The Monoterpene Carvacrol Generates Endoplasmic Reticulum Stress in the Pathogenic Fungus Candida albicans

Abstract

The monoterpene carvacrol, the major component of oregano and thyme oils, is known to exert potent antifungal activity against the pathogenic yeast Candida albicans. This monoterpene has been the subject of a considerable number of investigations that uncovered extensive pharmacological properties, including antifungal and antibacterial effects. However, its mechanism of action remains elusive. Here, we used integrative chemogenomic approaches, including genome-scale chemical-genetic and transcriptional profiling, to uncover the mechanism of action of carvacrol associated with its antifungal property. Our results clearly demonstrated that fungal cells require the unfolded protein response (UPR) signaling pathway to resist carvacrol. The mutants most sensitive to carvacrol in our genome-wide competitive fitness assay in the yeast Saccharomyces cerevisiae expressed mutations of the transcription factor Hac1 and the endonuclease Ire1, which is required for Hac1 activation by removing a nonconventional intron from the 3' region of HAC1 mRNA. Confocal fluorescence live-cell imaging revealed that carvacrol affects the morphology and the integrity of the endoplasmic reticulum (ER). Transcriptional profiling of pathogenic yeast C. albicans cells treated with carvacrol demonstrated a bona fide UPR transcriptional signature. Ire1 activity detected by the splicing of HAC1 mRNA in C. albicans was activated by carvacrol. Furthermore, carvacrol was found to potentiate antifungal activity of the echinocandin antifungal caspofungin and UPR inducers dithiothreitol and tunicamycin against C. albicans. This comprehensive chemogenomic investigation demonstrated that carvacrol exerts its antifungal activity by altering ER integrity, leading to ER stress and the activation of the UPR to restore protein-folding homeostasis.

FIG 1

Chemical-genetic profiling using HCGP assay identified key UPR regulators as required for carvacrol tolerance. (A) GO term enrichment of carvacrol sensitive mutants. The P value was calculated using the hypergeometric distribution. (B, C) Individual confirmations of the chemical-genetic screen by spot serial dilution assay. A total of three deletion mutants, the ire1, hac1, and aro2 mutants, were selected and spotted on YPD with DMSO (control), YPD containing 0.8 mM or 1 mM carvacrol (B) or 1.74 mM eugenol, 2.16 mM isopulegol, 2.22 mM l-(−)-carvone (vol/vol), or 2.22 mM d-(+)-carvone (C). Plates were incubated at 30°C for 2 days.

FIG 2

Carvacrol disrupts the morphology and the integrity of ER. Sec61-GFP fusion was used as an ER marker. (A) Fluorescence micrographs of Sec61 localization in control cells treated with DMSO. Cortical (cER) and nuclear ERs (nER) are labeled. (B) Reorganization of Sec61 localization in cells treated with 0.8 mM carvacrol. Bars, 4 μm.

FIG 3

The UPR pathway is important for carvacrol tolerance in the pathogenic yeast C. albicans. Growth assays of C. albicans bck1 (A), ire1 (B), and mkc1 (C) mutants and the WT strain SC5314 challenged with 1 mM carvacrol. Cells were grown in YPD at 30°C, and OD₅₉₅ readings were taken every 10 min. MIC values for each mutant and WT strain are indicated.

FIG 4

Genome-wide transcriptional profiling reveals that the monoterpene carvacrol induces the UPR in C. albicans. GO analysis of transcripts differentially regulated in C. albicans cells treated with carvacrol for 5 min (A) or 30 min (B) using BiNGO software (26). Results were charted using Cytoscape (25) and the Enrichment Map plug-in (52). (C) Heat map and two-dimensional hierarchical clustering of the transcriptional profiles of carvacrol- and thymol-treated cells. Upregulated and downregulated genes are indicated by red and green, respectively. Molecular structures of carvacrol and thymol are shown to emphasize the unique difference, which is the position of the hydroxyl group.

FIG 5

Carvacrol induces splicing of the transcription factor gene HAC1 mRNA. (A) Effect of carvacrol on HAC1 mRNA splicing in WT C. albicans. Cells were treated with carvacrol and at the indicated time samples were harvested and splicing of HAC1 was assessed using RT-PCR. nsHAC1, nonspliced HAC1; sHAC1, spliced HAC1. (B) Effect of tunicamycin on HAC1 splicing in the presence or absence of carvacrol. Cells were treated with tunicamycin (Tm) or with tunicamycin and carvacrol (Tm + Crv) and sampled at the indicated times to asses HAC1 splicing. As a control, splicing of HAC1 mRNA was also monitored in nontreated cells (Ctrl).