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

School of Biological Sciences, 1.800 Stopford Building, University of Manchester, Manchester M13 9PT, UK Correspondence Geoffrey D. Robson geoff.robson{at}man.ac.uk 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 H 2 O 2 (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. Abbreviations: PI, propidium iodide; PS, phosphatidylserine; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling