Objectives: Aspergillus fumigatus is a leading cause of opportunistic 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. fumigatus clinical isolates from patients failing echinocandin therapy. Echinocandin resistance in Candida is known to arise from amino acid substitutions in β-(1,3)-D-glucan synthase encoded by the fks1 gene. Yet, these clinical isolates did not contain mutations in fks1, indicating an undefined resistance mechanism. The objective of this study was to explore a novel mechanism of echinocandin resistance independent of fks1 mutations.
Methods: To explore this new mechanism, we used a lab-derived strain, RG101, which does not contain fks1 mutations but is resistant to caspofungin (CAS). Inhibition of glucan synthase enzyme isolated from RG101 grown in the absence and presence of CAS was determined in vitro using radioactive substrate and IC50 (drug concentration required for 50% reduction of activity) values were measured. Post-translational modifications (PTMs) potentially leading to resistance was explored by evaluating enzyme derived from RG101 grown in the absence and presence of CAS (1 µg/mL) using nano LC-MS/MS. A comprehensive lipidomics analysis was also performed to compare lipid profiles of glucan synthase containing fractions derived from RG101 grown in the absence and presence of CAS (1 µg/mL) using LC-MS/MS. To measure Reactive Oxygen Species (ROS) levels induced by different echinocandins, a 2’,7’-dichlorofluorescin diacetate (DCFDA)-based fluorescence assay was used, and ROS levels measured after one hour of drug exposure.
Results: 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 post-translational modifications, but found none linked to resistance. However, analysis of the lipid microenvironment of CASinduced resistant enzyme revealed a prominent increase in abundance of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to sensitive enzyme recapitulated the drug insensitivity of the CAS-derived enzyme. Further analysis demonstrated that CAS induces mitochondrial-derived ROS, and dampening ROS formation by antimycin A or thiourea eliminated drug-induced resistance.
Conclusion: We conclude that CAS-induces cellular stress, promoting formation of ROS, triggering an alteration in the composition of plasma membrane lipids within the local environment of glucan synthase, rendering it insensitive to echinocandins. We have discovered a new mechanism of resistance in A. fumigatus independent of the well-characterized FKS mutation mechanism observed in Candida. This study also identifies an off-target effect of CAS, ROS production in A. fumigatus, and integrates 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.
Full conference title:
- TIMM (2019)