ABSTRACTAspergillus fumigatusis a leading cause of invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high-minimum-effective-concentration (MEC)A. fumigatusclinical isolates from patients failing echinocandin therapy. Echinocandin resistance is known to arise from amino acid substitutions in β-(1,3)-d-glucan synthase encoded by thefks1gene. Yet these clinical isolates did not contain mutations infks1, indicating an undefined resistance mechanism. To explore this new mechanism, we used a laboratory-derived strain, RG101, with a nearly identical caspofungin (CAS) susceptibility phenotype that also does not containfks1mutations. Glucan synthase isolated from RG101 was fully sensitive to echinocandins. Yet exposure of RG101 to CAS during growth yielded a modified enzyme that was drug insensitive (4 log orders) in kinetic inhibition assays, and this insensitivity was also observed for enzymes isolated from clinical isolates. To understand this alteration, we analyzed whole-enzyme posttranslational modifications (PTMs) but found none linked to resistance. However, analysis of the lipid microenvironment of the enzyme with resistance induced by CAS revealed a prominent increase in the abundances of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to the sensitive enzyme recapitulated the drug insensitivity of the CAS-derived enzyme. Further analysis demonstrated that CAS induces mitochondrion-derived reactive oxygen species (ROS) and that dampening ROS formation by antimycin A or thiourea eliminated drug-induced resistance. We conclude that CAS induces cellular stress, promoting formation of ROS and triggering an alteration in the composition of plasma membrane lipids surrounding glucan synthase, rendering it insensitive to echinocandins.IMPORTANCEResistance to first-line triazole antifungal agents amongAspergillusspecies has prompted the use of second-line therapy with echinocandins. As the number ofAspergillus-infected patients treated with echinocandins is rising, clinical observations of drug resistance are also increasing, indicating an emerging global health threat. Our knowledge regarding the development of clinical echinocandin resistance is largely derived fromCandidaspp., while little is known about resistance inAspergillus.Therefore, it is important to understand the specific cellular responses raised byA. fumigatusagainst echinocandins. We discovered a new mechanism of resistance inA. fumigatusthat is independent of the well-characterizedFKSmutation mechanism observed inCandida. This study identified an off-target effect of CAS, i.e., ROS production, and integrated oxidative stress and sphingolipid alterations into a novel mechanism of resistance. This stress-induced response has implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.
Structural basis of HapEP88L-linked antifungal triazole resistance in Aspergillus fumigatus
