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First published online 4 March 2003
doi: 10.1242/jcs.00329

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Aspergillus fumigatus conidia survive and germinate in acidic organelles of A549 epithelial cells

¹ Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, B.C., Canada, V5A 1S6
² Department of Biological Sciences, Simon Fraser University, Burnaby, B.C., Canada, V5A 1S6

View larger version (46K): [in a new window]   Fig. 1. Co-localization of A. fumigatus conidia with endosomal markers in J774 cells. (A) J774 cells were infected with A. fumigatus conidia for 25 minutes (a) or 120 minutes (b) and then stained with an antibody to CD71 (a) or LAMP-1 (b). From left to right: fluorescence image showing the green channel (conidia); fluorescence image of the blue channel (red pseudocolor) (endosomes/lysosomes); and a merged overlay of both images. Bars, 10 µm. (B) The percentage of A. fumigatus phagosomes that contained either CD71 or LAMP-1 at 25, 45 or 120 minutes was scored by analysis of at least 100 internalized conidia in three separate experiments, each represented by a different symbol (circles, squares and diamonds).

View larger version (23K): [in a new window]   Fig. 2. Co-localization of A. fumigatus conidia with endosomal markers in A549 cells. (A) A549 cells were infected with A. fumigatus conidia for 3 (a,b) or 24 hours (c) and then stained with an antibody to CD71 (a), CD63(b) or cathepsin D (c). From left to right: fluorescence image showing the green channel (conidia); fluorescence image of the blue channel (red pseudocolor) (endosomes/lysosomes); and a merged overlay of both images. Bars, 10 µm. (B) The percentage of A. fumigatus phagosomes that contained either CD71, CD63, LAMP-1 or cathepsin D at 3, 6 or 24 hours was scored by analysis of at least 100 internalized conidia in three separate experiments, each represented by a different symbol (circles, squares and diamonds).

View larger version (15K): [in a new window]   Fig. 3. Co-localization of A. fumigatus conidia with acidic organelles in A549 and J774 cells. (A) A549 cells were incubated with A. fumigatus conidia for 6 (a) or 24 hours (b). J774 cells were incubated with A. fumigatus conidia for 6 hours (c). Following incubation times, cells were incubated with pre-warmed LysoTracker for 30 minutes and then unfixed cells were directly viewed on the confocal microscope. From left to right: fluorescence image showing the green channel (conidia), fluorescence image of the red channel (endosomes/lysosomes) and a merged overlay of both images. Bars, 10 µm. (B) The percentage of A. fumigatus phagosomes that co-localized with the LysoTracker probe was scored by analysis of at least 100 conidia in three separate experiments, each represented by a different symbol (circles, squares or diamonds).

View larger version (15K): [in a new window]   Fig. 4. Infection of cultured cells with A. fumigatus conidia does not induce cellular cytotoxicity. A549 (A) or J774 (B) cells were incubated with A. fumigatus conidia for 6, 12 or 24 hours and then the supernatant was assayed for LDH activity. Infected samples contained cells plus conidia and uninfected samples contained cells only. Lysed represents uninfected cells that were treated with 1% Triton X-100 prior to sampling. Samples were incubated with LDH substrate for 5 minutes and then the absorbance was read on a microplate reader at 490 nm. *P<0.05.

View larger version (67K): [in a new window]   Fig. 5. Intracellular germlings escape from the A549 phagosome and grow into hyphae in the extracellular medium. A549 cells were incubated with A. fumigatus conidia for 24 (a) or 36 (b) hours. Following infection, samples were processed for immunofluorescence. From left to right: differential interference contrast (DIC) image, fluorescence image showing the green channel (total conidia/hyphae), fluorescence image of the red channel (extracellular conidia), and a merged overlay of all images. Bars, 10 µm. The results are representative of two independent experiments. Note that some conidia/hyphae are seen in the DIC image but not the green channel since the fluorescent image represents a single 0.2 µm section that only detects signals from spores in the same plane.

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