Uptake of Aspergillus fumigatus Conidia by Phagocytic and Nonphagocytic Cells In Vitro: Quantitation Using Strains Expressing Green Fluorescent Protein

Abstract

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 μM cytochalasin D and by 65% with 5 μM 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.