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Background: The prevalence of azole resistance in clinical and environmental Aspergillus fumigatus isolates is rising over the past decades, but the molecular basis of the development of antifungal drug resistance is not well understood. This study focuses on the role of phenotypic plasticity in the evolution of azole resistance in A. fumigatus. When A. fumigatus is challenged with a new stressful environment, phenotypic plasticity may allow A. fumigatus to adjust their physiology to still enable growth and reproduction, therefore allowing the establishment of genetic adaptations through natural selection on the available variation in the mutational and recombinational gene pool. To investigate these short-term physiological adaptations, we conducted time series transcriptome analyses on three clinical A. fumigatus isolates, during incubation with itraconazole. Results: After analysis of expression patterns, we identified 3955, 3430, 1207, and 1101 differentially expressed genes (DEGs), after 30, 60, 120 and 240 min of incubation with itraconazole, respectively. We explored the general functions in these gene groups and we identified 186 genes that were differentially expressed during the whole time series. Additionally, we investigated expression patterns of potential novel drug-efflux transporters, genes involved in ergosterol and phospholipid biosynthesis, and the known MAPK proteins of A. fumigatus. Conclusions: Our data suggests that A. fumigatus adjusts its transcriptome quickly within 60 min of exposure to itraconazole. Further investigation of these short-term adaptive phenotypic plasticity mechanisms might enable us to understand how the direct response of A. fumigatus to itraconazole promotes survival of the fungus in the patient, before any "hard-wired" genetic mutations arise.

The filamentous fungus Aspergillus fumigatus is an opportunistic pathogen which causes life-threatening diseases in immunocompromised patients. Resistance of A. fumigatus against azole class compounds continues to pose a threat to human health worldwide. How this fungus maintains fitness before any resistance causing mutations arise is not well understood. In this study, we use RNA-seq to demonstrate how A. fumigatus exerts its phenotypic plasticity when exposed to itraconazole, and which important cellular processes contribute to its adaptation to a new, stressful environment containing azole compounds. We conducted an in vitro assay in which we exposed 24h old mycelia of clinical A. fumigatus isolates grown at 37°C, to sublethal concentrations of itraconazole which might permit gradual adaptation of the fungus to the new, stressful environment. Mycelia were then harvested after 30, 60, 120 or 240 minutes respectively, and relative expression was compared to the mycelia harvested directly after 24h without the addition of itraconazole