This study focuses on the role of phenotypic plasticity (PP) in the evolution of azole resistance, before any genetic mutations have arisen in Aspergillus fumigatus. When A. fumigatus is challenged with a new environment (stress), PP may allow A. fumigatus to adjust their physiology to still enable growth and reproduction, allowing natural selection to establish genetic adaptations from the mutational and recombinational gene pool. The capability of A. fumigatus to adapt in the human host during infection contributes to treatment failure and persistence of the fungus in patients. Therefore, it is important to understand the adaptation strategies of A. fumigatus when exposed to azoles. In this study, we perform RNAseq analysis to monitor the cellular events that take place directly after encountering itraconazole in the “environment”. 

Three sensitive clinical A. fumigatus isolates were inoculated for 24h in Aspergillus Minimal Medium. After 24h, itraconazole was added at IC50 concentrations. Strains were harvested through Miracloth after 0, 30, 60, 120 and 240 minutes and immediately frozen solid in liquid nitrogen. Subsequently, cells were lysed using a MagnaLyser and RNA was extracted using TRIzol reagent. TruSeq mRNA libraries were created for analysis on a NextSeq500  (Illumina). Paired-end reads of 2x75 bp were generated in High Output mode. Reads were aligned with STAR against the reference genome sequence of A. fumigatus Ensemble CADRE 30. Differentially expressed genes (DEGs) were identified (adjusted P value <0.1, log2FC >1.5, or < -1.5) by DESeq2 for R by Bioconductor. Functional categories of DEGs were grouped according to the Gene Ontology (GO) terms.

A total of 30 samples were obtained; three clinical isolates, harvested at five different time points, in duplicate. All samples showed at least 80% mapping to the Af293 reference genome, with an average read coverage of 65x the complete A. fumigatus genome. Our data showed the most differentially expressed genes after only 30 minutes of incubation, with decrease over time. The GO categories transport, response to stress and response to chemical substances were enriched in both up- and downregulated genes. The categories mostly enriched by downregulated genes include the carbohydrate metabolic process and the secondary metabolic process. The categories mostly enriched by upregulated genes include the DNA metabolic process, the RNA metabolic process, translation, ribosome biogenesis and organelle organisation. Furthermore, several putative transporters and transcription factors were upregulated within 60 minutes of incubation, which have never been correlated with itraconazole stress before in A. fumigatus. 

Our data suggests that A. fumigatus adapts its transcriptome enormously already within 60 minutes of exposure to itraconazole, after which the cells reach an improved homeostatic state. Transcriptomic analysis by RNAseq can give major insights in the differentially expressed genes, enabling us to understand how the direct response of A. fumigatus to itraconazole promotes survival of the fungus in the patient, before any stable genetic mutations arise.


Maintaining fitness through phenotypic plasticity in Aspergillus fumigatus; elucidating transcriptional differences between three clinical isolates exposed to itraconazole