Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-d-Glucan Synthase Cause Clinically Important Echinocandin Resistance in Aspergillus fumigatus

Abstract

Aspergillus fumigatus is 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. fumigatus clinical 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 the fks1 gene. Yet these clinical isolates did not contain mutations in fks1, 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 contain fks1 mutations. 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.IMPORTANCE Resistance to first-line triazole antifungal agents among Aspergillus species has prompted the use of second-line therapy with echinocandins. As the number of Aspergillus-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 from Candida spp., while little is known about resistance in Aspergillus. Therefore, it is important to understand the specific cellular responses raised by A. fumigatus against echinocandins. We discovered a new mechanism of resistance in A. fumigatus that is independent of the well-characterized FKS mutation mechanism observed in Candida 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.

Keywords: Aspergillus fumigatus; ROS; antifungal resistance; caspofungin; echinocandins; glucan synthase; glucan synthase inhibitors; lipids.

FIG 1

RG101 shows breakthrough growth in CAS. (A) Time-dependent changes in growth phenotypes of RG101 and ATCC 13073 in RPMI 1640 medium. At 24 h, the MEC of CAS for RG101 was 0.25 μg/ml, with the formation of characteristic rosettes indicating inhibition (red). However, breakthrough growth began to manifest at 0.5 μg/ml (green), and at between 1 and 8 μg/ml of CAS, this strain showed complete resistance. At 16 μg/ml, rosettes began to form again, indicative of growth inhibition. At 30 h, resistance of RG101 to CAS was seen at all concentrations of CAS tested. (B) Results of drug susceptibility testing of RG101 and ATCC 13073 at 24 h in the presence of different antifungals. RG101 was resistant only to CAS but was sensitive to other echinocandins, azoles, and polyenes. ATCC 13073 was sensitive to all antifungals.

FIG 2

RG101 is resistant to caspofungin and sensitive to micafungin in vivo. Resistance of RG101 to CAS in vivo was tested in an acute pulmonary aspergillosis model. Inbred DBA/2 mice were rendered neutropenic and infected with 1 × 10⁶ RG101 conidia intratracheally. The mice were then treated with CAS, micafungin, or vehicle daily for 5 days, and lung burdens were determined using qPCR. The average fungal lung burdens in animals treated with vehicle, CAS, and MFG were 25.8, 22.6, and 5.2 ng fungal DNA per lung, respectively. Statistical analysis showed no significant differences between the vehicle-treated and CAS-treated groups, whereas MFG-treated animals showed approximately 5-fold decrease in burden, suggesting that RG101 is resistant to CAS and sensitive to MFG in vivo.

FIG 3

CAS induces modification of β-glucan synthase in RG101 and several clinical isolates. Sensitivity of partially purified glucan synthase to different echinocandins was examined in vitro after isolating the enzyme from RG101 grown in different drug conditions. (A) Dose-dependent insensitivity of RG101 glucan synthase to CAS induced by CAS during mycelial growth of RG101. The IC₅₀ of glucan synthase for RG101 grown in the absence of CAS [IC₅₀ (no CAS)] was ∼0.01 ng/ml and for RG101 grown at 4 μg/ml CAS [IC₅₀ (4 μg/ml CAS)] was high at >10,000 ng/ml. Previous exposure to CAS in RG101 culture induced insensitivity to CAS at the enzyme level in a dose-dependent fashion. (B) Previous exposure to CAS in culture induced insensitivity to micafungin at the enzyme level [IC₅₀ (no CAS), <0.01 ng/ml; IC₅₀ (4 μg/ml CAS), >10,000 ng/ml], indicating potential CAS-induced cross-resistance in cells. (C to E) Enzyme profiles similar to those seen with CAS were observed in clinical isolates 117 (C), 2770 (D), and 32458 (E). IC₅₀ values for the three clinical isolates are as follows: for isolate 117, IC₅₀ (no CAS) = ∼0.01 ng/ml and IC₅₀ (4 μg/ml CAS) = >10,000 ng/ml; for isolate 2770, IC₅₀ (no CAS) = ∼0.01 ng/ml and IC₅₀ (4 μg/ml CAS) = >10,000 ng/ml; for isolate 32458, IC₅₀ (no CAS) = ∼10 ng/ml and IC₅₀ (4 μg/ml CAS) = ∼5,000 ng/ml.

FIG 4

CAS induces cross-resistance to other glucan synthase inhibitors in RG101. RG101 was grown for 24 h in RPMI 1640 media containing 1 μg/ml of different glucan synthase inhibitors, including micafungin (MFG), anidulafungin (ANF), rezafungin (RZF), and ibrexafungerp (IFG), in both the absence and presence of CAS (1 μg/ml). While RG101 was susceptible to these drugs in the absence of CAS, as seen by characteristic formation of rosettes, it showed complete resistance in the presence of CAS, as seen by the presence of extended mycelia.

FIG 5

Certain lipids affect the sensitivity of β-glucan synthase to CAS. (A and B) Lipidomics analysis of enriched glucan synthase from RG101 grown in the absence and presence (1 μg/ml) of CAS showed (A) >2-fold-increased abundance of dihydrosphingosine (DhSph) and (B) 10-fold-increased abundance of phytosphingosine (PhytoSph) in CAS-exposed cells. (C) Addition of DhSph and PhytoSph at a concentration of 150 μg/ml for 1 h to enriched glucan synthase extract in the enzyme assay made the enzyme insensitive to CAS [IC₅₀ (no lipid) = ∼0.01 ng/ml; IC₅₀ (150 μg/ml DhSph) = 0.1 ng/ml; IC₅₀ (150 μg/ml PhytoSph) = ∼100 ng/ml], whereas addition of other lipids such as phytoceramide did not alter the enzyme property [IC₅₀ (150 μg/ml C2 PhytoCer) = ∼0.01 ng/ml].

FIG 6

CAS induces ROS production. (A) Levels of ROS induced by different echinocandins as measured by a fluorescence assay using DCFDA dye. After 1 h of exposure of cells to echinocandins, CAS induced the highest levels of ROS production in the A. fumigatus RG101 and ATCC 13073 strains in comparison to other echinocandins. (B) Dose-dependent increase in ROS production induced by CAS. (C) Addition of thiourea, a ROS scavenger, at 15 mM reduced CAS-induced ROS production in RG101. (D) Addition of antimycin A, a mitochondrial respiration inhibitor, at 0.5 μg/ml reduced CAS-induced ROS production in RG101, indicating mitochondrion-associated ROS production induced by CAS. (E) Testing of susceptibility of ATCC 13073, RG101, and S679P strains with CAS (1 μg/ml) and thiourea (15 mM) revealed reversion of the resistance phenotype of RG101 but no change of the phenotype of ATCC 13073 and S679P.

FIG 7

Working model showing the non-fks1 mutation-mediated mechanism of resistance in RG101. (A) Addition of CAS during growth of RG101 altered the properties of glucan synthase, rendering it resistant to CAS, at both the cellular and enzyme levels. (B) Working model for CAS-induced resistance in RG101. CAS induces ROS production in cells. We hypothesize that high ROS levels alter the lipid composition in the microenvironment of glucan synthase, causing a conformational change in glucan synthase and leading to CAS resistance.