentous mycotoxigenic fungus, causes human disease through direct infection and devastating losses of agricultural crops by contaminating food stuffs with the notorious carcinogen, aflatoxin. As in other filamentous fungi, azole resistance in A. flavus has been associated with point mutations, particularly in the cyp51 gene. We observed that exposure of A. flavus to voriconazole (VRC) resulted in the evolution of a transient azole resistance. We hypothesized that A. flavus can acquire azole resistance arose through aneuploidy. A small number of clones that were resistant very high concentrations of VRC were obtained by subculturing a highly aflatoxigenic strain of A. flavus on VRC-containing media. Whole-genome sequencing of eight clones identified aneuploidy in three. Two of these aneuploid clones had a complete duplication of chromosome eight (Chr8) while the other clone displayed a segmental duplication of chromosome three (Chr3). These duplications did not coincide with known azole resistance genes, emphasizing the potential diversity of aneuploidy-mediated resistance mechanisms. Aneuploid clones returned to their original level of susceptibility following repeated transfers on drug-free media. Our results identify a novel mechanism of azole resistance in filamentous fungi.