Transcriptomic and metabolomic analysis of a non-cyp51A mutant azole-resistant Aspergillus fumigatus isolated from Ningxia, China.

Background: Azole-resistant A. fumigatus (Aspergillus fumigatus) has been extensively documented both domestically and internationally, with mutations in the cyp51A gene identified as the predominant mechanism of resistance. However, the incidence of clinical non-cyp51A mutation-resistant A. fumigatus has gradually increased in recent years, and the resistance mechanisms remain unclear. Methods: We isolated a non-cyp51A mutant azole-resistant strain of A. fumigatus, designated Af68, from Ningxia. The fungus was further characterized using MALDI-TOF mass spectrometry and DNA sequencing of the beta-tubulin gene and the calmodulin gene for molecular characterization. We analyzed the growth diameters and responses of the azole-sensitive strain Af293 and the azole-resistant strain Af68 to various concentrations of oxidative agents, including menadione, H2O2, sodium dodecyl sulfate (SDS), and Congo Red. Transcriptome RNA sequencing was performed to identify differentially expressed genes between Af293 and Af68. Furthermore, a liquid chromatography-mass spectrometry (LC-MS) system was utilized for a comparative metabolomic analysis between the two strains. The mRNA levels of cyp51A, cyp51B, MDR2, sitT, catA, and SOD2 were quantified using real-time quantitative PCR (qRT-PCR). Results: Compared to the wild-type A. fumigatus Af293, the strain Af68 exhibited a significantly increased growth diameter when exposed to various oxidative agents. However, no significant difference in radial growth was observed between the two strains when cultured in potato dextrose agar (PDA) medium at 37 °C on days 1 and 5. Transcriptional alterations between the two strains were analyzed using RNA-sequencing technology, revealing 594 genes with significant expression differences. The mRNA levels of C6 transcription factors, the bZIP transcription factor MeaB, and stress-activated MAP kinase interacting proteins were significantly reduced in Af68, while the mRNA levels of C2 domain-containing proteins, zinc metalloproteases, and MFS transporters were significantly increased. GO (Gene Ontology) analysis suggested that cellular processes, metabolic pathways, localization, and bioregulation collectively contribute to the biological processes underlying drug resistance. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis indicated that the differentially expressed genes are primarily associated with autophagy, amino sugar and nucleotide metabolism, ABC transporters, homologous recombination, and DNA mismatch repair. A total of 129 distinct metabolites were screened from the two groups of samples, of which 96 were found to be upregulated in the Af68. These metabolites encompass various categories, including organic acids, amino acid derivatives, peptides, natural products, and nucleotides. Furthermore, these metabolites are primarily enriched in metabolic pathways such as tyrosine metabolism, purine metabolism, D-amino acid metabolism, and glutathione metabolism. Conclusion: In conclusion, this study offers new insights and perspectives on the research of non-cyp51A mutation-related resistance mechanisms, utilizing phenotypic experiments, transcriptome sequencing, and metabolite analysis.