ABSTRACT: Persistence and tolerance allow susceptible microbial populations to survive antimicrobial exposure without changes in minimum inhibitory concentration, but these phenomena remain poorly characterized in fungi. We investigated the mechanisms underlying voriconazole (VOR) persistence in Aspergillus fumigatus. Transcriptional profiling of wild-type conidia surviving prolonged 4×MIC VOR exposure showed that persistence is not passive dormancy but an active program combining cellular reinforcement (protein and cell wall synthesis), metabolic adaptation (alternative energy sources, lipid remodeling), and stress mitigation (secondary metabolism, membrane transport), alongside broad repression of transcription, RNA processing, and DNA repair. Chemical inhibition of protein translation (puromycin), sphingolipid biosynthesis (myriocin), or cell wall remodeling (Calcofluor White/Congo Red) each reduced the persistence index, with sphingolipid inhibition producing the strongest effect. Screening 484 transcription factor deletion mutants identified two regulators, MtfA and AFUB_040000, whose loss increased VOR persistence and altered both germination kinetics and the basal transcriptional state of conidia. Titratable repression or overexpression of the essential regulators TorA and PkcA similarly enhanced persistence in both directions, indicating that maintenance within a defined physiological range, rather than pathway activity per se, governs the persistent phenotype. Extending this analysis to the core MAPK network, MpkA, MpkB, and MpkC, but not SakA, contributed to VOR persistence, with MpkC required for VOR-induced upregulation of MtfA and AFUB_040000. Together, these pharmacological and genetic data converge on a model in which protein translation, sphingolipid biosynthesis, and cell wall integrity/MAPK signaling coordinately determine A. fumigatus persistence under azole stress, nominating these pathways as candidate targets for combination antifungal strategies.
