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

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.

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.

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.

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.