Immobilization of Aspergillus fumigatus colonies in a soft agar matrix allows visualization of A549 cell detachment and death

A model to examine the effects of proliferating Aspergillus fumigatus ATCC 26933 colonies embedded in a thin layer of soft agar on a monolayer of A549 cells was developed and enabled an investigation of the response of cultured cells to fungal growth. This model simulates the conditions on the respiratory surface in patients with invasive aspergillosis and also in the mucus secretions of cystic fibrosis patients with allergic bronchopulmonary aspergillosis. Conidia of A. fumigatus adhering to A549 cells were immobilized in a thin layer of soft agar (0.6% (w/v)) and allowed to germinate at 37 degrees C. Fungal colonies greater than 5 mm in diameter caused rounding-up and detachment of A549 cells underneath the colony and towards the hyphal tips. As the fungal colony diameter increased, cell detachment occurred ahead of the hyphal tips. Cells detached for short periods (less than 6 h) showed no annexin-V (AV) or propidium iodide (PI) staining, suggesting no externalization of phosphatidylserine and an intact plasma membrane. Cells that had detached for periods greater than 6 h were positive for AV and PI indicating the rupture of the plasma membrane and cell death by necrosis. Chemical extraction and separation by thin layer chromatography of agar from zones of cell detachment around fungal colonies revealed the presence of three compounds that may play a role in inducing cell death