Screening of fluconazole resistance-related genes in Trichosporon asahii with induced fluconazole resistance in vitro

Abstract

Objective To screen the genes associated with fluconazole resistance in Trichosporon asahii to facilitate mechanistic studies of fluconazole resistance and the development of new antifungal agents. Methods Three clinical isolates of fluconazole-sensitive Trichosporon asahii were cultured in the presence of increasing concentrations of fluconazole until the drug concentration reached 128 μg/mL. After further culture for 30 generations in the absence of fluconazole, the MIC value of the induced bacteria was measured. The total RNA was extracted from both the sensitive and resistant strains of the bacteria for construction of the stranded RNA-Seq libraries and transcriptome sequencing to identify the differentially expressed genes (DEGs) in the resistant strains. The genomes of the 3 sensitive and resistant strains were sequenced for comparison of the sequence of the differentially expressed ERG11 gene. Results We successfully obtained stable Trichosporon asahii strains with fluconazole resistance. Transcriptome sequencing data showed that the genes associated with drug transport, ergosterol synthesis and antioxidation were up-regulated in the resistant strains, and among them ERG11 gene, the target gene of fluconazole, was up-regulated by 3.7 folds. Genomic sequencing proved no mutations in the sequence of ERG11 gene in the 3 resistant strains. Conclusion The molecular mechanism for drug resistance in Trichosporon asahii is complex. The upregulation of ergosterol synthesis pathway, especially ERG11 gene, along with other genes associated with efflux transfer and oxidative stress, is one of the main mechanisms for drug resistance in Trichosporon asahii.

Keywords

• trichosporon asahii
• fluconazole
• induction in vitro
• transcriptome
• drug resistance gene
• erg11