Oxidative and amphotericin B-mediated cell death in the opportunistic pathogen Aspergillus fumigatus is associated with an apoptotic-like phenotype

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Abstract
When protoplasts of the opportunistic fungal pathogen Aspergillus fumigatus were treated with low but toxic levels of hydrogen peroxide (0·1 mM) or amphotericin B (0·5 μg ml−1), loss of cell viability and death were associated with a number of phenotypic changes characteristic of apoptosis. The percentage of protoplasts staining positive with annexin V-FITC, an indicator of the externalization of phosphatidylserine and an early marker of apoptosis, rose to ∼55 % within 1 h. This was followed by a similar increase in apoptotic DNA fragmentation detected by the TUNEL assay, and led to a loss of cell permeability and death in ∼90 % of protoplasts, as indicated by the uptake of propidium iodide. The development of an apoptotic phenotype was blocked when protoplasts were pre-treated with the protein synthesis inhibitor cycloheximide, indicating active participation of the cell in the process. However, no significant activity against synthetic caspase substrates was detected, and the inclusion of the cell-permeant broad-spectrum caspase inhibitor Z-VAD-fmk did not block the development of the apoptotic-like phenotype. Higher concentrations of H2O2 (1·8 mM) and amphotericin B (1 μg ml−1) caused protoplasts to die without inducing an apoptotic phenotype. As predicted, the fungistatic antifungal agent itraconazole, which inhibits growth without causing immediate cell death, did not induce an apoptotic-like phenotype.