Deletion of AIF1 but not of YCA1/MCA1 protects Saccharomyces cerevisiae and Candida albicans cells from caspofungin-induced programmed cell death

Abstract

Caspofungin was the first member of a new class of antifungals called echinocandins to be approved by a drug regulatory authority. Like the other echinocandins, caspofungin blocks the synthesis of β(1,3)-D-glucan of the fungal cell wall by inhibiting the enzyme, β(1,3)-D-glucan synthase. Loss of β(1,3)-D-glucan leads to osmotic instability and cell death. However, the precise mechanism of cell death associated with the cytotoxicity of caspofungin was unclear. We now provide evidence that Saccharomyces cerevisiae cells cultured in media containing caspofungin manifest the classical hallmarks of programmed cell death (PCD) in yeast, including the generation of reactive oxygen species (ROS), the fragmentation of mitochondria, and the production of DNA strand breaks. Our data also suggests that deleting AIF1 but not YCA1/MCA1 protects S. cerevisiae and Candida albicans from caspofungin-induced cell death. This is not only the first time that AIF1 has been specifically tied to cell death in Candida but also the first time that caspofungin resistance has been linked to the cell death machinery in yeast.

Keywords: AIF1; Candida albicans; MCA1/YCA1; Saccharomyces cerevisiae; caspofungin; programmed cell death.

Figure 1. FIGURE 1: Saccharomyces cerevisiae cells cultured in caspofungin manifest numerous phenotypic markers associated with yeast programmed cell death.

Exponentially-growing wildtype cells from the W303-1A strain background were cultured in YPD containing 0.02µg/ml caspofungin for three hours. The cells were stained with 2.5µM CellROX Green to detect ROS (A, E) ; with a FLICA kit to detect activated caspase activity (B, F) ; and with the mitochondrial vital dye, JC-1, to assess their mitochondrial membrane potential (D, H) . To visualize the structure of their mitochondria, the cells were transformed with a p416 GPD-mtGFP plasmid constitutively expressing a mitochondria-localized GFP marker and their mitochondria were visualized after they had been treated with caspofungin as described above (C, G) . Representative images are shown (A-D) . Statistical significance for the graphs (E-H) was determined with the unpaired Student’s t-test (**: p<0.05; ***: p<0.005), and a minimum of 500 cells was counted for each assay.

Figure 2. FIGURE 2: Deletion of AIF1 but not YCA1/MCA1 protects Saccharomyces cerevisiae and Candida albicans cells from caspofungin-induced programmed cell death.

(A) A series of ten-fold dilutions of two cultures each of wildtype, ∆aif, and ∆yca1 W303-1A Saccharomyces cells were plated on YPD plates and YPD plates with 0.015 µg/ml caspofungin for two days. (B) A series of ten-fold dilutions of two independent cultures each of wildtype, aif∆/aif∆ and mca1∆/mca1∆ RBY1132 Candida cells were plated on YPD plates and YPD plates with the indicated concentrations of caspofungin for two days. Representative images are shown.