Farnesol, a fungal quorum-sensing molecule triggers Candida albicans morphological changes by downregulating the expression of different secreted aspartyl proteinase genes

Abstract

The aim of this study was to determine the effect of exogenous farnesol in yeast-to-hyphae morphogenesis, and Saps (2, 4, 5 and 6) mRNA expressions by a Candida strain that does not produce endogenous farnesol. C. albicans was cultured in the absence and presence of farnesol at various concentrations (10, 100, and 300 µM), in proteinase induction medium, and then used to determine yeast-to- hyphae changes, Candida ultrastructure and to determine Saps 2, 4, 5 and 6 expressions using q-TR-PCR and ELISA (for Sap2). Data demonstrated that farnesol greatly reduced the yeast-to-hyphae morphogenesis of a Candida strain that does not produce endogenous farnesol. Farnesol induced several ultrastructural alterations, including changes in the cell-wall shape, a visible disconnection between the cell wall and cytoplasm with an electron-lucent zone between them, and the presence of electron-dense vacuoles. Tested on gene expressions, farnesol was able to significantly (p < 0.01) decrease Sap2 secretion and mRNA expression. Farnesol downregulated also Sap4-6 mRNA expression. These results demonstrated for the first time that farnesol modules Candida morphogenesis through a downregulation of Saps 2, 4, 5 and 6 expressions. Overall these data point to the potential use of farnesol as an antifungal molecule.

Keywords: Candida; farnesol; saps.; transition.

Fig. (1)

Farnesol modulated Candida transition from blastospore to hyphae forms. C. albicans was cultured PIM containing or not BSA with and without farnesol at various concentrations (10, 100, and 300 µM) for 6 and 24 h at 37°C. After each time point, the cultures were observed under an inverted microscope and photographed. Panel A showed morphological changes at 6 h. The numbers of yeast and hyphal forms were then counted. The percentage of hyphae was obtained by dividing the number of hyphae by the total number of cells (blastospores and hyphae) in each culture. The means + SD relative values are shown in (B). The levels of significance were obtained by comparing the percentages of yeast-tohyphae transition in the presence/absence of farnesol. (a), untreated C. albicans ; (b), treated with 10 mM ; (c), treated with 100 mM ; (d), treated with 300 mM of farnesol.

Fig. (2)

Scanning electron microscopy (left column) and transmission electron microscopy (two right columns) micrographs of C. albicans with and without farnesol. Candida was untreated (Ctrl), treated with Ampho-B (positive control) or with farnesol at various concentrations for 24 h then subjected to scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses. (n), nucleus; (cm), continuous cytoplasmic membrane; (CW), Cell wall; (V), vacuole. Each experiment was repeated three and four times, for SEM and TEM, respectively.

Fig. (3)

Effect of farnesol on Sap2, 4, 5 and 6 genes expression. Following C. albicans culture in the presence of various concentrations of farnesol, total RNA was extracted from each cell culture and qRT-PCR was performed using specific primers for Sap2, 4, 5 and 6. ACT-1 was used as the housekeeping gene for internal control. The changes in mRNA levels are presented as the fold expression of the gene in the test sample compared to this gene’s expression in the control (without farnesol). Data are expressed as means + SD from triplicate assays of three different experiments.

Fig. (4)

Effect of farnesol on the secretion of Sap2 protein by C. albicans. Following Candida culture in the presence of various concentrations of farnesol for 24 h, the supernatants were collected and used to quantify Sap2 level by competitive binding inhibition ELISA. The bars represent the means + SD of Sap2 concentration on three separate experiments. The levels of significance were obtained by comparing the values of farnesol stimulated cultures with control cultures.