Early Cellular Responses of Neurospora crassa to Azole Stress: Osmotic Adjustment, Redox Buffering, Sterol-Pathway Feedback, and Cell Wall Remodelling

Background/Objectives: Azole resistance in fungal pathogens is a growing clinical and environmental concern. However, short-term cellular responses during the first hours of azole exposure remain insufficiently characterised, particularly in filamentous fungi. This study used Neurospora crassa as a genetically tractable model to investigate early cellular responses to azole stress. Methods: Exponentially growing mycelia were exposed to conidium-derived MIC80 reference concentrations of fluconazole, voriconazole, ravuconazole, and ketoconazole. Early responses were assessed by monitoring radial growth, intracellular glycerol, H2DCF-DA microscopy, antioxidant enzyme activities, Calcofluor White staining, and RT-qPCR analysis of genes associated with osmoregulation, sterol homeostasis, oxidative stress, and cell-wall remodelling. Results: Azole exposure was associated with reduced net post-transfer radial growth and rapid treatment- and time-dependent changes in intracellular glycerol. Induction of hog1 together with treatment-dependent changes in gpd1 and glycerol accumulation was consistent with an early osmoregulatory response. Sterol-homeostasis genes showed selective feedback regulation, and several cell-wall-remodelling genes underwent treatment-dependent transcriptional changes. Qualitative H2DCF-DA microscopy showed limited oxidant-associated fluorescence during azole challenge. Catalase activity remained close to control levels, whereas representative SOD-activity experiments showed larger fold changes after voriconazole and ravuconazole exposure. Representative Calcofluor White micrographs showed irregular septation and localised regions of enhanced cell-wall-associated staining. Conclusions: Neurospora crassa shows multiple concurrent early responses to azole stress involving osmotic adjustment, sterol-pathway feedback, antioxidant responses, and cell-wall remodelling. These findings provide a framework for future studies examining how such stress responses contribute to recovery, tolerance, or longer-term adaptation.