Accelerated mutator phenotype in a clinical Aspergillus fumigatus isolate contributes to adaptive evolution

The opportunistic pathogen Aspergillus fumigatus represents a major threat to immunocompromised individuals and is increasingly resistant to antifungal therapies. Resistance selection primarily takes place through environmental selection to azole fungicides, but in-host resistance may develop in patients with chronic aspergillosis receiving azole therapy. In this study, we examine clinical A. fumigatus isolates that exhibit irregular growth and accumulated mutations rapidly during antifungal treatment. Whole-genome sequencing of serial isolates revealed an accelerated mutation rate as the likely driver of the observed phenotype. The mutation frequency of this isolate was approximately 15-times higher than other A. fumigatus strains. We identified non-synonymous single nucleotide polymorphisms (SNPs) as potential loci involved in the increased mutation rate. Using CRISPR/Cas9 gene editing and comprehensive genomic analysis, we show that a mutation in mre11, a gene critical for genomic stability during DNA replication, is responsible for this elevated mutation rate. Mutations within mre11 result in a 27% reduction in radial growth, highlighting the fitness cost associated with the higher mutation rate. All mre11-mutant isolates in this study belong to clade B, a lineage that rarely carries environmental azole-resistance mutations, potentially supporting in-host adaptation. The Phe332Leu allele was observed both in clinical and environmental isolates, suggesting that the mutator phenotype may represent a general adaptive strategy, allowing A. fumigatus to persist under prolonged azole pressure. We hypothesize that this heightened mutation background could facilitate the rapid spread of antifungal resistance alleles within A. fumigatus populations.