Two clinical isolates of C. albicans 0304103 and 01010 (both with FLC MIC₈₀ > 64 μg/mL and BBR MIC₈₀ > 16 μg/mL; MIC₈₀ was determined as the lowest concentration of the drug that inhibited cell growth by 80%) were used in this study. C. albicans cells were routinely propagated in yeast-peptone-dextrose (YPD) liquid medium (1% w/v yeast extract, 2% w/v peptone, and 2% w/v dextrose) at 30°C in a shaking incubator. FLC (Pfizer-Roerig Pharmaceuticals, New York, NY, USA) was prepared in sterile H₂O, BBR (Sigma–Aldrich, St Louis, MO, USA) was prepared in dimethyl sulfoxide (DMSO), and ergosterol (Sigma–Aldrich) was prepared in Tween 80–ethanol in 1:1 (v/v) ratio as 5 mM stock (Zhang et al., 2010; Zavrel et al., 2013).

Total sterols were extracted from whole cells according to a previous report with slight modifications (Liang et al., 2009). Briefly, FLC (64 μg/mL), and/or BBR (16 μg/mL) was added to exponentially growing C. albicans cells for 16 h. The same volume of DMSO was added to the control group. The cells were then harvested and washed twice with phosphate-buffered saline [PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride, and 137 mM sodium chloride (pH 7.4)]. The net weight of the cell pellet (approximate 0.5 g) was determined. PBS (2.5 mL) and 6 mL of 15% alcoholic sodium hydroxide solution (15 g NaOH and 10 mL of sterile distilled water, brought to 100 mL with 100% ethanol) were added to each pellet and mixed well by vortexing. The cell suspensions were incubated in an 80°C water bath for 1 h and then cooled to room temperature. Total sterols were extracted thrice with 6 mL of petroleum ether. Sterile distilled water (6 mL) was added to the combined upper phases and swirled for 1 min. Water was removed, and the tubes were incubated in a 60°C water bath until petroleum ether layer volatilized completely. The resulting samples were then dissolved in 0.5 mL of cyclohexane and used for gas chromatography (GC) quantification. Ergosterol standard (0.5 mg/mL), also dissolved in cyclohexane, and the sample-derivatized sterols were run through a VF-5MS fused silica column in a gas chromatograph (Agilent GC 7890B, Palo Alto, CA, USA) interfaced to a flame ionization detector. The injector temperature was 250°C. Moreover, the oven temperature was programmed to constant 100°C for 1 min, followed by a temperature increase of 10°C/min to a final temperature of 300°C. This final temperature was maintained for 10 min. Helium was used as the carrier gas. The linear velocity was 1 mL/min with a split ratio 10:1. The results were obtained by calibrating from the standard curve of ergosterol.

The hot-phenol method was used to extract total RNA samples from C. albicans isolates (Schmitt et al., 1990), which were treated with DNase I (TaKaRa Biotechnology, Dalian, China) to remove genomic DNA contamination. Reverse transcription was performed in a total volume of 20 μL with Avian Myeloblastosis Virus Reverse Transcriptase (TaKaRa Biotechnology), Random Primer (6-mer) (TaKaRa Biotechnology), and 1 μg of total RNA, followed by an exposure to 30 °C for 10 min, 45°C for 15 min, and 99°C for 2 min, as recommended by the manufacturer.

Real-time RT-PCR reactions were performed with SYBR Green I (TaKaRa Biotechnology), using a LightCycler Real-Time PCR system (Roche Molecular Biochemical, Mannheim, Germany). Gene-specific primers were designed using the Discovery Studio Gene software (Accelrys, Inc., San Diego, CA, USA). The primers are shown in Table 1. The thermal cycling conditions were as follows: an initial step at 95°C for 10 s, followed by 40 cycles at 95°C for 10 s, 62°C for 20 s, and 72°C for 15 s. The system software was used to monitor the change in fluorescence of SYBR Green I in every cycle, and then the threshold cycle (C_T) was measured. For internal control, 18S rRNA was used. The formula 2^-ΔΔCT was used to calculated the gene expression level of the drug-treated cells relative to that of the control cells, where ΔC_T was the C_T-value of genes of interest minus that of the internal control, and ΔΔC_T was the mean ΔC_T value of the drug-treated cells minus that of the control cells.

Exponentially growing C. albicans cells were harvested and resuspended in fresh YPD medium to 1 × 10⁵ CFU/mL. Various concentrations of BBR and ergosterol (alone or in combination) were added (Zavrel et al., 2013) to the cells. The cells were cultured at 30°C with constant shaking (200 rpm), and the OD₆₀₀ was measured at designated time points after culture (0, 3, 6, 9, 12, and 24 h). The same volume of solvents (DMSO, Tween 80, and ethanol) was added to the untreated control group. Three independent experiments were performed.

Intracellular BBR concentration was detected according to a previously described protocol (De et al., 2007; Pereira et al., 2007), with a few modifications. Exponentially growing C. albicans cells were harvested, washed twice with PBS, and resuspended in RPMI 1640 medium at a cell density of 1 × 10⁷ CFU/mL. Then, different concentrations of BBR and ergosterol were added. The same volume of solvents (DMSO, Tween 80, and ethanol) was added to the untreated control group. Each sample (1 mL) was cultured at 30°C with constant shaking (200 rpm) for 0, 2, 4, 6, 8, and 10 h; centrifuged; washed twice; and resuspended in 1 mL of PBS at 1 × 10⁷ CFU/mL. Then, 100 μL of each disposed sample was transferred into a black, clear-bottomed, 96-well microplate (Greiner, Pleidelsheim, Germany). An Infinite 200 Universal Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland) was used to measure the fluorescence of BBR at 405-nm excitation and 520-nm emission wavelengths.

Assays were carried out in 96-well microtiter plates (Greiner) according to a broth microdilution protocol of the Clinical and Laboratory Standards Institute M27-A3 method, with a few modifications (Quan et al., 2006; Xu et al., 2009; Li et al., 2013). The initial concentration of fungal suspension in RPMI 1,640 medium was 1 × 10³ CFU/mL. The final concentrations ranged from 0.125 to 64 μg/mL for FLC and 1 to 32 μg/mL for BBR. The plates were incubated at 35°C for 24 h. MIC₈₀s were determined as the lowest concentration of the drugs (alone or in combination) that inhibited growth by 80% compared with that in drug-free wells. The FIC index was defined as the sum of the MIC₈₀ of each drug when used in combination divided by the MIC₈₀ of the drug used alone. Synergy and antagonism were defined by FIC indices of ≤ 0.5 and >4, respectively. An FIC index of > 0.5 but ≤4 was considered indifferent (Odds, 2003).

Exponentially growing C. albicans cells treated with 10 μg/mL of FLC and/or 16 μg/mL of BBR for 16 h were washed twice with PBS and fixed in 500 μL of a fixative solution (sodium cacodylate buffer, pH 7.2, containing 4% polyoxymethylene) at 4°C for 24 h. The samples were then washed with saline and postfixed with 1% phosphotungstic acid for 90 min. The fixed cells were then dehydrated through a graded series of ethanol and embedded in EPON-812. Ultrathin sections were prepared, double stained with 3% uranyl acetate (Biopeony, Beijing, China) and 3% lead citrate (Sbjbio, Nanjing, China), and observed under a transmission electron microscope (Hitachi H-800, Tokyo, Japan) at a magnification of ×10,000.

Data were expressed as the mean ± SD of the independent assays in triplicate. The Student's t-test was used to assess the significance of the differences. A P < 0.05 or < 0.01 was considered statistically significant.

In this study, we investigated the mechanism underlying BBR tolerance in FLC-resistant C. albicans isolates and the effect of FLC on BBR tolerance. First, the cellular ergosterol level was measured using GC analysis in the FLC-resistant clinical isolate 0304103 after FLC and/or BBR treatment. Interestingly, the ergosterol content significantly increased in the cells treated with 16 μg/mL BBR compared with the control group (0.536 vs. 0.298 mg/mL), but was barely detected in the cells treated with 64 or 64 μg/mL FLC combined with 16 μg/mL BBR (Figures 1A–F). The findings suggested that BBR monotreatment resulted in marked augmentation of cellular ergosterol content in FLC-resistant C. albicans. However, this effect could be obliterated by the presence of FLC.

To further investigate the effect of BBR on the expression of ergosterol synthesis genes, the real-time RT-PCR analysis was performed in isolate 0304103 treated or untreated with 16 μg/mL BBR for 6 h. The same volume of DMSO was added in the BBR-untreated group. The results revealed a global upregulation of ergosterol synthesis genes in response to BBR treatment. Figure 2 shows a significant increase (more than 10-fold) in the expression of genes ERG1, ERG2, ERG3, ERG4, ERG7, and ERG24 (11.75- to 21.06-fold), and a modest increases in the expression of genes ERG5, ERG6, ERG10, ERG11, ERG13, and ERG25 (1.35- to 9.50-fold). These results indicated that BBR monotreatment increased the mRNA level of genes related to ergosterol synthesis, which was consistent with the result of ergosterol content measurement.

We hypothesized that the increase in cellular ergosterol content might be involved in BBR tolerance in FLC-resistant C. albicans. To test this hypothesis, the effect of feeding exogenous ergosterol on the susceptibility of isolate 0304103 to BBR was examined. The result of growth curve assay indicated no obvious effect on the growth of this isolate treated with 50 μM ergosterol, whereas the growth rates of the cells slowed down significantly when treated with high doses of BBR (64 and 128 μg/mL) (Figure 3). Interestingly, feeding 50 μM ergosterol markedly increased the growth rates of BBR-treated cells (Figure 3), suggesting that the fungistatic effect of BBR can be weakened in the presence of exogenous ergosterol.

To further test our hypothesis that the augmentation of ergosterol might contribute to the tolerance of FLC-resistant C. albicans to BBR, we evaluated the intracellular concentration of BBR with or without feeding exogenous ergosterol by fluorescence measurement. A strong fluorescence was detected in BBR-treated cells due to the higher treatment doses of BBR (32 and 64 μg/mL) (Figure 4). As expected, the feeding of 50 μM ergosterol markedly decreased the intracellular BBR fluorescence (Figure 4), indicating that the exogenous ergosterol could reduce the intracellular accumulation of BBR in FLC-resistant C. albicans.

The efflux of intracellular BBR was reported to be associated with the drug efflux pump in many species (Maeng et al., 2002; Li et al., 2013; Budeyri et al., 2016). Therefore, the mRNA levels of drug efflux pump genes MDR1, CDR1, and CDR2 were analyzed by real-time RT-PCR in isolate 0304103 exposed to 16 μg/mL BBR and/or 50 μM ergosterol for 6 h. The same volume of solvents (DMSO, Tween 80, and ethanol) was added to the untreated control group. Figure 5 shows that BBR treatment upregulated the expression of genes MDR1, CDR1, and CDR2 by 2.90-, 2.77-, and 1.92-fold, respectively. However, ergosterol treatment downregulated the expression of genes MDR1, CDR1, and CDR2 by 56, 25, and 55%, respectively. Notably, the expression of these three genes significantly increased after BBR–ergosterol combination treatment (4.67-, 6.54-, and 5.17-fold increase for gene MDR1, CDR1, and CDR2, respectively). The results suggested that feeding ergosterol could further upregulate the drug efflux pump genes to increase the efflux of intracellular BBR, which might be one of the reasons for the decrease in intracellular BBR concentration in FLC-resistant C. albicans after exposure to exogenous ergosterol.

Since the major antifungal mechanism of action of FLC was its inhibitory effect on cellular ergosterol synthesis, we postulated that FLC could sensitize the FLC-resistant isolates to BBR by decreasing ergosterol content. To validate this hypothesis, the antifungal efficacy of BBR combined with other antifungal agents in both isolates 0304103 and 01010 was investigated using checkerboard microdilution assay. As shown in Table 2, all the antifungal agents inhibiting ergosterol biosynthesis or binding to ergosterol, including sulconazole (SCZ), miconazole (MCZ), ketoconazole (KCZ), itraconazole (ITZ), amphotericin B (AMB), and nystatin (NST), demonstrated synergism with BBR against both isolates (FIC index ranged from 0.03 to 0.27). However, the antifungal agents that did not act on ergosterol, including N-myristoyl transferase inhibitor (N-MY) and 5-flucytosine (5-FC) (Weston et al., 1998; Vermes et al., 2000), were not synergistic with BBR (FIC index ranging from 1.02 to 2.02). The results indicated that the susceptibility of FLC-resistant C. albicans to BBR could be increased by antifungals acting on ergosterol, well supporting our hypothesis.

Since ergosterol was also present in many other organelle membranes (Lv et al., 2016), the ultrastructural changes in the cells treated with BBR and/or FLC in isolate 0304103 were observed under an electron microscope. Figure 6 shows that the cells in the untreated control group were well preserved, with an intact cell wall, a normal-shape plasma membrane, and a homogeneous cytoplasm. Cells treated with 16 μg/mL BBR also exhibited normal morphology of cell wall and plasma membrane. Although isolate 0304103 was highly resistant to FLC, 10 μg/mL of FLC could modestly damage the cell membrane (black arrow). BBR + FLC treatment severely damaged the cells, as indicated by a detachment of the cell membrane from the cell wall (black arrow) and extensive solubilization in the cytoplasm. Interestingly, oversized vacuoles appeared in the cells subjected to BBR monotreatment (white arrow), indicating that the vacuoles fused after treatment with BBR. It was reported that the membrane fusion was enhanced in ergosterol-enriched vacuoles (Kato and Wickner, 2001; Jones et al., 2010; Tedrick et al., 2012). Therefore, the results demonstrated that BBR treatment could elevate the cellular ergosterol content in FLC-resistant C. albicans.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.