Genome-wide discovery and phenotyping of non-coding transcripts in A. fumigatus reveals lncRNAs with a role in antifungal drug sensitivity

Recent data suggests one fungal species, Aspergillus fumigatus , causes more deaths each year than HIV or malaria combined. Coupled with the rapid emergence of antifungal drug resistance in A. fumigatus strains most associated with pathogenesis amounts to one of the major challenges in infectious diseases in recent years. The very limited range of effective treatments for aspergillosis and high prevalence of AMR result in mortality after infection of > 50%. In recent years, long-noncoding RNAs (lncRNAs) involved in drug resistance and virulence have been identified in pathogenic yeasts such as Candida sp. and non – pathogenic filamentous moulds such as Neurospora crassa showing that there is considerable untapped genetic material present in these genomes. So far, there is very limited knowledge and a lack of systematic annotation of lncRNAs in human pathogenic moulds, such as A. fumigatus . Here we exploited in-house generated transcriptomics data of A. fumigatus exposed to different environments to annotate over 8000 transcribed features, mapping to 2388 genomic loci. We manually curated the output and generated an annotated database of over 1000 lncRNAs finding that the majority of lncRNAs are only conserved within species, and we identify the few that are genus-specific. We further observed that the lncRNAs display orchestrated transcriptional profiles upon drug treatment and co-localise with genes involved in azole sensitivity. The complete set of intergenic lncRNAs was knocked out to create mutant strains that can be used in functional genomics approaches. We then optimized automated fitness measurements on solid medium for this mould, carried out a large-scale phenotypic analysis and identified 60 lncRNA mutants displaying condition-dependent fitness changes with 11 mutants exhibiting a positive effect on growth under azole stress. Overall, this study has generated and experimentally validated an important resource that will enable the wider research community to increase systematic understanding of the functional importance of lncRNAs in A. fumigatus , including their involvement in drug sensitivity.