High throughput assay to detect compounds that enhance the proton permeability of Candida albicans membranes

We describe a 96-well microtiter plate format assay to detect changes in pr oton permeability in membranes of the pathogenic yeast, Candida albicans. C andida albicans cells were incubated with the lipophilic ester of 2',7'-bis -(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH-sensitive fluo rescein derivative. Inside the cells, BCECF was released and trapped in the vacuole. Compounds that destroyed membrane integrity increased the pH valu e of the vacuole due to proton leakage into the cytoplasm. This was paralle led by an increase in BCECF fluorescence intensity, which could be quantifi ed. The test assay was validated with amphotericin B, as well as with other membrane-active compounds known to increase membrane permeability. Possibl e applications and limitations of this assay in the held of antifungal drug discovery are discussed.