Accelerated mutator phenotype in a clinical <i>Aspergillus fumigatus</i> isolate contributes to adaptive evolution

The opportunistic pathogen <i>Aspergillus fumigatus</i> 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 <i>A. fumigatus</i> 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 <i>A. fumigatus</i> 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 <i>mre11</i>, a gene critical for genomic stability during DNA replication, is responsible for this elevated mutation rate. Mutations within <i>mre11</i> result in a 27% reduction in radial growth, highlighting the fitness cost associated with the higher mutation rate. All <i>mre11</i>-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 <i>A. fumigatus</i> to persist under prolonged azole pressure. We hypothesize that this heightened mutation background could facilitate the rapid spread of antifungal resistance alleles within <i>A. fumigatus</i> populations.