The trans, trans-farnesol was purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). Farnesol (1 M) stock was prepared fresh in methanol prior to each experiment. The original flask containing farnesol was de-gassed with nitrogen gas after each use to prevent farnesol degradation by oxygen. In this study, farnesol was used at 10, 100 and 300 µM, These concentrations were comparable to those previously used in different studies [26].

We selected C. albicans (ATCC 10231) which is known to be a nonproducer of endogenous farnesol [30]. This allows us to have specific effect, if any, related to exogenous farnesol. C. albicans was cultured for 24 h on Sabouraud dextrose agar plates (Becton Dickinson, Oakville, ON, Canada) at 30°C. For the C. albicans suspensions, one colony was used to inoculate 10 ml of Sabouraud liquid medium supplemented with 0.1% glucose, pH 5.6. The cultures were grown to the stationary phase in a shaking water bath for 18 h at 30°C. The yeast cells were then collected, washed with phosphate buffered saline (PBS), counted by means of a haemocytometer and adjusted to 10⁷/ml prior to use.

To determine the effect of farnesol on the yeast-to-hyphae transition, C. albicans (10⁴ cells) was grown in 10 ml of proteinase induction medium (PIM: 2% glucose, 0.1% KH2PO4, 0.05% MgSO4; adjusted to pH 4.0) supplemented with 10% (v⁄v) bovine serum albumin (BSA, Sigma) as previously reported [31] for 24 h at 37°C. Farnesol was added with and without fetal bovine serum (FBS) at various concentrations (10, 100, and 300 µM). The cultures were incubated at 37°C, observed microscopically at 6 and 24 h, and photographed to record C. albicans morphology. To calculate the percentage of yeast cells that underwent the morphological transition, three aliquots from each culture (n = 5) were used to determine the number of yeast cells and hyphae by means of a haemocytometer and an optical microscope, as previously described [32]. The percentage of cells that underwent morphological transition was determined using the following formula: (number of hyphae divided by the number of yeast cells and hyphae) × 100.

C. albicans (10⁶ cells) was cultured in Sabouraud liquid medium in the presence of farnesol at 10, 100, and 300 µM. C. albicans cultured in the presence of 0.1% methanol and in the presence of 5 µg/ml of amphotericin-B (Amph-B) constituted the negative and positive controls, respectively. Following a 24-h culture, pellets of C. albicans were prepared and used for scanning and transmission electron microscopy analyses.

C. albicans cells were fixed in 3% (v/v) gluteraldehyde in PBS (pH 7.4) and dehydrated in increasing concentrations of ethanol (10%, v/v, increments to 100%). A drop was placed onto a round glass cover slip for critical-point drying (CPD 030; Balzers, Liechtenstein, Germany). The sample was mounted onto an aluminium slab using silver paint (Agar Scientific, Stansted, UK). Sputter coating was performed (S150B, Edwards, London, UK) and the image was viewed using the scanning electron microscope model Jeol JSM 6360 LV (Soquelec, Montréal, QC, Canada). The experiment was repeated three times.

C. albicans was fixed in cacodylate buffer, pH 6.9, containing 1% (v/v) paraformaldehyde and 2% (v/v) gluteraldehyde, for 1 h at 4°C. The suspensions were then centrifuged and dehydrated in successive washes of ethanol for 30 min. The C. albicans pellets were embedded in Spurr resin and ultra-thin sections were obtained (Ultratome III, LKB, Bromma, Sweden). These sections were then mounted onto a 0.5% Pioloform (in chloroform)-coated 3.05 mm copper grid and stained with 3% aqueous uranyl acetate and 0.1% lead citrate prior to imaging under a JEOL 1200 transmission electron microscope (Soquelec, Montréal, QC, Canada). The experiment was repeated four times.

RNA (1 µg of each sample) was reverse transcripted into cDNA using Maloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen Life Technologies, Mississauga, ON, Canada) and random hexamers (Amersham Pharmacia Biotech, Inc., Baie d’Urfé, QC, Canada). RT conditions were 10 min at 65°C, 1 h at 37°C, and 10 min at 65°C. Quantitative PCR was carried out as described [33, 34]. Amounts of mRNA transcripts were measured using the Bio-Rad CFX96 real-time PCR detection system (Bio-Rad, Mississauga, ON, Canada).

Reactions were performed using a PCR supermix from Bio-Rad (iQ SYBR Green supermix). Primers Table 1 were added to the reaction mix at a final concentration of 250 nM. Five microliters of each cDNA sample were added to a 20 µl PCR mixture containing 12.5 µl of iQ SYBR Green supermix (Bio-Rad) and 0.5 µl of specific primers (Sap2, Sap4, Sap5, Sap6, and ACT-1) (Medicorp, Inc., Montréal, QC, Canada) and 7 µl of Rnase- and Dnase-free water (MP Biomedicals, Solon, OH, USA). Each reaction was performed in a Bio-Rad MyCycler^TM Thermal Cycler (Bio-Rad). For the qPCR, the CT was automatically determined using the accompanying Bio-Rad CFX manager. Thermocycling conditions for the Sap quantitation were 15 min at 95°C, followed by 45 cycles for 15 sec at 94°C and 1 min at 60°C, then 30 s at 72°C, with each reaction done in triplicate. The specificity of each primer pair was verified by the presence of a single melting temperature peak. ACT-1 produced uniform expression levels varying by less than 0.5 CTs between sample conditions and was therefore used as a reference gene for this study.

To determine farnesol’s effect on C. albicans Sap production, 100 ml of a 10⁷ yeast cell suspension were grown in the presence or absence of farnesol in proteinase induction medium PIM supplemented with 10% bovine serum albumin. Candida cultures in the absence of farnesol and presence of pepstatine-A at 50 µg/ml were used as negative (no Sap activity) controls. Candida cultures without farnesol and with PIM that contains or not methanol served as positive controls, whereas the cultures without farnesol and BSA were used as internal negative controls (no Sap production). Each concentration was simultaneously tested in triplicate (in separate tubes). These were incubated for 8 days at 37°C under agitation. Farnesol concentrations were refreshed every 24 h. This relatively long experimental duration time has previously been shown to be required for sufficient Sap production [31]. On the last day of culture, the supernatants were collected, centrifuged at 1200 g for 10 min, and used to determine Sap2 concentration in each sample by ELISA.

Anti-Sap2 IgG antibody was obtained by immunizing male Balbc/c mice with recombinant Saps2 protein [35]. Sera of immunized and sham-immunized mice were collected after 21 days and used to purify anti-Sap2 IgG antibody. Following purification, the reactivity of the anti-Sap2 IgG antibody was determined by ELISA using recombinant Sap proteins rSap1, rSap2, rSap3, rSap5, rSap6 (a kind gift from Dr. Bernard Hube, Hans Knoell Institute, Jena, Germany) and native Sap2 (Brenntag, Denmark). This showed that our anti-Sap2 IgG reacted with both the recombinant and native Sap2 and, to a lesser extent, Sap1 and Sap3. Anti-Sap2 IgG did not react with other Saps. Anti-Sap2 IgG antibody was thus used to measure the Sap2 production by C. albicans following treatment with farnesol. Polystyrene microtitre plates (Nunc) were coated with 50 µl of 5 µg/ml rSap2 in 0.06 M sodium carbonate buffer (pH 9.6) and incubated at 4°C. The wells were then saturated for 2 h at room temperature with 2% BSA in Tris-Buffered Saline and Tween 20 (TBST). The plates were washed three times with TBST, after which time 100 μl of each incubation mixture (prepared as follows) were plated in duplicate and incubated for 1 h at 37°C. The incubation mixtures, prepared in TBST 2% BSA, consisted of serial dilutions of rSap2, starting at 500 μg/ml (to obtain a Sap2 standard curve), or samples to be titrated, which were incubated in polystyrene tubes for 1 h at room temperature with an equal volume of anti-Sap2 IgG (diluted 1:75 in TBST 2% BSA). After washing, the bound antibodies were detected by adding alkaline phosphatase-coupled monoclonal goat anti-mouse IgG (Southern Biotechnology Associates) for 30 min at room temperature. Substrate solution containing p-nitrophenyl phosphate (Sigma) was then added after washing and the reaction was stopped with the addition of 0.1 M EDTA pH 8.0. Absorbance at 405 nm was inversely proportional to the amount of Sap present, and inhibition results were converted into μg/ml of Sap by means of the obtained standard curve, which was linear over the concentration range used.

Each experiment in this study was performed three times or more. Experimental values are given as means + SD. The statistical significance of differences between the control values and the test values was evaluated using a one-way ANOVA. Posteriori comparisons were done using Tukey’s method. Normality and variance assumptions were verified using the Shapiro-Wilk test and the Brown and Forsythe test, respectively. All of the assumptions were fulfilled. Data were analyzed using the SAS version 8.2 statistical package (SAS Institute Inc., Cary, NC, USA). Results were considered significant at p < 0.05.

As shown in Fig. (1), germ tube formation was reduced in the farnesol-treated C. albicans cells compared to that observed in the untreated control cultures Fig. (1A). The inhibition of germ tube formation was significant at both low and high concentrations of farnesol Fig. (1B) and as early as 6 h after contact. The inhibitory effect of farnesol on the yeast-to-hyphae transition was maintained after 24 h Figs. (1A and 1B). Only few, if any germ tubes were observed in the absence of BSA, either with or without farnesol (data not shown). The effect of farnesol against C. albicans transition could be through ultrastructural changes.

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.

The aerobic growth of C. albicans cells showed typical yeast cells displaying the characteristic bud scars (Fig. (2), SEM, Ctrl). No development of pseudohyphae (chains of elongated unseparated blastospores) was observed in the cultures with or without farnesol or Amph-B. Following the addition of farnesol, the external morphology of the cells did not appear as smooth as that of the untreated cells, which indicates a possible loss of cytosolic volume. Indeed, farnesol basically distorted the cell wall surface at high concentrations (Fig. (2), SEM). Nevertheless, the cell wall of C. albicans cultured in the presence of farnesol did not resemble to that of C. albicans treated with Amph-B. To confirm these results we performed transmission electron microscopy analyses. Incubation of C. albicans in the presence of farnesol resulted in notable alterations in the internal morphology, when compared to control cells (Fig. (2), TEM). Non-treated C. albicans cells displayed a normal cellular morphology with typical dense cytoplasm and a distinct cell wall. Furthermore, untreated Candida showed continuous cytoplasmic membrane (cm) lining a homogeneous and electron-dense cytoplasm that contains visible nucleus. However, in the presence of farnesol, large and irregular cytoplasmic vacuoles were detected. Peripheral vacuoles present in the farnesol-treated cells were not visible in organisms from the control cultures. Treatment with farnesol also resulted in an increased granulation of the cytoplasm, diminished definition of the cell membrane as well as a loss of cell-wall integrity (Fig. (2), TEM). A similar effect was obtained when C. albicans was treated with Amph-B.

As shown in Fig. (3), farnesol significantly (p< 0.01) affected Sap2 expression, showing decreased Sap2 mRNA expression at all of the tested concentrations. The decrease varied between 4- and 6-fold, compared to non-treated C. albicans cultures Fig. (3A). The same observations were made with Saps4-6. Farnesol significantly (p< 0.05) downregulated mRNA expression of Saps4-6. The important effects were obtained with Sap5 and Sap6. With Sap5, the decrease varied between 2 and 5 folds Fig. (3C). With Sap6, the decrease was about 10 folds following C. albicans treatment with 100 and 300 µM of farnesol Fig. (3D). With 10 µM of farnesol no effect was observed basically for Sap4 and Sap6.

Whenever possible, mRNA expression should be confirmed by using protein expression analysis. As farnesol clearly downregulated Sap2 expression, and taking into account that Sap2 is the predominant Sap gene produced in vitro under most proteinase-inducing conditions, the effect of this quorum-sensing molecule on Sap2 protein production was thus investigated. To do so, anti-Sap2 IgG antibodies were produced to determine, by means of competitive binding inhibition ELISA, the concentration of Sap2 produced by C. albicans following treatment with farnesol. As shown in Fig. (4), Sap2 production was significantly reduced in the presence of farnesol. This observed reduction ranged between 4- and 16-fold and was dose-dependent. These results are in agreement with those of the Sap2 mRNA levels following farnesol treatment Fig. (3).