The Paradoxical Effect of Echinocandins in Aspergillus fumigatus Relies on Recovery of the β-1,3-Glucan Synthase Fks1

Abstract

Echinocandins target the fungal cell wall by inhibiting biosynthesis of the cell wall carbohydrate β-1,3-glucan. This antifungal drug class exhibits a paradoxical effect that is characterized by the resumption of growth of otherwise susceptible strains at higher drug concentrations (approximately 4 to 32 μg/ml). The nature of this phenomenon is still unknown. In this study, we analyzed the paradoxical effect of the echinocandin caspofungin on the pathogenic mold Aspergillus fumigatus Using a conditional fks1 mutant, we show that very high caspofungin concentrations exert an additional antifungal activity besides inhibition of the β-1,3-glucan synthase. This activity could explain the suppression of paradoxical growth at very high caspofungin concentrations. Additionally, we found that exposure to inhibitory caspofungin concentrations always causes initial growth deprivation independently of the capability of the drug concentration to induce the paradoxical effect. Paradoxically growing hyphae emerge from microcolonies essentially devoid of β-1,3-glucan. However, these hyphae expose β-1,3-glucan again, suggesting that β-1,3-glucan synthesis is restored. In agreement with this hypothesis, we found that expression of the β-1,3-glucan synthase Fks1 is an essential requirement for the paradoxical effect. Surprisingly, overexpression of fks1 renders A. fumigatus more susceptible, whereas reduced expression leads to hyphae that are more resistant to the growth-inhibitory and limited fungicidal activity of caspofungin. Upregulation of chitin synthesis appears to be of minor importance for the paradoxical effect, since paradoxically growing hyphae exhibit significantly less chitin than the growth-deprived parental microcolonies. Our results argue for a model where the paradoxical effect primarily relies on recovery of β-1,3-glucan synthase activity.

Keywords: Aspergillus fumigatus; caspofungin; echinocandin; fks1; glucan synthase; paradoxical effect.

FIG 1

Growth of A. fumigatus in the presence of different caspofungin concentrations followed over time. (A and B) Wild-type conidia were inoculated in AMM in a 24-well plate (5 × 10³ conidia per well). The medium was supplemented with the indicated amounts of caspofungin (CS). Representative dark-field images were taken after 1, 2, 3, 4, and 5 days of incubation at 37°C. (B) Magnifications of selected dark-field images depicted in panel A.

FIG 2

Paradoxically growing hyphae expose β-1,3-glucan. (A and B) Conidia of the wild type expressing GFP and fks1_tetOn were inoculated in AMM on coverslips. The medium was supplemented with the indicated amounts of CS. After 1 day of incubation (A) or 3 days of incubation (B), hyphae were fixed, stained with aniline blue, and immediately analyzed with a fluorescence microscope. (Left) GFP fluorescence. (Right) Glucan-specific (green) and nonspecific (blue) aniline blue fluorescence. Scale bars, 50 μm (A) and 200 μm (B) for all images. The arrow in panel B indicates paradoxically growing hyphae.

FIG 3

Expression of β-1,3-glucan synthase is required for paradoxical growth. Conidia of the wild type and the fks1_tetOn strain were inoculated in AMM in a 24-well plate (5 × 10³ conidia per well). The medium was supplemented with the indicated amounts of CS. Where indicated (+ Doxy), The medium was supplemented with 10 μg ml⁻¹ doxycycline. Representative dark-field images were taken after 1, 3, and 5 days of incubation at 37°C.

FIG 4

Very high caspofungin concentrations exert additional antifungal activity beyond inhibition of the β-1,3-glucan synthase. Conidia of the indicated strains were inoculated in AMM in a 24-well plate (5 × 10³ conidia per well). The medium was supplemented with the indicated amounts of CS. Representative dark-field images were taken after 3 and 5 days of incubation at 37°C.

FIG 5

Expression of fks1 increases the fungicidal and growth-inhibitory activity of caspofungin. (A) Conidia of the indicated strains were inoculated in AMM supplemented with 1 μg ml⁻¹ caspofungin. Where indicated (+ Doxy), the medium was supplemented with 10 μg ml⁻¹ doxycycline. After 3 days of incubation at 37°C, representative dark-field images were taken. (B) Conidia of the indicated strains were spread (4 × 10⁴ conidia per well) (top row) or spotted (1.5 × 10³ conidia per well) (bottom row) on AMM agar. The medium was supplemented with the indicated amounts of CS and doxycycline (Doxy). The images were taken after 2 days of incubation at 37°C. (C) Representative bright-field image of a dead and a viable wild-type microcolony raised in AMM in the presence of 1 μg ml⁻¹ caspofungin for 2 days at 37°C. The primary criterion for viability was light refraction; viable hyphae were bright, and dead hyphae were dark. (D) Conidia of the wild type and the fks1_tetOn strain were inoculated in AMM supplemented with 1 μg ml⁻¹ caspofungin in a 24-well plate (4 × 10³ conidia per well). The medium was supplemented with the indicated amounts of Doxy. After 48 h of incubation at 37°C, the percentage of surviving microcolonies was determined. Experiments were performed in triplicate. Statistical significance (*, P ≤ 0.05) was calculated with a two-tailed unpaired (assuming equal variances) Student t test. The error bars indicate standard deviations.

FIG 6

Paradoxically growing hyphae exhibit significantly less chitin than their echinocandin-inhibited progenitor microcolonies. Conidia of the wild type expressing GFP and fks1_tetOn were inoculated in AMM on coverslips. The medium was supplemented with 1 μg ml⁻¹ or 8 μg ml⁻¹ CS. After 1 day (A and B) and 3 days (C and D) of incubation at 37°C, hyphae were fixed, stained with calcofluor white, and analyzed with a confocal laser scanning microscope. (A and C) The chitin content was quantified by comparing the calcofluor white fluorescence signal intensities of at least 7 and up to 17 (8 μg ml⁻¹ caspofungin; 3 days) hyphal sections per strain and condition. The signal intensities of the wild type were normalized to those of the fks1_tetOn strain under repressive conditions (baseline correction; the fluorescence intensity of fks1_tetOn was set to 100%). The statistical significance (***, P ≤ 0.001) was calculated with a two-tailed unpaired (assuming equal variances) Student t test. The error bars indicate standard deviations. (B and D) Representative images of GFP fluorescence and calcofluor white (CFW) fluorescence after the indicated times of incubation. Scale bars, 25 μm.