Farnesol-induced apoptosis in Candida albicans

Abstract

Farnesol, a precursor in the isoprenoid/sterol pathway, was recently identified as a quorum-sensing molecule produced by the fungal pathogen Candida albicans. Farnesol is involved in the inhibition of germination and biofilm formation by C. albicans and can be cytotoxic at certain concentrations. In addition, we have shown that farnesol can trigger apoptosis in mammalian cells via the classical apoptotic pathways. In order to elucidate the mechanism behind farnesol cytotoxicity in C. albicans, the response to farnesol was investigated, using proteomic analysis. Global protein expression profiles demonstrated significant changes in protein expression resulting from farnesol exposure. Among the downregulated proteins were those involved in metabolism, glycolysis, protein synthesis, and mitochondrial electron transport and the respiratory chain, whereas proteins involved in folding, protection against environmental and oxidative stress, actin cytoskeleton reorganization, and apoptosis were upregulated. Cellular changes that accompany apoptosis (regulated cell death) were further analyzed using fluorescent microscopy and gene expression analysis. The results indicated reactive oxygen species accumulation, mitochondrial degradation, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in the farnesol-exposed cells concurrent with increased expression of antioxidant-encoding and drug response genes. More importantly, the results demonstrated farnesol-induced upregulation of the caspase gene MCA1 and the intracellular presence of activated caspases. In conclusion, this study demonstrated that farnesol promotes apoptosis in C. albicans through caspase activation, implying an important physiological role for farnesol in the fungal cell life cycle with important implications for adaptation and survival.

FIG. 1.

Percentage of killed C. albicans cells in total of 2 × 10⁷ cells/ml following 24-h exposure to farnesol (0 to 300 μM), as determined by MTS metabolic assay. Error bars indicate standard errors of the means.

FIG. 2.

Representative two-dimensional gels of extracted proteins from C. albicans grown in the presence of 0 (0F), 40 (40F), or 200 (200F) μM farnesol, demonstrating differential protein expression. Forty-five protein spots (marked with numbered arrows) displayed consistent alterations upon farnesol treatment. Panels A to D represent the corresponding sections marked on the two-dimensional gels. Information on the identities and functions of these spots are listed in Table 2. MW, molecular weight.

FIG. 3.

Representative confocal scanning laser fluorescence images of farnesol-treated and untreated C. albicans cells, revealing the presence of (A) ROS accumulation, indicated by green fluorescence, and necrotic or dead cells, indicated by red fluorescence; (B) mitochondrial degradation in the farnesol-exposed cells, indicated by green fluorescence, with healthy mitochondria appearing as red aggregates; and (C) activation of intracellular caspases in the farnesol-exposed cells, indicated by green fluorescence. Minimal patchy fluorescence was observed in the cells treated with 40 μM farnesol, with increasing fluorescence seen with increasing concentrations of farnesol. The amount of farnesol is indicated in each panel by a number and the letter “F.” The bar represents 20 μm.

FIG. 4.

TUNEL assay showing a dose-dependent increase in apoptosis, indicated by appearance of green fluorescence in farnesol-treated C. albicans cells compared to untreated cells. The amount of farnesol is indicated in each panel by a number and the letter “F.” The bar represents 20 μm.