Table 1.
Percentage (± SE) of germ tube formation after 4, 6 or 8 h swelling in Czapek-Dox medium (CZD) medium at 37°C.
Fig 1.
Colony size of A. fumigatus isolates grown for 2 days on potato dextrose agar (PDA) at 37°C.
Table 2.
Highest frequency of the diameter (μm) of conidia (S1 Fig) and average diameter of colony (mm) (± SE) after 2 days of growth (Fig 1).
Fig 2.
Oxidative stress resistance of A. fumigatus strains.
Sensitivity of five A. fumigatus isolates for hydrogen peroxide (A, 500 mM H2O2) (A) or menadione (B, 1 mM Menadione) as determined in an agar plate diffusion assay. Bars represent the average inhibition zone based on biological and technical triplicates (± SE). * indicates statistical significance, based on a one-way ANOVA with a p-value ≤ 0.05.
Fig 3.
Association (A) and internalization (B) of conidia after 4 h of incubation with type II A549 lung epithelial cells. CalcoFluorWhite (CFW) was used to distinguish between external and internalized fungi. CFW only interacts with chitin in the fungal cell wall if the fungi are is not internalized in A549 cells. Bars represent the average (± SE) of three independent experiments. * indicates statistical significance, for the association data based on a one-way ANOVA, internalization is based upon a Kruskal Wallis test both with a p-value ≤ 0.05.
Fig 4.
Germination of A. fumigatus during coincubation with epithelial cells.
Percentage of hyphae after 12 h of incubation with type II A549 lung epithelial cells (A). Internalized conidia or hyphae after 12 hours of incubation with the A549 lung epithelial cells (B). Bars represent the average (± SE) of three independent experiments. * indicates statistical significance, based on a Kurskal Wallis test with a p-value ≤ 0.05.
Fig 5.
Host cell damage and immune response of A549 cells during confrontation with A. fumigatus.
LDH release (mU mL-1) (A) and IL-8 production (pg mL-1) (B) of the A549 cells after 4 (light grey) and 12 (dark grey) h of infection. Percentage of cells which are alive (green), apoptotic (orange) or necrotic (red) after 4 (C) or 12 (D) h of infection with A. fumigatus conidia. Bars represent the average of three independent experiments (± SE). * indicates statistical significance, based on a one-way ANOVA with a p-value ≤ 0.05.
Fig 6.
Uptake of swollen conidia by P. aurantium after 2 h of incubation.
DIC (A) and FITC (B) channel of phagocytosed and non-phagocytosed pre-incubated conidia (4.5 h) and their merge (C). FITC signal of the phagocytosed conidia is lower due to the acidification of the phagolysosome. D) Percentage (± SE) of amoeba that had phagocytosed one or more conidia. Bars represent three individual experiments. * indicates statistical significance, based on a Kruskal Wallis test with a p-value ≤ 0.05.
Fig 7.
Resistance of A. fumigatus to the predatory phagocyte P. aurantium.
Survival, measured by the conversion of resazurin to resorufin (A), hyphal growth (B) and hyphal length (μm; C) of conidia following pre-incubation for 4 (light grey), 6 (grey) or 8 h (dark grey) and coincubation with P. aurantium for 18 h. Bars represent the average (± SE) of three independent experiments. * indicates statistical significance, survival and hyphal growth is based on a Kruskal Wallis test and the hyphal length is based on a one-way ANOVA, both with a p-value ≤ 0.05.
Fig 8.
Amoeba survival after 18 hours of co-incubation with conidia following pre-incubation for 4 (light grey), 6 (grey) or 8 (dark grey) hour swollen conidia. Bars represent 3 individual experiments ± SE. * indicates a statistical difference, based upon the Kruskal Wallis test.
Fig 9.
Survival of A. fumigatus (A-C) and G. melonella larvae (D-F) after infection. Bar graph of A. fumigatus survival and Kaplan-Meier survival plots of G. melonella over 3 or 7 days of incubation with 105 (A, D), 106 (B, E) and 107 (C, F) conidia mL-1, respectively. Bars represent the average (± SE) of three independent experiments. Each line consists of three biological replicates. * indicates statistical significance, fungal survival is based on a Kruskal Wallis test and the larvae survival on a non-parametric log rank test, both with a p-value ≤ 0.05.
Fig 10.
Kaplan-Meier survival plot of the zebrafish embryos injected with 100 conidia of different A. fumigatus isolates.
Each line consists of three biological replicates. * indicates a statistical difference, based on a non-parametric log rank test with a p-value ≤ 0.05.
Fig 11.
Venn diagram of all SNPs (low, moderate and high impact) in the virulence related genes listed by [41].
The genome of Af293 is used as a reference genome.
Fig 12.
Phylogenetic tree based on SNPs compared to the Af293 reference genome.
Strains that were isolated from an invasive infection are marked red, from a non-invasive infection marked blue and strains isolated from the environment marked green. Strains used for the experiments in this study are indicated in bold with an *.
Fig 13.
A complete overview can be found in S5 Table.
Table 3.
Strains used in this study.