Elevated mutation rates in multi-azole resistant Aspergillus fumigatus drive rapid evolution of antifungal resistance

Abstract

The environmental use of azole fungicides has led to selective sweeps across multiple loci in the Aspergillus fumigatus genome causing the rapid global expansion of a genetically distinct cluster of resistant genotypes. Isolates within this cluster are also more likely to be resistant to agricultural antifungals with unrelated modes of action. Here we show that this cluster is not only multi-azole resistant but has increased propensity to develop resistance to next generation antifungals because of variants in the DNA mismatch repair system. A variant in msh6-G233A is found almost exclusively within azole resistant isolates harbouring the canonical cyp51A azole resistance allelic variant TR₃₄/L98H. Naturally occurring isolates with this msh6 variant display up to 5-times higher rate of mutation, leading to an increased likelihood of evolving resistance to other antifungals. Furthermore, unlike hypermutator strains, the G233A variant conveys no measurable fitness cost and has become globally distributed. Our findings further suggest that resistance to next-generation antifungals is more likely to emerge within organisms that are already multi-azole resistant due to close linkage between TR₃₄/L98H and msh6-G233A, posing a major problem due to the prospect of dual use of novel antifungals in clinical and agricultural settings.

Fig. 1. Variants in MMR are significantly overrepresented in clade A.

Occurrence of MMR variant alleles in clade A (red) and clade B (blue) for a msh2 (E812G), b msh6 (G233A) and c pms1 (E444G). d An unrooted maximum-likelihood phylogenetic tree using genome-wide SNPs relative to Af293 of 218 WGS UK isolates. The presence of variants in msh2, msh6 and pms1 are highlighted in the black boxes, the presence of cyp51A resistance variants and clade are coloured. e Domain structures of Msh6 in A. fumigatus, G233A variant labelled, labels show predicted domain positions in protein sequence. f G233 locus homology across Trichocomaceae, alignments of 140 isolates spanning Aspergillus ssp., 27 Talaromyces and Paecilomyces, and 122 Penicillium Msh6 protein sequences. G233 highlighted in yellow, G233A variants are only present in Aspergillus fumigatus. Cladogram shows the hierarchical clustering of Msh6 protein sequences. Source data are provided as a Source Data file.

Fig. 2. MutS and MutL null mutants significantly elevate mutation rates.

a Workflow of fluctuation tests to measure mutation rates in A. fumigatus. Clonal isolates were cultured in the absence of antifungal selection to generate genetic diversity. Resistant mutants were selected on lethal concentrations of antifungals. Counts of resistant mutants were fitted to the Luria–Delbrück distribution to calculate the number of mutational events. b Mutation rates for resistance to voriconazole for MMR-deficient mutants. Each point shows the calculated mutation rate from a single independent fluctuation test using 12 replicate cultures. Error bars show 95% confidence intervals, cross bars show the median mutation rate across fluctuation tests. Fold differences show median fold change in mutation rate from the parental MFIG001 strain. Triangle points represent mutation rates measured using independently constructed deletion mutants. c Mutational frequency to olorofim resistance. d Mutational frequency to itraconazole resistance. Each point shows the mutational frequency of an individual population (N = 6). Error bars show SEM and cross bars show median mutational frequency. Fold differences show median fold change in mutation frequency from the parental MFIG001 strain. Source data are provided as a Source Data file.

Fig. 3. Deletion of MMR genes carries a significant cost to fitness.

a The fraction of Δmsh2, Δmsh6 and Δpms1 through time when in direct competition with the parental MFIG001 strain on solid agar faceted by media type (rich = aspergillus complete media, minimal = aspergillus minimal media). The coloured lines show the mean of 6 independent competitions, presented by individual grey lines. The horizontal dashed lines show the starting fraction of the MMR deletion strain. b Mean fitness of Δmsh2, Δmsh6 and Δpms1 relative to MFIG001 across the five transfers presented in panel a, the cross bar shows the median (N = 6 independent competitions), the lower and upper hinges correspond to the first and third quartiles and the whiskers extend to 1.5*IQR. Box plots coloured by media type. The horizontal dashed line shows equal fitness of zero, p-values show significant difference from zero using two-sided t-tests, using Holm correction for multiple testing, Δmsh2 rich P = 0.00023, minimal P = 4.8e⁻⁶, Δmsh6 rich P = 1, minimal P = 0.97, Δpms1 rich P = 0.025, minimal P = 0.00012, asterisks represent significance (*p < 0.05, **p < 0.01, ***p < 0.0001). Source data are provided as a Source Data file.

Fig. 4. msh6-G233A elevates mutation rates in neutral background and in natural isolates from clade A.

a Each point shows the calculated mutation rate from a single independent fluctuation test using 12 replicate cultures. Each of the three points for the MFIG001 msh6-G233A variant is a separate independent transformant. Error bars show 95% confidence intervals. Fold change shows the median fold change in mutation rate from the parental MFIG001 strain. b Mean fitness of msh6-G233A relative to MFIG001 across five transfers, the cross bar shows the median (three independent transformants, to replicates each for a total of N = 6), the lower and upper hinges correspond to the first and third quartiles and the whiskers extend to 1.5*IQR. Box plots coloured by media type. The horizontal dashed line shows equal fitness. c Mutation rate of natural genotypes to olorofim. Each point shows the calculated mutation rate from a single independent fluctuation test using 12 replicate cultures. Error bars show 95% confidence intervals. Fold difference shows the median fold change in mutation rate of isolates with G233A allele from clade B isolates. Points coloured by clade, red clade A, blue clade B. Key below plot shows the presence of azole resistance mutation cyp51A TR₃₄/L98H in orange, TR₄₆/Y121F/T289A in blue, and the presence of msh6-G233A in grey. Source data are provided as a Source Data file.