Investigation into mechanisms for antifungal resistance in Aspergillus fumigatus

Abstract

Aspergillus fumigatus is a filamentous saprophytic mold that is found abundantly in the biosphere. A. fumigatus is also an airborne human pathogen and is considered the major cause of aspergillosis, infections caused by inhalation of conidia. In immunocompetent individuals, the spores rarely cause any harm as they are cleared by innate pulmonary defences; however, in immunocompromised patients, the host immune system can fail to clear the inhaled conidia and aspergillosis may develop. Indeed, aspergillosis represents a major cause of morbidity and mortality in these populations. Aspergillosis is commonly treated using triazole and amphotericin B (AMB) antifungal agents. However, the increasing prevalence of triazole resistant strains and emergence of AMB resistance has become a challenge in treatment. To further expand our knowledge on the mechanisms of antifungal resistance in the species, we tested previously known or associated genes for antifungal resistance as well as investigated novel mechanisms via multiple genome-wide association studies (GWAS), which used a total of 211 genomes from A. fumigatus strains in 12 countries. Our results identified many novel mutations related to triazole and AMB resistance. Specifically, using stepwise GWAS analyses, we identified 6 and 18 missense variants to be significantly associated with itraconazole and voriconazole resistance, respectively. A linkage disequilibrium analysis identified six additional missense variants associated with triazole resistance, with two of these six being consistently associated with pan-azole resistance across subsets of samples. Furthermore, examination of known mutation sites and genes overexpressed with triazole exposure found a total of 65 SNPs implicated in triazole resistance. For the AMB study, we identified a total of 34 mutations associated with AMB tolerance using a GWAS. Subsequent analysis with 143 progeny strains, generated from a laboratory cross and genotyped with PCR-RFLP, identified epistatic interactions between five of these SNP sites that impacted growth in different concentrations of AMB. With the expanding immunocompromised population and increasing frequency of antifungal resistance, our results will help in investigating novel resistance mechanisms in A. fumigatus and in expanding the molecular diagnostic toolset in resistance screening, to enable rapid and accurate diagnosis and treatment decision-making.