RNA-seq of Aspergillus felis CCF5624 treated with itraconazole/voriconazole/posaconazole against untreated control
Biological Relevance and Intent: Cryptic species within the Aspergillus section Fumigati, such as the Aspergillus viridinutans species complex, are increasingly recognized as important opportunistic pathogens in both human and veterinary medicine. These species often exhibit an intrinsic reduction in susceptibility to standard azole antifungals. The intent of this experiment was to determine how the tolerant veterinary isolate Aspergillus felis CCF5624 responds to acute azole stress (tolerance is transient state, not genetically determined like mutations). This study aimed to map the active cellular survival pathways and identify regulatory networks that could be targeted to bypass this protective tolerance machinery.  Experimental Workflow Overview: Conidia suspensions of Aspergillus felis CCF5624 were inoculated onto pure RPMI medium covered with sterile cellophane and cultivated for 40 hours at 37 °C to establish vegetative colonies. To capture the immediate transcriptomic shifts associated with drug tolerance, the grown biomass was transferred directly to new RPMI plates supplemented with either a control solvent (DMSO) [abbreviated as \"A\"] or one of three clinical azole compounds (itraconazole, voriconazole, or posaconazole) [abbreviated as B, C,D, respectively] at concentrations corresponding to EUCAST resistance breakpoints. Following an acute 4-hour exposure, fungal biomass was harvested and flash-frozen in liquid nitrogen. Total RNA was isolated using a combined TRI reagent and spin-column methodology. High-throughput RNA sequencing (RNA-seq) was performed by Novogene Limited UK. Poly-A enriched mRNA libraries were prepared and sequenced on the Illumina NovaSeq X Plus platform using a 150 bp paired-end (PE150) strategy, yielding a minimum sequencing depth of 3 Gb (approximately 10 million paired-end reads) per individual sample.  Reference gemone Aspergillus pseudoviridinutans IFM_55266 https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/018/340/605/GCF_018340605.1_Asppvi_assembly01/GCF_018340605.1_Asppvi_assembly01_genomic.fna.gz https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/018/340/605/GCF_018340605.1_Asppvi_assembly01/GCF_018340605.1_Asppvi_assembly01_genomic.gtf.gz
Biological Relevance and Intent: Cryptic species within the Aspergillus section Fumigati, such as the Aspergillus viridinutans species complex, are increasingly recognized as important opportunistic pathogens in both human and veterinary medicine. These species often exhibit an intrinsic reduction in susceptibility to standard azole antifungals. The intent of this experiment was to determine how the tolerant veterinary isolate Aspergillus felis CCF5624 responds to acute azole stress (tolerance is transient state, not genetically determined like mutations). This study aimed to map the active cellular survival pathways and identify regulatory networks that could be targeted to bypass this protective tolerance machinery.  Experimental Workflow Overview: Conidia suspensions of Aspergillus felis CCF5624 were inoculated onto pure RPMI medium covered with sterile cellophane and cultivated for 40 hours at 37 °C to establish vegetative colonies. To capture the immediate transcriptomic shifts associated with drug tolerance, the grown biomass was transferred directly to new RPMI plates supplemented with either a control solvent (DMSO) [abbreviated as \"A\"] or one of three clinical azole compounds (itraconazole, voriconazole, or posaconazole) [abbreviated as B, C,D, respectively] at concentrations corresponding to EUCAST resistance breakpoints. Following an acute 4-hour exposure, fungal biomass was harvested and flash-frozen in liquid nitrogen. Total RNA was isolated using a combined TRI reagent and spin-column methodology. High-throughput RNA sequencing (RNA-seq) was performed by Novogene Limited UK. Poly-A enriched mRNA libraries were prepared and sequenced on the Illumina NovaSeq X Plus platform using a 150 bp paired-end (PE150) strategy, yielding a minimum sequencing depth of 3 Gb (approximately 10 million paired-end reads) per individual sample.  Reference gemone Aspergillus pseudoviridinutans IFM_55266 https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/018/340/605/GCF_018340605.1_Asppvi_assembly01/GCF_018340605.1_Asppvi_assembly01_genomic.fna.gz https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/018/340/605/GCF_018340605.1_Asppvi_assembly01/GCF_018340605.1_Asppvi_assembly01_genomic.gtf.gz