Proteomic Study Of Azole Resistance In Aspergillus Fumigatus Biofilm In Response To Itraconazole

ObjectiveThis study aimed to investigate proteome alterations in Aspergillus fumigatus biofilms under azole drug pressure,identify differentially expressed proteins,and uncover potential key proteins and novel mechanisms involved in drug resistance.MethodsA TMT-based comparative proteomic study on the biofilm of both the wild-type and azole-resistant strains of A.fumigatus under azole pressure.ResultsA total of 2,377 proteins were identified,with 480 and 604 differentially expressed proteins observed in the wild-type and azole-resistant A.fumigatus strains exposed to itraconazole,respectively(fold change>2 or<0.5,P-value<0.05).A substantial proportion of regulated proteins were localized in the cytoplasm,nucleus,and mitochondria.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses indicated significant enrichment of metabolic processes and ribosome pathways.Altered proteins were implicated in ergosterol synthesis(e.g.,Cyp51A,Cyp51B,Erg6),overexpression of efflux pumps(e.g.,Mdr1),oxidative stress counteraction(e.g.,Aspf3,Hsp90),DNA repair(e.g.,Tell),DNA replication(e.g.,Adkl),and transcription(e.g.,Tif6).Parallel reaction monitoring(PRM)was utilized to measure 18 target proteins exhibiting significant changes in the resistant strain Shjt42b biofilm exposed to itraconazole.At least 15 proteins displayed regulation types consistent with omics analysis(P-value<0.05).ConclusionTMT-based proteomics,combined with PRM-targeted validation,identified differentially expressed proteins in wild-type and resistant A.fumigatus biofilms under itraconazole pressure.Several known resistance-related proteins were validated,and potential key proteins involved in azole drug tolerance were discovered.Altered proteins in biofilms were associated with ergosterol synthesis,oxidative stress,efflux pumps,DNA repair,DNA replication,and transcription.