Abstract
Cryptococcus neoformans, designated as a "critical priority" pathogen by the World Health Organization, poses significant therapeutic challenges with only three drug classes currently available for cryptococcosis treatment. The emergence of antifungal resistance, compounded by cross-resistance, further limits therapeutic efficacy. Aneuploidy, known for its potential to induce diverse traits, including antifungal resistance, remains poorly documented in C. neoformans. Utilizing tunicamycin, a well-established ER stress inducer, we investigated its impact on aneuploidy formation in C. neoformans. Our findings reveal that both mild and severe ER stress induced by tunicamycin result in the formation of aneuploidy in C. neoformans. These aneuploid strains exhibit diverse karyotypes, with select karyotypes conferring resistance or cross-resistance to fluconazole and 5-flucytosine. Additionally, these aneuploid strains display instability, spontaneously losing extra chromosomes in the absence of stress. Transcriptome analysis unveiled simultaneous upregulation of multiple drug resistance-associated genes in aneuploid strains, highlighting the genome plasticity of C. neoformans as a major mechanism contributing to non-antifungal-induced antifungal resistance.