Discovery of a new azole resistance mechanism in Aspergillus fumigatus through whole genome sequencing and sexual crossing
Azole compounds are the primary therapy for patients with diseases caused by Aspergillus fumigatus. However, prolonged treatment may cause resistance to develop, which is associated with treatment failure. The azole target cyp51A is a hotspot for short nucleotide polymorphisms (SNP) that confer phenotypic resistance, but in an increasing number of resistant isolates the underlying mechanism is unknown. Here, we report the discovery of a novel resistance mechanism, caused by a mutation in the CCAAT-binding transcription factor complex subunit HapE. We obtained four isogenic A. fumigatus clinical isolates, which developed an azole resistant phenotype during prolonged azole therapy. Because the resistant isolates contained a wild type cyp51A gene and the genetic background of the isolates was similar, we sequenced the complete genomes of the last susceptible isolate and the first resistant isolate (taken 17 weeks apart) using Illumina technology. By comparing the genome sequences to each other and to the reference genomes of A. fumigatus isolates Af293 and A1163, we identified several potential non-synonymous mutations in protein-coding regions, six of which could be validated by conventional PCR and Sanger sequencing. Subsequent sexual selection experiments showed that resistant progeny always contained a P88L substitution in HapE, while the other mutations did not correlate with resistance. Cloning the mutated hapE gene into the azole susceptible akuBKU80 strain showed that the SNP alone could confer the resistant phenotype. This is the first time that whole genome sequencing and sexual crossing strategies were used to find the genetic basis of a trait of interest. The discovery may help understand alternate pathways for azole resistance in A. fumigatus with implications for the molecular diagnosis of resistance and drug discovery.