The GET Pathway Modulates Azole Susceptibility through Sterol Metabolic Remodeling and Protein Trafficking in Filamentous Fungi

ABSTRACT: Azole antifungals are widely used to control fungal infection, yet resistance mechanisms beyond drug efflux and target modification remain insufficiently understood. Here, we identified the Guided Entry of Tail-anchored proteins (GET) pathway as a conserved regulator of azole susceptibility in Neurospora crassa and Aspergillus fumigatus. Deletion or dysfunction of the core GET components Get-3 or Get-4 confers resistance to multiple azoles without impairing hyphal growth or sporulation. Mechanistically, GET deficiency neither reduced intracellular accumulation nor altered drug efflux and target expression or total ergosterol levels. Instead, GET-deficient strains displayed significantly reduced accumulation of the toxic sterol intermediate 14α-methyl-3,6-diol under azole stress, indicating a potential bypass of azole-induced sterol toxicity. Proximity labeling and functional validation indicated that GET deficiency perturbs the trafficking of multiple azole-resistance-associated proteins, including the key tail-anchored protein transporter Emp-47 and mitochondrial Cox subunits Cox-4 and Cox-15, which deletion enhanced azole resistance. Transcriptomic and functional analysis further revealed that GET disruption altered the expression of some azole resistance-associated genes, which involve in membrane transport, metabolism, and cell wall organization, independent of canonical stress pathways. In this study, a total of 17 new genes that modulate azole susceptibility were identified. Collectively, our findings uncover a non-classical mechanism of azole resistance mediated by GET-dependent protein trafficking and metabolic adaptation, conserved across filamentous fungi.

ORGANISM(S): Neurospora Crassa Or74a

SUBMITTER: Xianyun Sun  

PROVIDER: PXD076447 | iProX | Wed Apr 01 00:00:00 GMT+01:00 2026

REPOSITORIES: iProX