Nootkatone antifungal activity and calcium signaling-mediated resistance in Aspergillus fumigatus

Tobacco is rich in natural bioactive compounds, yet the antifungal potential of its essential oil remains largely unexplored. In this study, we demonstrate for the first time that tobacco essential oil exhibits significant antifungal activity against the opportunistic human pathogen Aspergillus fumigatus. At high concentrations, the oil effectively inhibits hyphal growth and suppresses conidial production. Chemical profiling identified nootkatone as the principal active compound. Nootkatone exerts a concentration-dependent dual antifungal effect, simultaneously inhibiting hyphal elongation and spore formation. Mechanistic analyses revealed that the calcium signaling pathway, particularly the crzA and cchA genes, plays a central regulatory role in mediating fungal resistance to nootkatone. Deletion of either gene (ΔcrzA or ΔcchA) led to significantly increased sensitivity to nootkatone, highlighting their role in nootkatone stress response. Furthermore, under nootkatone stress, CrzA was found to regulate the expression of multiple efflux pump genes (abcC, abcE, atrA, mdr1, and atrB), uncovering a previously unrecognized link between calcium signaling and nootkatone efflux. In addition to targeting cellular signaling, nootkatone also disrupted fungal cell wall integrity and triggered the release of host immune mediators, suggesting a synergistic antifungal mechanism that combines structural disruption with immune activation. In the Galleria mellonella infection model, nootkatone significantly enhanced host survival and reduced fungal burden. Notably, treatment of ΔcrzA-infected larvae resulted in a 70% survival rate, markedly higher than that observed in wild-type (WT) infections. Moreover, nootkatone retained strong inhibitory activity against itraconazole-resistant isolates, underscoring its potential as a novel, broad-spectrum antifungal agent. KEY POINTS: • Nootkatone inhibits fungal growth-suppresses Aspergillus fumigatus hyphae and spores via dual concentration-dependent action • Targets calcium signaling-key genes (crzA/cchA) regulate resistance; deletion enhances susceptibility and links to efflux pump activation. • Dual antifungal mechanisms and drug resistance overcoming-disrupts cell wall integrity, activates host immunity, and is effective against azole-resistant strains, boosting survival in infection models.