New antifungal agents are urgently required to combat life-threatening infections caused by opportunistic fungal pathogens like Candida albicans. The manipulation of endogenous fungal programmed cell death responses could provide a basis for future therapies. Here we assess the physiology of death in C. albicans in response to environmental stresses (acetic acid and hydrogen peroxide) and an antifungal agent (amphotericin B). Exposure of C. albicans to 40-60 mM acetic acid, 5-10 mM hydrogen peroxide, or 4-8 microg.ml-1 amphotericin B produced cellular changes reminiscent of mammalian apoptosis. Nonviable cells that excluded propidium iodide displayed the apoptotic marker phosphatidylserine (as shown by annexin-V-FITC labeling), were terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive (indicating nuclease-mediated double-strand DNA breakage), and produced reactive oxygen species. Ultrastructural changes in apoptotic cells included chromatin condensation and margination, separation of the nuclear envelope, and nuclear fragmentation. C. albicans cells treated at higher doses of these compounds showed cellular changes characteristic of necrosis. Necrotic cells displayed reduced TUNEL staining, a lack of surface phosphatidylserine, limited reactive oxygen species production, and an inability to exclude propidium iodide. Necrotic cells lacked defined nuclei and showed extensive intracellular vacuolization. Apoptosis in C. albicans was associated with an accumulation of cells in the G2/M phase of the cell cycle, and under some apoptosis-inducing conditions, significant proportions of yeast cells switched to hyphal growth before dying. This is a demonstration of apoptosis in a medically important fungal pathogen.
