Identification of ATM1 gene involved in antifungal resistance based on CRISPR/Cas9 technology in Cryptococcus gattii

Cryptococcus gattii is a fungal pathogen that poses significant threats to human health, affecting both immunocompromised and immunocompetent individuals. Treatment of C. gattii infections typically involves the use of antifungal agents, such as azoles. However, the increasing emergence of antifungal resistance in C. gattii is a growing concern, highlighting the critical need for novel therapeutic strategies. In our previous study, we identified a mitochondrial ATP-binding cassette (ABC) transporter, Atm1, as potentially involved in antifungal resistance in C. gattii through transcriptome sequencing, but its function remains unclear and requires additional confirmation and investigation. In this study, we developed a "suicide" clustered regularlyinterspaced short palindromic repeats-CRISPR-associated protein 9 system in C. gattii, based on the system used in C. neoformans, and successfully validated its functionality by targeting the ADE2 gene. We subsequently generated C. gattii mutants lacking ATM1 and assessed their growth under various stress conditions. Our data suggest that Atm1 is involved in the iron-sulfur cluster biosynthesis process. Besides, disruption of ATM1 resulted in various growth impairments, including reduced stress tolerance, impaired capsule formation, and diminished virulence. Importantly, we observed compromised antifungal drug resistance in the atm1∆ mutant and performed RNA sequencing-based transcriptome analysis and gene ontology analysis with and without antifungal treatment for further investigation. In conclusion, our findings indicate that ATM1 plays a role in iron homeostasis and is critical for antifungal resistance in C. gattii, offering new insights into potential drug development strategies for the clinical treatment of cryptococcosis.