Myricetin Disturbs the Cell Wall Integrity and Increases the Membrane Permeability of Candida albicans

The fungal cell wall and membrane are the principal targets of antifungals. Herein, we report that myricetin exerts antifungal activity against Candida albicans by damaging the cell wall integrity and notably enhancing the membrane permeability. In the presence of sorbitol, an osmotic protectant, the minimum inhibitory concentration (MIC) of myricetin against C. albicans increased from 20 to 40 and 80 μg/ml in 24 and 72 h, respectively, demonstrating that myricetin disturbs the cell wall integrity of C. albicans. Fluorescence microscopic images showed the presence of propidium iodidestained C. albicans cells, indicating the myricetin-induced initial damage of the cell membrane. The effects of myricetin on the membrane permeability of C. albicans cells were assessed using crystal violet-uptake and intracellular material-leakage assays. The percentage uptakes of crystal violet for myricetin-treated C. albicans cells at 1×, 2×, and 4× the MIC of myricetin were 36.5, 60.6, and 79.4%, respectively, while those for DMSO-treated C. albicans cells were 28.2, 28.9, and 29.7%, respectively. Additionally, myricetin-treated C. albicans cells showed notable DNA and protein leakage, compared with the DMSO-treated controls. Furthermore, treatment of C. albicans cells with 1× the MIC of myricetin showed a 17.2 and 28.0% reduction in the binding of the lipophilic probes diphenylhexatriene and Nile red, respectively, indicating that myricetin alters the lipid components or order in the C. albicans cell membrane, leading to increased membrane permeability. Therefore, these data will provide insights into the pharmacological worth of myricetin as a prospective antifungal for treating C. albicans infections.


Reagents
Myricetin was obtained commercially from Tauto Biotech (China). DMSO (Dimethyl Sulfoxide), amphotericin B, Calcofluor White (CFW) M2R, PI, crystal violet, DPH and Nile red were purchased from Sigma (USA). Phosphate Buffered Saline (PBS, pH 7.4) was procured from Gibco (USA) and Qubit dsDNA BR kit was purchased from Invitrogen (USA). Bradford reagent was obtained from Bio-Rad (USA). In addition, myricetin (20 mg/ml) was dissolved in DMSO, aliquoted, and stored at -20°C in the dark until use. Crystal violet (0.1 mg/ml) was dissolved in water, filtered, and stored at -20°C. Nile red (1 mg/ml) was dissolved in acetone and stored at 4°C.

Antifungal Susceptibility Testing
Antifungal susceptibility to myricetin was evaluated for each strain by the standard broth microdilution CLSI M27-A3 method [27], using resazurin as a cell growth indicator [28].
Briefly, the two-fold serial dilutions of myricetin or amphotericin B (100 μl) were added to the wells of a roundbottom 96-well microplate containing RPMI-1640 medium. The inoculum suspension (100 μl) containing 0.1 mg/ml resazurin was added to attain a final cell density of 1 × 10 3 -5 × 10 3 cells/ml, and the plate was incubated at 35°C for 24 h. Colorimetric MIC end-points were considered as the lowest sample concentration at which the solution remained blue, or the first sample whose color changed from blue to purple [28]. DMSO, which was the solvent used for the preparation of myricetin solution, was included as a growth control; no growth-inhibitory effects of DMSO were detected up to the concentration of 1%. Amphotericin B was used as a positive control.

Sorbitol Protection Assay
To ascertain whether myricetin affects the C. albicans cell wall structure, the sorbitol protection assay [29] was performed using modified CLSI M27-A3 protocol, with resazurin [30]. In a 96-well round bottom microplate, two-fold dilutions of myricetin and two-fold dilutions of myricetin with 0.8 M sorbitol were added in two separate rows. All the wells were inoculated with C. albicans cell suspensions, and the plate was incubated at 35°C. The MIC values were evaluated at the 24-and 72-h time points.

Microscopic Analysis
To examine the effect of myricetin on C. albicans cells, the myricetin-treated cells were observed using a confocal laser scanning microscope (CLSM) and a fluorescence microscope, respectively. First, log-phase C. albicans cells 1 × 10 8 cells /ml were grown in the presence of 20 μg/ml myricetin or 1 μg/ml amphotericin B in YM broth at 35°C with agitation at 200 rpm. The cells were harvested either at 2.5 or 4 h by centrifugation at 12,000 ×g for 1 min and stained with 10 μg/ml PI in PBS. The cells were observed using a CLSM. Secondly, C. albicans SC5314 cells (1 × 10 8 cells) were grown in the presence of 2 μl of DMSO or 40 μg myricetin per ml of YM broth at 35°C with shaking at 200 rpm for 2.5 h. The cells were harvested by centrifugation at 12,000 ×g for 1 min and stained with 10 μg/ml PI and 0.01% CFW in PBS. Then, they were observed using a fluorescence microscope equipped with triple RGB filters or a bright-field microscope.

Crystal Violet-Uptake Assay
To evaluate the effects of myricetin on membrane permeability, the crystal violet-uptake assay was performed according to the method described by Vaara and Vaara [31] with slight modifications. Log-phase C. albicans SC5314 cells were harvested by centrifugation at 12,000 ×g for 5 min, and then washed and resuspended in PBS. Cell suspensions (5 × 10 7 cells/ml) were treated with 1×, 2×, or 4× the MIC of myricetin and incubated at 35°C with shaking at 200 rpm for 30 and 60 min. Solvent (DMSO) controls for each treatment of myricetin were included. Then, the cell suspensions (0.9 ml) were harvested by centrifugation at 12,000 ×g for 5 min and washed in PBS. The cells were suspended in 1 ml of PBS containing 10 μg/ml crystal violet and incubated at 35°C with shaking at 200 rpm for 15 min. Further, the cells were precipitated by centrifugation at 12,000 ×g and 4°C for 20 min, and the supernatant (0.2 ml) was placed in quadruplicate into a 96-well flat-bottom microplate. The amount of crystal violet remaining in the supernatant was measured as the absorbance at 590 nm (A 590 ) using a spectrofluorometer (Tecan, Austria). The optical density values of the initial solution of crystal violet used in the assay were regarded as 100%. The percentage of crystal violet uptake was calculated using the following formula: uptake of crystal violet (%) = 1-A 590 of the sample/A 590 of crystal violet solution × 100.

Leakage of Intracellular Materials
An evaluation of myricetin-induced nucleotide and protein leakage was performed using a fluorometric and spectrophotometric method, respectively [32]. An overnight culture of C. albicans SC5314 cells was diluted by 1:5 into fresh YM broth and incubated further at 35°C with shaking at 200 rpm for 3 h. The cells were harvested by centrifugation at 12,000 ×g for 5 min, washed with PBS, and resuspended in PBS to achieve a cell density of 1 × 10 8 cells/ml. The cell suspensions were then incubated with 40 and 80 μg/ml myricetin at 35°C with agitation at 200 rpm for 30 or 60 min. DMSO controls for each myricetin treatment were included. The cell suspensions (0.8 ml) were centrifuged at 13,200 ×g at 4°C for 20 min, and the supernatants were saved for further analysis. For the nucleotide leakage analysis, the Qubit dsDNA BR assay kit and a Qubit 4 Fluorometer was used; this kit measures the levels of double-stranded DNA over RNA highly selectively. The supernatant (20 μl) were mixed with 180 μl of working solution in triplicate and the fluorescence of these mixtures was measured. The concentrations of the nucleotides in the samples were calculated using the dilution calculator feature of the Qubit 4 fluorometer. For protein leakage analysis, the Bradford assay [33] was performed according to the manufacturer's instructions. Diluted Bradford concentrate (150 μl) mixed with 50 μl of supernatant or PBS was added to a 96-well clear flat-bottom microplate in quadruplicate, and the absorbance of the samples at 590 nm was measured using a spectrofluorometer. The amount of protein leakage was calculated as the A 590 of the sample − the A 590 of Bradford solution containing PBS.

DPH-Binding Assay
To monitor whether myricetin affects the lipid components or order in the C. albicans cell membrane, the DPHbinding assay was performed [32]. C. albicans SC5314 cells (1 × 10 8 cells/ml) at the log phase were incubated with myricetin or DMSO at 35°C with shaking at 200 rpm for 30 min. The cells (0.9 ml) were then harvested by centrifugation at 12,000 ×g for 5 min, washed with PBS, and resuspended in 0.9 ml of PBS containing 50 μM DPH. The cell suspension (0.2 ml) was then transferred in quadruplicate to a 96-well black flat-bottom microplate, followed by incubation for 10 min in the dark at room temperature. The amount of DPH binding to the C. albicans cell membrane was measured using a spectrofluorometer (Tecan, Austria) at 360 nm (bandwidth, 35 nm) and 460 nm (bandwidth, 10 nm) as the excitation and emission wavelengths, respectively. The DPH-binding percentage was calculated using the following formula: relative DPH binding (%) = (F myricetin -F PBS containing DPH )/ (F DMSO control -F PBS containing DPH ) × 100; F represents the fluorescence intensity.

Nile Red-Binding Assay
The Nile red-binding assay was performed as follows: C. albicans SC5314 cells in the exponential growth phase were harvested by centrifugation at 12,000 ×g and the precipitate was suspended in PBS. The cell suspension (2 × 10 7 cells/ml) was exposed to myricetin (from 20 to 80 μg/ml) or an equivalent amount of DMSO at 35°C with agitation at 200 rpm for 1 h. Then, the cells (0.9 ml) were harvested by centrifugation at 12,000 ×g for 15 min, washed with PBS, and suspended in 0.9 ml of PBS containing 0.25 mg/ml Nile red solution. The cell suspension (0.2 ml) was transferred in quadruplicate to a 96-well black flat-bottom microplate, followed by incubation for 5 min in the dark at room temperature. The amount of Nile red binding to C. albicans cells was measured using a spectrofluorometer at 488 nm (with a bandwidth of 20 nm) and 580 nm (with a bandwidth of 20 nm) as the excitation and emission wavelengths, respectively. The Nile red-binding percentage was calculated using the following formula: relative Nile red binding (%) = (F myricetin -F PBS containing Nile red )/(F DMSO control -F PBS containing Nile red ) × 100; F represents the fluorescence intensity.

Statistical Analysis
All experiments were performed at least twice in triplicate or quadruplicate. For each outcome, the data were represented as mean ± standard deviation. The effect of myricetin compared with controls was analyzed using SigmaPlot 13.0. A p value less than 0.05 was regarded as statistically significant.

Antifungal Susceptibility Testing
Higher plants defend against pathogens with secondary metabolites or antimicrobial compounds including polyphenols, such as myricetin. The MIC of myricetin against C. albicans SC5314, a strain used for routine assays in several fungus-related studies, was 20 μg/ml. The MIC values of myricetin against C. glabrata ATCC 2001, C. krusei ATCC 6258, and C. parapsilosis ATCC 22019 were 1.3, 5, and 5 μg/ml, respectively (Table 1). In contrast, the MIC values of amphotericin B against the tested Candida species ranged from 0.5 to 1 μg/ml. Myricetin appears to have moderate anticandidal activity and the data agree reasonably with other researcher's MIC values of 16-64 μg/ml against C. albicans, 3.9 μg/ml against C. glabrata, 64 μg/ml against C. krusei, and 54 μg/ml against C. tropicalis [25]. The reason why the MIC value of myricetin is considerably higher (20 μg/ml) than that of amphotericin B (1 μg/ml) against C. albicans SC5314 is due to their differences in cellular targets and structures, although both amphotericin B and myricetin induce increased membrane permeability to result in cell death.

Sorbitol Protection Assay
The fungal cell wall surrounding cell membrane affords cells strength and rigidity and maintains osmotic support from the turgor pressure of protoplasts. Impairments in cell wall components by antifungals will result in cell lysis, but cells can survive in the presence of an appropriate osmotic protectant in the medium [29]. To examine whether the antifungal activity of myricetin is related to the alteration of the fungal cell wall structure, the sorbitol protection assay was performed using the CLSI M27-A3 microdilution assay with myricetin against C. albicans cells with or without 0.8 M sorbitol (Table 2). In the presence of sorbitol, the MIC values of myricetin against C. albicans increased from 20 to 40 and 80 μg/ml in 24 and 72 h, respectively. The increase in the MIC values in the sorbitol protection assay indicates that myricetin is involved in disrupting the integrity of the C. albicans cell wall.

Microscopic Analysis
PI can bind to DNA and RNA through compromised cell membranes, but it is mostly eliminated from live cells. Therefore, PI can enter dead or dying cells with defective cell membranes and emit a red fluorescence signal, while live cells with intact cell membranes are not stained with PI [34]. As can be seen in Fig. 1B2, CLSM images show the presence of red PI-stained myricetin-treated C. albicans cells, suggesting that the C. albicans cells showed an initial impairment of the cell membrane after treatment with 20 μg/ml myricetin for 2.5 h. Although cell lysis was not detectable in the cells treated with myricetin for 2.5 h, they were noticeable after myricetin treatment for 4 h, as indicated by arrows in Fig. 1C3. Amphotericin B-treated C. albicans cells, the positive controls, were seen as fluorescent red cells with an intact form (cell wall), indicating that amphotericin B is involved in damaging the C. albicans cell membrane (Fig. 1D2). Amphotericin B, which is a polyene macrolide, is reported to participate in the formation of protein-like ion channels in the cell membrane [35,36] or the disruption of the polar head group region of biomembranes [37]. Lysed cell debris (Fig. 1C3), a characteristic of cells with a damaged cell wall, is easily found in myricetin-treated C. albicans cells. Thus, the results of the sorbitol protection assay and confocal laser microscopic analysis demonstrated that myricetin disrupts the cell wall integrity and injures the C. albicans cell membrane.
In addition, C. albicans cells treated with DMSO or 40 μg/ml myricetin for 2.5 h were stained with both CFW and PI. CFW is a fluorescent dye that stains fungal cell walls, which are composed of cellulose, chitin, and other β-1,4-carbohydrates [38]. As seen in Fig. 2C, the control C. albicans cells showed fluorescent blue cell walls stained with CFW, and no significant red fluorescence was detected, demonstrating that the cells had intact cell walls and membranes. In contrast, red fluorescent aggregates were found in case of myricetin-treated C. albicans cells (Fig. 2D). Furthermore, these PI-stained cells looked atrophied and formed cell aggregates, as indicated by red arrows in a bright-field image (Fig. 2B). These aggregates or clumps were generally detected when C. albicans cells were exposed to relatively high myricetin concentrations, such as 2× or 4× the MIC of myricetin, or sublethal concentrations of myricetin for a long time (> 4 h). We assume that large membranous clumps and cell aggregates may be formed by membrane fusion and ionic interactions between protoplasts, respectively, in case of myricetintreated C. albicans cells because they have a compromised cell wall.

Crystal Violet-Uptake Assay
Crystal violet or gentian violet exists as a lipophilic cation at neutral pHs. Although it does not penetrate cells with intact cell membranes, crystal violet enters cells with damaged cell membranes. Hence, the crystal violetuptake assay is generally used for the detection of membrane impairment. As myricetin-treated C. albicans cells were seen as PI-stained fluorescent red cells, these were identified as membrane-damaged cells (Figs. 1 and 2). Therefore, the crystal violet-uptake assay was performed to ascertain whether myricetin affects the membrane permeability of C. albicans cells.
C. albicans cells treated with 1×, 2×, and 4× the MIC of myricetin or an equivalent amount of DMSO for 30 min were subjected to the crystal violet-uptake assay; the cell supernatants were placed in a 96-well microplate, as shown in Fig. 3A. There was a notable difference in color between the supernatants of cells treated with each concentration of myricetin supernatant and the supernatants of those treated with equivalent amounts of DMSO (control); this difference was concentration-dependent (Fig. 3A). The percentages of crystal violet uptake by C. albicans cells treated with 1×, 2×, and 4× the MIC of myricetin for 30 min were 36.5, 60.6, and 79.4%, while those by C. albicans cells treated with the corresponding amounts of DMSO were 28.2, 28.9, and 29.7%, respectively (Fig. 3B). The difference between each myricetin-treated sample and an equivalent DMSO control was statistically significant (p < 0.001), and these data clearly demonstrate that myricetin markedly increases the membrane permeability of C. albicans cells.

Leakage of Intracellular Materials
Since the notable enhancement of the membrane permeability in myricetin-treated C. albicans cells was displayed via the crystal violet-uptake assay, whether the treatment of C. albicans cells with myricetin causes the leakage of nucleotides and proteins was examined using the fluorometric and spectrophotometric method, respectively. C. albicans cells treated with DMSO or myricetin for 30 or 60 min were centrifuged and the supernatants were subjected to an analysis of the leakage of intracellular materials. As seen in Fig. 4A, DNA leakage levels of 0.139 and 0.241 μg/ml were found in case of the C. albicans cells treated with 2× and 4× the MIC of myricetin for 30 min, respectively, but a negligible amount and 0.104 μg/ml of DNA leakage were detected in the C. albicans cells treated with equivalent amounts of DMSO (0.2 and 0.4% DMSO), respectively. The difference observed between the DNA leakage levels in the myricetin-treated cells and the corresponding DMSO-treated controls was significant (p < 0.001).
For the protein leakage analysis, the A 590 was measured after Bradford reagent was mixed with the supernatant (Fig. 4B). The absorbance values at 590 nm at 30 min were 0.030 and 0.040 in case of the control cells treated with 2× and 4× the MIC of DMSO, respectively, but were 0.218 and 0.281 in case of the C. albicans cells treated with 2× and 4× the MIC of myricetin, respectively. The difference between the A 590 values of each myricetin-treated cell sample and the corresponding DMSO-treated control cell sample was also significant (p < 0.001). As revealed by the results of the crystal violet-uptake assay (Fig. 3) and the analysis of the leakage of intracellular materials (Fig. 4), the antifungal effects of myricetin against the membrane permeability of C. albicans cells were remarkable.

The Binding of DPH into C. albicans Cell Membranes
To keep the viability of C. albicans cells, maintaining the integrity of the cell membrane is critical. Cells can regulate membrane function through regulating membrane fluidity and membrane protein arrangement. Therefore, changes in membrane permeability are related to alterations in membrane fluidity, which occur via changes in the lipid composition or order or pore formation. Hence, whether the increase of membrane permeability is associated with the lipid composition or order of C. albicans cell membrane was investigated using the DPH-or Nile red-binding assay.
DPH is almost non-fluorescent in water, but it shows a strong fluorescence after intercalation into membranes. Therefore, it can be used as a probe for viscosity, polarity, and lipid order [39]. DMSO-or myricetin-treated C. albicans cells were incubated with PBS containing DPH, and the fluorescence intensity of each sample was measured. The relative percentages of DPH binding to C. albicans cells treated with 1×, 2×, and 4× the MIC of myricetin for 30 min were 82.8 ± 0.9, 73.2 ± 0.7, and 73.8 ± 0.5%, respectively, compared to the corresponding DMSO controls (Fig. 5). The difference between each myricetin-treated cell sample and the corresponding DMSO-treated control cells was significant (p < 0.001), and there was no significant difference between the DPH binding (%) of the cells treated with 2× and 4× the MIC of myricetin. Consequently, decreased DPH entry into myricetin-treated C. albicans cell membranes suggest that myricetin causes alterations of the lipid components or order of C. albicans cell membranes.

The Binding of Nile Red to C. albicans Cell Membranes
Nile red is a fluorescent probe that is used as a lipid stain to visualize bacterial cell membranes [40]. This dye displays low fluorescence in a polar environment, but selectively binds to lipids and emits strong fluorescence when incorporated into hydrophobic cell membranes [41,42]. The penetration depth and the orientation of Nile red may vary in membranes with different lipid compositions; this could affect its fluorescence-emission ratios [43]. Therefore, Nile red can be used for monitoring the organization, fluctuation, and heterogeneity in membranes, specifically for membranes containing cholesterol [41,44]. Accordingly, it was assumed that the  binding of Nile red to cells would be changed if myricetin influences the lipid composition or organization of the cell membrane in C. albicans cells. As shown in Table 3, the relative binding of Nile red to myricetin-treated C. albicans cells was drastically reduced. After treatment with 20, 40, and 80 μg/ml myricetin for 1 h, the relative percentages of Nile red binding to C. albicans cells were 72.0, 64.6, and 55.7%, respectively, compared with the case for the corresponding DMSO-treated control cells. Consequently, a significant reduction of Nile red binding after myricetin treatment suggests that myricetin is induces alterations in the lipid composition or arrangement, such as the loosening of the packing of the lipid bilayer in cell membrane, leading to the enhanced membrane fluidity of C. albicans cells. Interestingly, treatment with 0.1 to 0.4% DMSO increased the fluorescence intensity of C. albicans cells (Table 3), since DMSO is a stain carrier that helps Nile red penetrate through the cell wall and cell membrane in microorganisms [45]. Nile red is also used to visualize and quantify lipid droplets, especially, droplets of neutral lipids within cells in oleaginous microorganisms such as Candida spp. [42,46]. Oleaginous yeasts can accumulate lipids in the range of 20 to 70% of their biomass under appropriate cultivation conditions [47]. Therefore, the reduced binding of Nile red to C. albicans cells does not indicate the only changes in membrane fluidity, but the significant reduction of the binding of both DPH and Nile red to myricetin-treated C. albicans cells suggests that myricetin induces changes in lipid components, such as ergosterol, phospholipids, or sphingolipids, in the cell membrane of myricetin-treated C. albicans cells. Thus, the enhanced membrane permeability and notable reduction of the entry of DPH into, and the binding of Nile red to, myricetin-treated C. albicans cells imply that myricetin increases membrane fluidity by inhibiting the biosynthetic pathways or functions of lipid components in the cell membrane, leading to perturbations in the structure and function of the cell membrane. To clarify this hypothesis, further studies will be needed. In conclusion, myricetin acts as an antifungal against C. albicans through a combined action of membrane disturbance by enhancing membrane permeability and cell wall damage. Thus, the presence of dual targets of myricetin against C. albicans indicates its potential as a therapeutic agent to treat infections caused by Candida spp. C. albicans cells treated with myricetin or an equivalent amount of DMSO were incubated in PBS containing 0.25 mg/ml Nile red. The amount of Nile red binding to C. albicans cells was measured at 488 nm (bandwidth, 20 nm) and 580 nm (bandwidth, 20 nm), as the excitation and emission wavelengths, respectively, using a spectrofluorometer. The data represent the means ± standard deviations obtained from one of three independent experiments. AU: arbitrary units.