Dual adaptive strategies in Candida glabrata under tunicamycin stress: petite mutations and chromosome C aneuploidy drive transient drug resistance.

Background: Candida glabrata is an opportunistic fungal pathogen known for its ability to rapidly develop resistance to antifungal agents. Tunicamycin (TUN), an inhibitor of N-linked glycosylation, induces Endoplasmic Reticulum (ER) stress, but the adaptive mechanisms enabling C. glabrata to survive TUN exposure remain poorly understood. Objective: This study aimed to identify and characterize the genetic and phenotypic adaptations that confer TUN resistance in C. glabrata and evaluate their stability in the absence of drug pressure. Methods: We exposed C. glabrata strain BG2 to sub-inhibitory (0.5 μg/mL) and inhibitory (1-8 μg/mL) TUN concentrations and isolated resistant mutants. Phenotypic characterization included growth assays, mitochondrial function tests (YPG medium), and fluconazole (FLC) susceptibility testing. Whole-genome sequencing assessed chromosomal alterations, and serial passaging in drug-free medium evaluated adaptation stability. Results: Under TUN stress, C. glabrata adopted two distinct resistance strategies: (1) mitochondrial dysfunction (petite formation), which conferred cross-resistance to FLC, and (2) aneuploidy, particularly disomy of chromosome C (ChrCx2), often accompanied by additional chromosomal gains in high-TUN conditions. However, both adaptations exhibited significant trade-offs: petite mutants retained irreversible respiratory deficiency but lost TUN and FLC resistance upon passaging, while aneuploid strains rapidly reverted to euploidy in non-selective conditions, abolishing TUN resistance. Conclusion: C. glabrata survives TUN stress through unstable genetic adaptations-petite formation and aneuploidy-that are rapidly selected against in drug-free environments. These findings highlight the evolutionary constraints of antifungal resistance mechanisms and suggest that intermittent therapy may help counteract resistance development.