Uptake of Aspergillus fumigatus Conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains expressing green fluorescent protein

Abstract

Several pathogenic fungal organisms enter eukaryotic cells and manipulate the host cell environment to favor their own growth and survival. Aspergillus fumigatus is a saprophytic fungus that causes invasive lung disease in the immunocompromised host. To determine whether A. fumigatus could enter eukaryotic cells, we studied the uptake of two different GFP-expressing A. fumigatus strains into A549 lung epithelial cells, human umbilical vein endothelial (HUVE) cells, and J774 murine macrophages in vitro. A549 cells internalized 30% of the bound conidia whereas HUVE and J774 cells internalized 50 and 90%, respectively. Conidia within A549 cells remained viable for 6 h; however, 60 to 80% of conidia within J774 cells were killed after only 4 h. Live and heat-killed conidia were internalized to the same extent by A549 cells. After 6 h, almost none of the conidia inside A549 cells had germinated, whereas extracellular conidia had developed germ tubes. Internalization of conidia by A549 cells was a temperature-dependent process and required rearrangement of the underlying host cell cytoskeleton; uptake was inhibited by 75% with 0.5 microM cytochalasin D and by 65% with 5 microM colchicine. Fluorescent labeling of infected A549 cells with rhodamine phalloidin provided visible evidence of cytoskeletal alteration as many of the intracellular conidia were contained in actin-coated phagosomes. These data provide evidence that significant numbers of A. fumigatus conidia can be internalized by nonprofessional phagocytes in vitro and these cells may serve as reservoirs for immune cell evasion and dissemination throughout the host.

FIG. 1.

Detection of GFP protein in conidia of transformants by immunoblotting. Wild-type (a) or gGFP-transformed (b) conidia from strain 13073 were lysed, and proteins were separated on an SDS-12% PAGE gel and transferred to polyvinylidene difluoride membrane. The membrane was probed with an anti-GFP antibody diluted 1:100 followed by a goat anti-rabbit peroxidase-labeled secondary antibody diluted 1:1,000. Development of the membrane with the DAB substrate revealed a band of 32 kDa in the transformed strain. Molecular mass indicators (in kilodaltons) are labeled on the left.

FIG. 2.

Detection of GFP fluorescence in conidia by epifluorescence. Wild-type (a) or gGFP-transformed (c) conidia from strain 13073 were fixed onto poly-l-lysine-coated coverslips and viewed by epifluorescence using fluorescein filters. (b and d) The corresponding DIC images are shown. Bars = 10 μm.

FIG. 3.

Immunofluorescence microscopy demonstrates phagocytosis of A. fumigatus conidia by A549, HUVE, and J774 cells. A549 (A), HUVE (B), or J774 (C) cells were seeded overnight (A549 and HUVE cells) or for 2 h (J774 cells only) onto 12-mm-diameter coverslips in 24-well plates. Cells were incubated with 10⁶ A. fumigatus 13073 conidia/ml in MEM-10% FBS for 3 h (A549 and HUVE cells) or 1 h (J774 cells) at 37°C. Following infection, samples were washed with PBST and processed for immunofluorescence. From left to right, panels show DIC image, fluorescence image showing the green channel (total bound conidia), fluorescence image of the red channel (extracellular conidia), and a merged overlay of all images. The results are representative of two independent experiments. Bars = 10 μm.

FIG. 4.

Internalized conidia do not germinate in A549 cells. A549 cells were infected with 10⁶ A. fumigatus 13073 conidia/ml in MEM-10% FBS for 6 h at 37°C and then fixed with 4% paraformaldehyde. The following day, samples were immunolabeled and then viewed by DIC and fluorescence microscopy. From left to right, panels show DIC image, fluorescence image showing the green channel (total bound conidia), fluorescence image of the red channel (extracellular conidia/germlings), and a merged overlay of all images. Bars = 10 μm.

FIG. 5.

Internalization of A. fumigatus conidia in the presence of phagocytosis inhibitors. A549 cells were infected with 10⁶ A. fumigatus 13073 conidia/ml in MEM-10% FBS containing no inhibitors (37°C) or 0.5 μM cytochalasin D or 5 μM colchicine. Invasion assays were also carried out with no inhibitors at 4°C to determine whether uptake was temperature dependent. The number of conidia internalized by the A549 cells was determined using the nystatin protection assay (see Materials and Methods). The results are representative of two independent experiments and are expressed as the means + standard deviations (error bars) of three replicates. ∗, P < 0.05.

FIG. 6.

Rearrangement of actin during the internalization of A. fumigatus conidia by A549 cells. A549 cells were infected with 10⁶ conidia/ml for 160 min. Following infection, actin microfilaments were stained with rhodamine phalloidin. Fluorescence from the red (a) and green (b) channels was collected separately on a Zeiss LSM confocal microscope and then overlaid in Adobe PhotoShop (c). Bars = 2 μm.