Genome-Wide Association for Itraconazole Sensitivity in Non-Resistant Clinical Isolates of Aspergillus fumigatus

Abstract

Aspergillus fumigatus is a potentially lethal opportunistic pathogen that infects over [~]200,000 people and causes [~]100,000 deaths per year globally. Treating A. fumigatus infections is particularly challenging because of the recent emergence of azole-resistance. The majority of studies focusing on the molecular mechanisms underlying azole resistance have examined azole-resistant isolates. However, isolates that are susceptible to azoles also display variation in their sensitivity, presenting a unique opportunity to identify genes contributing to azole sensitivity. Here, we used genome-wide association (GWA) analysis to identify loci involved in azole sensitivity by analyzing the association between 68,853 SNPs and itraconazole (ITCZ) minimum inhibitory concentration (MIC) in 76 clinical isolates of A. fumigatus from Japan. Population structure analysis suggests the presence of four distinct populations, with ITCZ MICs distributed relatively evenly across populations. We independently conducted GWA when treating ITCZ MIC as a quantitative trait and a binary trait and identified two SNPs with strong associations that were identified in both analyses. These SNPs fell within the coding regions of Afu2g02220 and Afu2g02140. We functionally validated Afu2g02220 by knocking it out using a CRISPR/Cas-9 approach, because orthologs of this gene are involved in sterol modification and ITCZ targets the ergosterol pathway. Knockout strains displayed no difference in growth compared to the parent strain in minimal media, yet a minor but consistent inhibition of growth in the presence of 0.15 ug/ml ITCZ. Our results suggest that GWA paired with efficient gene deletion is a powerful and unbiased strategy for identifying the genetic basis of complex traits in A. fumigatus. ImportanceAspergillus fumigatus is a pathogenic mold that can infect and kill individuals with compromised immune systems. The azole class of drugs provide antifungal activity against A. fumigatus infections and have become an essential treatment strategy. Unfortunately, A. fumigatus azole resistance has recently emerged and rapidly risen in frequency making treatment more challenging. Our understanding of the molecular basis of azole sensitivity has been shaped mainly through candidate gene studies. Unbiased approaches are necessary to understand the full repertoire of genes and genetic variants underlying azole resistance and sensitivity. Here, we provide the first application of genome-wide association analysis in A. fumigatus in the identification of a gene (Afu2g02220) that contributes to itraconazole susceptibility. Our approach, which combines association mapping and CRISPR/Cas-9 for functional validation of candidate genes, has broad application for investigating the genetic basis of complex traits in fungal systems.