C. albicans strain SC5314 was obtained from professor Dominique Sanglard (Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland). All clinical C. albicans isolates are obtained from Changhai Hospital of Shanghai, China. C. albicans cells were routinely maintained on Sabouraud dextrose agar (1% w/v peptone, 4% w/v dextrose, and 1.8% w/v agar) and grown in YPD liquid medium (1% yeast extract, 2% peptone, and 2% dextrose) at in an orbital shaker at 30°C (Zhu et al., 2013). For all the experiments in vitro, 6.4 mg/ml SK (purity > 98%, National Institutes for Food and Drug Control, Peking, China) in dimethyl sulfoxide and 25 mM farnesol (Sigma-Aldrich, United States) in methanol were used as stock solutions and added to the culture suspensions to obtain the required concentrations. Spider and Lee’s media used for hyphae formation were made as previously described (Gimeno et al., 1992; Liu et al., 1994). RPMI-1640 medium (Gibco, United States) was used for biofilm formation experiments.

Antifungal susceptibility testing was performed in 96-well tissue culture plates (Corning Inc., NY, United States) using a broth microdilution protocol of the Clinical and Laboratory Standards Institute M27-A3 method, with a few modifications (Quan et al., 2006). In brief, the initial concentration of fungal cells in RPMI-1640 medium was 5 × 10³ CFU/ml, and the final concentrations ranged from 0.125 to 64 μg/ml for SK and fluconazole. Plates were incubated at 35°C for 24 h. The MIC₈₀ was determined as the lowest concentration of the drugs that inhibited growth by 80%.

The in vitro biofilm formation assay was carried out as described previously (Ramage et al., 2001). In brief, C. albicans cells (1.0 × 10⁶ cells/ml) in RPMI-1640 medium were added to a 96-well tissue culture plate (Corning Inc., Corning, NY, United States) for 90 min of adhesion at 37°C. For RNA extraction, cells were introduced into a 75 cm² tissue culture flask (Thermo Fisher Scientific Inc., Waltham, MA, United States). Following the initial 90 min of adhesion, the medium was aspirated and non-adherent cells were removed and then fresh medium was added to the adherent cells. The plate was further incubated at 37°C for 24 h until formation of mature biofilms. To test the effect of the compounds (SK, farnesol) on C. albicans biofilm formation, different concentrations of the compounds were added to fresh RPMI-1640 after 90 min of adhesion. To detect the effect of SK on mature C. albicans biofilms, biofilms were formed at 37°C for 24 h as described above. The biofilms were washed with PBS, then fresh RPMI-1640 medium containing different concentrations of SK was added. The plates were incubated at 37°C for a further 24 h to detect the antibiofilm effect of SK.

The growth of biofilms was measured with a 2,3-bis-(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay, a reaction catalyzed by mitochondrial dehydrogenases (Ramage et al., 2001). In brief, biofilm cells were washed with PBS and then incubated with 0.5 mg/ml of XTT and 1 mM of menadione in PBS at 37°C for 90 min. The optical density (OD) was determined at 490 nm using a microtiter plate reader. The SMIC₈₀ was determined as the lowest concentration of the drugs that inhibited mature biofilm by 80%.

The measurement of biofilm biomass was conducted as described previously (Nobile et al., 2006) with slight modifications. Silicone disks (1.5 × 1.5 cm, Bentec Medical Corp., United States) were weighed before inoculation. C. albicans cells (1.0 × 10⁶ cells/ml) in RPMI-1640 medium were added to a 12-well tissue culture plate with one prepared silicone disk in each well. The inoculated plate was incubated with for 90 min at 37°C. Then the medium was aspirated, non-adherent cells were removed and fresh medium was added to the adherent cells. The plate was further incubated gentle agitation at 37°C for 24 h until the formation of mature biofilms. To test the effect of SK on biomass production, different concentrations of SK were added to fresh RPMI-1640 after 90 min of adhesion. The silicone disks with attached biofilms were removed from the wells, dried overnight and weighed. The total biomass of biofilm was calculated by subtracting the weight of the silicone prior to biofilm growth from the weight of the silicone after the drying period, with adjustment for the weight of control silicone squares exposed to no cells.

Three hypha-inducing media (Lee’s medium, Spider medium and YPD medium supplemented with 10% fetal bovine serum) was used to test the effect of SK on hyphal formation. C. albicans (5.0 × 10⁵ cells/ml) were suspended in the hypha-inducing media containing 0.5 μg/ml of SK and added to 24-well tissue culture plates. The plates were incubated at 37°C for 3 h. The morphology of the cells was photographed under microscope.

To observe the effect of SK on C. albicans biofilms formation with CLSM, siliconedisks (Bentec Medical Corp., United States) were used to develop biofilms. Biofilms were washed and incubated for 45 min at 37°C in PBS containing the fluorescent stains FUN-1 (10 μM) (Molecular Probes, Eugene, OR, United States) and concanavalin A-Alexa Fluor488 conjugate (ConA) (25 μg/ml) (Molecular Probes, Eugene, OR, United States). FUN-1 is converted to an orange-red cylindrical intravacuolar structure by metabolically active cells, whilst ConA binds to the glucose and mannose residues of cell wall polysaccharides and emits a green fluorescence. After incubation with the dyes, the disks were flipped and the stained biofilms were observed with a Leica TCS SP2 confocal laser scanning microscope equipped with argon and HeNe lasers.

Farnesol was extracted and measured as previously described (Hornby et al., 2001) with some modificaton. In brief, the supernatant of C. albicans biofilm treated with SK for 24 h was collected. The biofilm cells were scraped from the culture plate and filtered through filters (0.45 μm) and washed three times with ultrapure sterilized water. Cells were dried at room temperature to constant weight and weighed using an electronic balance. Farnesol in the supernatants was extracted using ethyl acetate and detected with HPLC (Agilent, United States). The detection was performed by absorbance at 210 nm. A 5 mm C18 reversed-phase column (4.6 by 250 mm) was utilized with a mobile phase of 4:1 methanol-H₂O eluted at a flow rate of 1 ml/min. Six different concentrations of farnesol standard samples were used to create a standard curve of integral areas eluting at its retention time. The extracted farnesol was calculated based on the HPLC integral area compared with a standard curve. Farnesol concentrations obtained from each culture were also normalized on the biofilm dry weight assays.

The cellular surface hydrophobicity (CSH) of C. albicans biofilms was determined by a water-hydrocarbon two-phase assay as described previously (Klotz et al., 1985). In brief, the formed C. albicans biofilms were removed from the culture plate surface with a sterile scraper to obtain a cell suspension (OD₆₀₀ = 1.0 in YPD medium). A total of 1.2 ml of the suspension was pipetted into a clean glass tube and overlaid with 0.3 ml of octane. The mixture was vortexed for 3 min and then stood at room temperature for phase separation. Soon after the two phases had separated, the OD₆₀₀ of the aqueous phase was determined, and the OD₆₀₀for the group without the octane overlay was used as the control. Relative CSH was calculated as follows: [(OD₆₀₀ of the control-OD₆₀₀ after octane overlay)/OD₆₀₀of the control] × 100.

RNA isolation and real-time RT-PCR were performed as described previously (Li et al., 2014). Total RNA was isolated with fungal RNAout kit (TIANZ, Beijing, China) according to the manufacturer’s protocol. The isolated RNA was resuspended in diethyl pyrocarbonate-treated water. The OD was measured at 260 nm and 280 nm. The integrity of the RNA was visualized by subjecting 2–5 μl of the samples to electrophoresis through a 1% agarose-MOPS gel. The cDNA was obtained using the cDNA Synthesis Kit for RT-PCR (TaKaRa Biotechnology, Dalian, China). Real-time PCR was performed with a 7500 real-time PCR system (Applied Biosystems). SYBR green I (TaKaRa Biotechnology, Dalian, China) was used to monitor the amplified products in real time according to the manufacturer’s protocol. The primers are shown in Table 1. The PCR protocol consisted of denaturation program (95°C for 10 s), 40 cycles of amplification and quantification program (95°C for 10 s, 60°C for 20 s, 72°C for 15 s with a single fluorescence measurement), melting curve program (60–95°C with a heating rate of 0.1°C per second and a continuous fluorescence measurement) and finally a cooling step to 40°C. The expression of each gene was normalized to that of 18S rRNA. The relative expression of each target gene was calibrated against the corresponding expression in untreated biofilms.

A mouse vaginal model was used to demonstrate the antifungal effect of SK in vivo according to the previous procedures with slight modification (Yano and Fidel, 2011). Briefly, 3 days prior to inoculation, female KM clean mice (Shanghai SLAC Laboratory Animal Center, China) were injected with 0.1 mg of β-estradiol (Sigma-Aldrich, Shanghai, China) subcutaneously in the lower abdomen. Estrogens injections were administered every other day for three times in a week. The intravaginal inoculation with 4 × 10⁶ C. albicans cells in 20 μl of PBS was injected into vaginal lumen of the estrogen-treated mice. After the establishment of murine model, SK (dissovled in sesame oil) was delivered intravaginally on days 1, 2, 3, and 4 after inoculation. Sesame oil was employed as a negative control. On the fifth day, the vaginal lumens of the mice were lavaged by 100 μl of PBS with repeated aspiration and agitation with a pipette tip. The lavage fluids were picked and cultured on the YPD plates. The CFU was counted to analyzed the infection burdens. The animal experiments were approved by the Animal Ethics Committee of the Second Military Medical University (Shanghai, China).

Statistics were calculated in GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, United States), in which P-value of < 0.05 or < 0.01 was considered statistically significant.

We first determined the MIC (minimum inhibitory concentration) values of SK on C. albicans strains, including a standard strain SC5314 and several clinical isolates. As shown in Table 2, SK could inhibit both planktonic and biofilm cells. The MIC_80s of SK against plantonic C. albicans cells ranged from 2 to 4 μg/ml. It should be noted that SK exhibited similar antifungal activity on the fluconazole-resistant strains as the fluconazole-sensitive strains. The SMIC_80s of SK against C. albicans mature biofilms ranged from 4 to 16 μg/ml while the SMIC_80s of fluconazole were over 64 μg/ml. Figure 1A showed that SK inhibited biofilm formation in a dose-dependent manner when this compound was added to C. albicans cells after 90 min of adhesion. More specifically, 1 μg/ml of SK inhibited biofilm formation by 17.3%, and the inhibitory effect on biofilm formation was enhanced when the concentration of SK was increased. 4 μg/ml of SK inhibited biofilm formation by 65.4%, while the biofilm growth was almost totally inhibited upon exposure to 32 μg/ml of SK. The effect of SK against mature C. albicans biofilms was shown in Figure 1B. Although the mature biofilm cells were hardly affected at the SK concentrations of less than 1 μg/ml, almost half of the mature biofilm was destroyed when SK was added at the concentration of 4 μg/ml. SK at the concentration of 32 μg/ml destroyed mature biofilms by 92.8%.

Consistent with the results of the XTT reduction assay, biofilm biomass determination revealed that SK could inhibit biomass production (Figure 1C). Addition of 1 μg/ml of SK led to a significant drop of biomass level as compared to the control group without SK treatment. The inhibitory effect on biomass production was enhanced when the concentration of SK was increased, while almost no biomass was produced in the presence of 32 μg/ml of SK.

Confocal laser scanning microscopy (CLSM) analysis further confirmed the antibiofilm activity of SK. In the SK-free group, C. albicans biofilm exhibited atypical three-dimensional nature, composed mainly of true hyphae. However, the structure of C. albicans biofilm was destroyed in the presence of SK. At the concentration of 4 μg/ml of SK, the biofilm was predominantly composed of yeast cells and pseudohyphae, while true hyphae were rarely observed. When 8 μg/ml of SK was added, the C. albicans biofilm displayed a poor architecture, with a complete disappear of true hyphae or pseudohyphae (Figure 1D).

Since yeast-to-hypha growth is a key factor in C. albicans biofilm formation and virulence, we tested the effect of SK on filamentous growth. C. albicans cells were cultured in various hypha-inducing media, including Lee’s, Spider and YPD liquid media containing 10% fetal bovine serum. As shown in Figure 2, in the absence of SK, C. albicans underwent hyphal initiation, produced elongated and regular hyphal cells. When 0.5 μg/ml SK was added, the filamentous growth was remarkably inhibited. Specifically, the filamentation in Lee’s media was completely inhibited by 0.5 μg/ml of SK as only yeast cells were observed.

The CSH is an important factor for cell adhesion and biofilm formation, so we determined the CSH of C. albicans biofilms upon SK treatment. As shown in Figure 3, a negative correlation was observed between SK concentrations and the CSH of C. albicans biofilms. The relative CSH of untreated C. albicans biofilm was high (about 91.7%). SK decreased the CSH of biofilms in a dose-dependent manner. When the biofilm was treated with 2 μg/ml of SK, the relative CSH was significantly reduced, to approximately 70.3%. The relative CSH of the biofilm almost reached zero following the treatment of 32 μg/ml of SK.

In view of the inhibition of SK on C. albicans filamentous growth and adhesion, we investigated the expression of several well-known genes involved in hyphae formation and adhesion. In this experiment, 2 μg/ml of SK was used to inhibit biofilm formation for 8 h. The real-time reverse transcription-PCR (RT-PCR) results showed that ECE1, HWP1, EFG1, CPH1, RAS1, ALS1, ALS3, and CSH1 were downregulated. The upregulated genes included TUP1, NRG1, and BCR1 (Figure 4).

It has been reported that farnesol, one of the quorum sensing molecules in C. albicans, played an important role in yeast-to-hypha transition and biofilm formation (Ramage et al., 2002). In view of the inhibition of hyphae and biofilm formation upon SK treatment, we hypothesized that SK might exert this effect through modulating the level of farnesol. As expected, our results revealed that SK increased the production of farnesol in a dose-dependent manner. The level of farnesol was four times higher in 8 μg/ml of SK-treated biofilm than that in SK-free biofilm (Figure 5A). Consistent with this, DPP3, which encodes a phosphatase and has been demonstrated to be responsible for farnesol synthesis by converting farnesyl pyrophosphate to farnesol (Navarathna et al., 2007), was remarkably upregulated upon SK treatment (Figure 4).

Since the level of farnesol was increased upon SK treatment, we investigated the effect of farnesol on the antibiofilm activity of SK. To this end, the concentrations of SK (0.5 and 1 μg/ml) that had only a slight impact on biofilm formation were used. Similarly, 25 μM of farnesol used alone had no significant effect on biofilm. When SK was used in combination with farnesol, the formation of biofilms was significantly inhibited. More specifically, the combined treatment of 1 μg/ml of SK and 25 μM of farnesol resulted in approximately 40% biofilm formation rate as compared to drug-free biofilm (Figure 5B).

Since C. albicans is believed to be commonly occurred in vulvovaginal candidiasis (VVC) and VVC infections mainly rely on biofilm formation (Sobel et al., 1998; Fazly et al., 2013), here the in vivo activity of SK was investigated using a mouse vaginal infection model. As shown in Figure 6, although treatment of 2 mg/kg of SK had a slight impact on the fungal burden, 4 mg/kg of SK-treated group showed approximately 3 log10 CFU/ml reduction of fungal burden as compared to the control (SK-free) group. There was an even remarkable drop of fungal burden when SK was administrated at the dose of 8 mg/kg, while the fungal cells seemed to be wiped out.