Azole-induced cell wall carbohydrate patches kill Aspergillus fumigatus

Abstract

Azole antifungals inhibit the fungal ergosterol biosynthesis pathway, resulting in either growth inhibition or killing of the pathogen, depending on the species. Here we report that azoles have an initial growth-inhibitory (fungistatic) activity against the pathogen Aspergillus fumigatus that can be separated from the succeeding fungicidal effects. At a later stage, the cell wall salvage system is induced. This correlates with successive cell integrity loss and death of hyphal compartments. Time-lapse fluorescence microscopy reveals excessive synthesis of cell wall carbohydrates at defined spots along the hyphae, leading to formation of membrane invaginations and eventually rupture of the plasma membrane. Inhibition of β-1,3-glucan synthesis reduces the formation of cell wall carbohydrate patches and delays cell integrity failure and fungal death. We propose that azole antifungals exert their fungicidal activity by triggering synthesis of cell wall carbohydrate patches that penetrate the plasma membrane, thereby killing the fungus. The elucidated mechanism may be potentially exploited as a novel approach for azole susceptibility testing.

Fig. 1

Multiple manifestations of voriconazole-induced death are linked to cell wall integrity failure. a–d A. fumigatus wild-type conidia expressing mitochondria-targeted GFP were inoculated in Sabouraud medium and incubated at 37 °C. After 9 h, medium was supplemented with 0.4 µg ml⁻¹ (a), 1.27 µg ml⁻¹ (b, c) or the indicated amount (d) of voriconazole. The fate of individual hyphae was followed over time with confocal laser scanning microscopy. a–c Exemplary bright field and time-lapse GFP fluorescence images (green) of optical stacks covering the entire hyphae in focus are depicted. d Quantitative analysis of three voriconazole-induced fungal death manifestations. Approximately 160 hyphal compartments were analyzed per condition for 13 h. Bars represent means of the six individual data points, error bars indicate standard deviations. Shown are results representative of two independent time-lapse microscopy experiments per condition. e A. fumigatus conidia harboring a luciferase-based cell wall salvage reporter were inoculated in a 96-well plate in Sabouraud medium and incubated at 37 °C. After 7 h, luciferin and the indicated amount of voriconazole were added. Upper panel, luciferase activity over time after addition of voriconazole. Lower panel, exemplary microscopic dark-field images of hyphae after 17 h co-incubation. Data are representative of three independent experiments. f Conidia of wild-type, Δrho4 and rho4 expressing cytosolic GFP were inoculated in Sabouraud medium. After 11 h incubation at 37 °C, medium was supplemented with 1.27 µg ml⁻¹ voriconazole. After 5 h co-incubation, hyphae were stained with trypan blue to quench the GFP signal in lysed compartments. The percentage of viable microcolonies was determined (graph). Data points represent means, the error bars indicate standard deviations. Data are representative of six independent blinded experiments. An exemplary overlay fluorescence image of optical stacks covering a partially viable wild-type hypha (green, GFP; red, trypan blue) is depicted on the left. a–c, f Bars represent 10 μm and are applicable to all subpanels

Fig. 2

Inhibition of the lanosterol 14α-demethylase triggers excessive synthesis of cell wall carbohydrates at defined foci. a–c Conidia of A. fumigatus wild-type expressing mitochondria-targeted GFP (a), cytosolic GFP (b), or no GFP (c) were inoculated in Sabouraud medium on cover slips and incubated at 37 °C. The experiment depicted in b was additionally inoculated with conidia of a conditional β-1,3-glucan synthase mutant under repressive conditions (fks1_tetOn; staining control). a When indicated ( + Vori), medium was supplemented with 0.4 or 1.27 µg ml⁻¹ voriconazole 10 h after inoculation. After a total of 15 h incubation, hyphae were fixed, stained with calcofluor white and analyzed with a confocal laser scanning microscope. Depicted are representative images of optical stacks of mitochondria (GFP; left panels), chitin (calcofluor white; middle panels) and an overlay (right panels) that cover the entire hypha in focus. b After 8 h incubation, medium was supplemented with 1.27 µg ml⁻¹ voriconazole. After additional 9 h incubation at 37 °C, hyphae were fixed, stained with aniline blue and immediately analyzed with a fluorescence microscope. Left, glucan-specific (green) and nonspecific (fks1_tetOn; blue) aniline blue fluorescence. Right, bright field microscopy. Arrow heads indicate glucan patches. c After 7 h incubation, medium was supplemented with the indicated amount of voriconazole. After 2, 3, and 5 h co-incubation, samples were fixed and stained with calcofluor white. The percentage of microcolonies with chitin patches was determined with a fluorescence microscope and plotted in the depicted graph. Bars represent means of the indicated data points, error bars indicate standard deviations. Data are representative of three independent blinded experiments. d Wild-type conidia expressing mitochondria-targeted GFP were stained with calcofluor white, either directly (resting conidia; left panel) or after 45 h incubation at 37 °C in Sabouraud medium supplemented with 1.27 µg ml⁻¹ voriconazole (right panel). Depicted are representative bright field images (left), images of single GFP (middle left) and calcofluor white (middle right) fluorescence cross sections and an overlay of both (right). a, b, and d Bars represent 5 μm and are applicable to all subpanels

Fig. 3

Azole-triggered cell death and synthesis of cell wall carbohydrate patches are linked to inhibition of the lanosterol 14α-demethylase. a–d Conidia of the conditional cyp51A_tetOn∆cyp51B strain (a– d) and wild-type (d) that express mitochondria-targeted GFP were inoculated under induced conditions in Sabouraud medium supplemented with 15 µg ml⁻¹ doxycycline. After 9 h incubation at 37 °C, hyphae were shifted to repressive conditions by substitution of the medium without doxycycline. a–c The fate of individual hyphae was followed over time with a confocal laser scanning microscope. Depicted are exemplary bright field and time-lapse GFP fluorescence images (green) of optical stacks covering the entire hyphae in focus. d After 5 h incubation under repressive conditions, hyphae were fixed, stained with calcofluor white and subjected to confocal laser scanning microscopy. Depicted images represent optical stacks of GFP fluorescence (left panels), calcofluor white fluorescence (middle panels) and an overlay (right panels) that cover the entire hypha in focus. a–d Bars represent 10 μm and are applicable to all subpanels

Fig. 4

Multiple azole-induced cell wall carbohydrate patches invaginate the plasma membrane. A. fumigatus conidia expressing mitochondria-targeted red fluorescence protein (RFP) and GFP-tagged membrane-anchored Wsc1 were inoculated in Sabouraud medium. After 9 h incubation at 37 °C, medium was supplemented with 0.53 µg ml⁻¹ voriconazole. Fluorescence was analyzed with a confocal laser scanning microscope. The micrographs show bright field (left), fluorescence cross sections (green, RFP; glow dark color scheme, GFP) and an overlay of the two fluorescence cross sections of a representative hypha incubated for 4 h in the presence of voriconazole. The GFP fluorescence intensity is additionally visualized with a heatmap color scheme (right micrograph), the accumulation of Wsc1-GFP at sites of plasma membrane invaginations are indicated with arrow heads. The bar represents 5 μm

Fig. 5

The fungicidal activity of azoles depends on the functionality of the conventional mitochondrial electron transport chain. a 1 × 10⁶ conidia of wild-type (wt) and of the conditional cytochrome c (cycA_tetOn) and complex III (rip1_tetOn) mutants were spread on Sabouraud agar plates. When indicated, medium was additionally supplemented with 15 µg ml⁻¹ doxycycline to induce the conditional promoter ( + Doxy). Voriconazole Etest strips were applied and plates were incubated at 37 °C. Representative photos were taken after approximately 48 h. The panels next to the macroscopic Etest strip photos show magnifications of the framed sections. b, c Conidia of the indicated strains were inoculated in Sabouraud medium supplemented with resazurin (cell viability marker, 0.1 µg ml⁻¹) and the indicated amount of voriconazole and incubated at 37 °C. Macroscopic (b) and microscopic (c) images were taken after 42 h

Fig. 6

Fungistatic azole concentrations do not trigger cell wall carbohydrate patch formation in mutants affected in the conventional mitochondrial electron transport chain. a–c Conidia of wild-type (wt; a), cycA_tetOn (b) and ript1_tetOn (c) were inoculated in Sabouraud medium and incubated at 37 °C. After 10 h incubation at 37 °C, the medium was supplemented with the indicated amount of voriconazole. After additional 10 h incubation, hyphae were stained with calcofluor white, fixed and analyzed with a confocal laser scanning microscope. Depicted are representative images of optical stacks of the calcofluor white fluorescence (chitin) that cover the hyphae in focus. The lower panels show magnifications of the framed sections in the upper panels. Bars represent 50 μm and are applicable to all respective subpanels

Fig. 7

The presence and size of the cell wall carbohydrate patch correlate with death of individual hyphae. a–d Conidia of the conditional complex III mutant (rip1_tetOn) expressing mitochondria-targeted GFP were inoculated in Sabouraud medium under repressed conditions. After 10 h of incubation at 37 °C, medium was supplemented with 2.4 µg ml⁻¹ voriconazole and incubated for another 15 h. Hyphae were stained with calcofluor white, and analyzed with time-lapse laser scanning microscopy. a Representative images of dead and living hyphae. Left, calcofluor white fluorescence (patches), right, GFP fluorescence (mitochondria). Dead hyphae (arrows) were characterized by multiple cell wall carbohydrate patches, arrest of mitochondrial dynamics (compare Supplementary Movie 7), fragmentation of the tubular mitochondrial network and fading of the GFP fluorescence. Living hyphae show highly dynamic and tubular mitochondria (compare Supplementary Movie 7) and less or no cell wall carbohydrate patches. The bar indicates 50 µm. b–c Short time-lapse sequences of multiple hyphae were taken and each hyphal compartment was analyzed for viability, compartment length, and cumulative diameter of the containing cell wall carbohydrate patches. The depicted results are based on time-lapse microscopy data obtained from three technical replicates in one experiment. Two-hundred and ninety-four hyphal compartments were analyzed in total. Very similar results were obtained in two independent experiments with the cycA_tetOn strain under similar conditions (Supplementary Fig. 1). b Absolute and relative numbers for living and dead compartments that exhibit patches or no patches. A significant number of living compartments exhibit no patches, while almost all dead compartments have patches. c The graphs indicate the cumulative patch diameter and compartment length for each living (blue) and dead (red) compartment. d Dead compartments exhibit a significantly higher ratio of the cumulative patch diameter and compartment length (patch index; depicted as box-and-whiskers graph). Statistical significance (***p ≤ 0.001) was calculated with a Mann–Whitney test

Fig. 8

Inhibition of β-1,3-glucan synthesis attenuates the fungicidal activity of voriconazole. a, b Conidia of A. fumigatus wild-type (a) or wild-type expressing mitochondria-targeted GFP (b) were inoculated in Sabouraud medium on cover slips (a) or in an eight-well live cell microscopy slide (b) and incubated at 37 °C. After 9 h incubation, medium was supplemented with 1.27 µg ml⁻¹ voriconazole (Vori) and, when indicated, 30 min later additionally with 4 µg ml⁻¹ caspofungin ( + Caspo). a After 4 h co-incubation with antifungals, hyphae were fixed, stained with calcofluor white and analyzed with a confocal laser scanning microscope. Depicted are representative calcofluor white fluorescence images of optical stacks that cover the entire hypha in focus. Bar represents 10 μm and is applicable to both subpanels. b After 5 and 6 h co-incubation with antifungals, the mitochondrial morphology and dynamics of at least 975 hyphal compartments per condition were analyzed with a confocal laser scanning microscope in seven independent experiments. The box-and-whiskers graph shows the percentage of viable compartments under each condition. Statistical significance (**p < 0.01) was calculated with a two-tailed paired Student’s t-test (assuming equal variances). c Conidia of the conditional β-1,3-glucan synthase mutant (fks1_tetOn) were inoculated in Sabouraud medium in 24-well plates and incubated at 37 °C under repressive conditions for 11 h. When indicated, media were subsequently supplemented with 1.27 µg ml⁻¹ voriconazole (Vori), or additionally with 10 µg ml⁻¹ doxycycline (Vori + Doxy) to induce expression of the β-1,3-glucan synthase Fks1. After additional 6 h incubation at 37 °C, medium was discarded. The wells were washed and supplemented with fresh medium without antifungals and doxycycline and the plates incubated for additional 10 to 30 h at 37 °C. The percentage of surviving microcolonies was microscopically determined and shown in the depicted box-and-whiskers graph. Criteria for viability were continuation of growth combined with light refraction; viable hyphae were bright, and dead hyphae were dark. Exemplary bright-field image of dead and viable microcolonies as assessed after the indicated additional incubation time are shown on the right, dead microcolonies are indicated with arrow heads. The depicted experimental results are representative of four independent blinded experiments under similar conditions. Statistical significance (***p ≤ 0.001) was calculated with a two-tailed unpaired (assuming equal variances) Student’s t-test

Fig. 9

Azoles trigger patch formation in azole-susceptible but not in azole-resistant A. fumigatus clinical isolates. Conidia of three azole-susceptible (2017–323, 2017–350, 2017–351) and two azole-resistant (2014–065, 2016–364) clinical isolates of A. fumigatus were inoculated in Sabouraud medium. After 10 h incubation at 37 °C, medium was supplemented with the indicated amount of voriconazole. After additional 10 h incubation, hyphae were stained with calcofluor white, fixed and analyzed with a confocal laser scanning microscope. Depicted are representative images of optical stacks of the calcofluor white fluorescence (chitin) that cover the hyphae in focus. The right panels show magnifications of the framed sections in the panels shown on the left. Bars represent 50 μm and are applicable to all respective subpanels

Fig. 10

Proposed model for the fungicidal activity of azole antifungals against A. fumigatus. Azole enters the growing Aspergillus hypha (I) and binds to the target enzyme lanosterol 14α-demethylase (CYP51). The inhibition of CYP51 results in the depletion of ergosterol and suppression of growth in less than 1 h (II). This is followed by extensive delivery of membrane protein-loaded transport vesicles to the cell membrane (accentuation in pink) and the induction of excessive β-1,3-glucan and chitin synthesis at defined spots along the hyphae (III). The continually increasing cell wall carbohydrate patches vigorously invaginate the plasma membrane, eventually resulting in membrane integrity failure and death of hyphae. Septa adjacent to damaged compartments are sealed off by Woronin bodies, specialized organelles that plug the septal pores, and improve survival of azole-exposed hyphae (IV). Inhibition of β-1,3-glucan synthesis, either by echinocandin antifungals or by reduced expression of the β-1,3-glucan synthase Fks1, attenuates the patch formation and delays the fungicidal activity of the azole. Mutants with a dysfunctional respiratory chain are strongly inhibited in growth but do not form cell wall patches, and thus do not die in the presence of lower inhibitory concentrations of azoles