Floricolin C elicits intracellular reactive oxygen species accumulation and disrupts mitochondria to exert fungicidal action

Floricolin C elicits intracellular reactive oxygen species accumulation and disrupts mitochondria... Abstract Candida albicans, one of the most prevalent fungal pathogens, causes severe mucosal and invasive infections in predisposed individuals. The rise of fungal infection and drug resistance demands the development of novel antifungal agents. In this study, we observed that floricolin C (FC), a p-terphenyl pigment from an endolichenic fungus, killed C. albicans cells in the planktonic state or within biofilms through reactive oxygen species (ROS) accumulation. Further tests revealed that FC could directly damage the mitochondria to cause ROS accumulation. In addition, FC can quench thiol-based agents through a Michael reaction involving the α,β-unsaturated carbonyl group, whose effect may chelate intracellular thiol-based molecules or proteins in C. albicans, resulting in an imbalance in redox homeostasis. Increased ROS generation led to mitochondrial dysfunction, nuclear dispersion and consequently cell death. We further demonstrated that FC could prevent biofilm formation of other Candida species and eradicate their pre-formed biofilms. An in vivo study demonstrated that FC prolonged the survival of C. albicans-infected Caenorhabditis elegans. Taken together, our study provides the basis for the application of FC to combat Candida infections. Candida albicans, floricolin C, mitochondria, ROS, biofilms INTRODUCTION Candida albicans, a commensal fungus that inhabits the human gastrointestinal tract and skin, can cause severe mucosal infections as well as fatal invasive infections in predisposed individuals, including those suffering from immune deficiencies due to cancer chemotherapy or immunosuppression due to transplantation of solid organs or stem cells (Ganguly and Mitchell 2011, Rajendran et al.2015). During infection, C. albicans has a propensity to form biofilms on a variety of surfaces ranging from human tissues to implanted medical devices, such as intravascular catheters, urinary catheters, acrylic surfaces, heart valves and orthopaedic implants (Kojic and Darouiche 2004, Ramage, Martinez and Lopez-Ribot 2006; Ramage, Wickes and Lopez-Ribot 2007). Unfortunately, clinically important antifungal agents such as fluconazole, flucytosine, itraconazole, ketoconazole and miconazole are either ineffective in eradicating C. albicans biofilms or must be used in very high concentrations (Braga et al.2008; Monteiro et al.2011). Moreover, as the result of overuse, even abuse, of antifungal agents in the clinical setting, resistance has emerged (Müller et al.2000). The continued emergence of fungal infections and drug resistance requires the development of new effective antifungal agents. Natural products are a source of bioactive molecules that have been explored for novel antimicrobial/antifungal properties (Newman 2008). The chemical investigation of p-terphenyl pigments dates to 1877 (Liu 2006), and these compounds have been reported to possess a range of biological properties, including immunosuppressive, antioxidative, neuroprotective, antimicrobial and cytotoxic activities (Holzapfel, Kilpert and Steglich 1989; Yonezawa et al.1998; Kuhnert et al.2015). We previously reported that floricolin C (FC), a p-terphenyl pigment, exhibited potent antifungal activity against C. albicans via disruption of the cytoplasmic membrane (Li et al.2016). In this study, we observed that FC could directly damage the mitochondria to cause reactive oxygen species accumulation, which in turn led to mitochondrial dysfunction, nuclear dispersion and consequently cell death. MATERIAL AND METHODS Strains and cultures Candida albicans strains SC5314 and TDH3-GFP-CAI4 (Chang et al.2015), clinical isolates of C. albicans (CA10, CA137, 24D, 28I), C. krusei (CK1), C. tropicalis (CT2) and C. glabrata (CG1) were used in this study (Table 1). Candida albicans (24D, 28I) were kindly donated by Dr Q. Qingguo from the School of Stomatology in Shandong University, and other clinical isolates were kindly provided by the Shandong Provincial Qianfoshan Hospital. Cells from stocks stored at −80°C were routinely propagated on YPD agar plates (yeast extract 1%, peptone 2%, dextrose 2% and agar 2%) and incubated overnight at 30°C in YPD broth (yeast extract 1%, peptone 2% and dextrose 2%) before each experiment. The overnight fungal cells were harvested by centrifugation and washed twice in phosphate-buffered saline (PBS) before use. FC used in this study was previously isolated in our lab from the endolichenic fungus Floricola striata, and its chemical structure is shown in Fig. 1A. Table 1. The MIC80 values of FC against fluconazole-resistant C. albicans strains and other Candida species. Strain  MIC80 (μg/ml)  Wild type C. albicans  SC5314  8a    CA10  8  Fluconazole-resistant C. albicans  CA137  16    24D  8    28I  8    CT2  8  Other Candida species  CG1  16    CK1  16  Strain  MIC80 (μg/ml)  Wild type C. albicans  SC5314  8a    CA10  8  Fluconazole-resistant C. albicans  CA137  16    24D  8    28I  8    CT2  8  Other Candida species  CG1  16    CK1  16  aThe MIC80 was defined as the first well with an approximate 80% reduction in growth compared to the growth of the vehicle well. The MIC value has previously been reported in our publication (Li et al.2016). View Large Table 1. The MIC80 values of FC against fluconazole-resistant C. albicans strains and other Candida species. Strain  MIC80 (μg/ml)  Wild type C. albicans  SC5314  8a    CA10  8  Fluconazole-resistant C. albicans  CA137  16    24D  8    28I  8    CT2  8  Other Candida species  CG1  16    CK1  16  Strain  MIC80 (μg/ml)  Wild type C. albicans  SC5314  8a    CA10  8  Fluconazole-resistant C. albicans  CA137  16    24D  8    28I  8    CT2  8  Other Candida species  CG1  16    CK1  16  aThe MIC80 was defined as the first well with an approximate 80% reduction in growth compared to the growth of the vehicle well. The MIC value has previously been reported in our publication (Li et al.2016). View Large Figure 1. View largeDownload slide The effect of FC on intracellular ROS generation. (A) The structure of floricolin C (FC). (B, C) Yeast cells were exposed to increasing concentrations of FC for 6 h. After staining with 40 μg/ml 2΄,7΄-dichlorodihydrofluorescein diacetate (DCFHDA), the samples were detected by flow cytometry and visualized by CLSM. BF, bright field. The bars indicate 10 μm. (D) FC-treated C. albicans mature biofilms were stained with MitoSOX Red (20 μM) and visualized by CLSM. The bars indicate 20 μm. (E) Effect of thiourea on the fungicidal activity of FC. Candida albicans cells were treated with FC (8 μg/ml) for 3 h after pre-incubation with 5 mM thiourea for 2 h. The number of viable cells was determined by a colony-counting method. Figure 1. View largeDownload slide The effect of FC on intracellular ROS generation. (A) The structure of floricolin C (FC). (B, C) Yeast cells were exposed to increasing concentrations of FC for 6 h. After staining with 40 μg/ml 2΄,7΄-dichlorodihydrofluorescein diacetate (DCFHDA), the samples were detected by flow cytometry and visualized by CLSM. BF, bright field. The bars indicate 10 μm. (D) FC-treated C. albicans mature biofilms were stained with MitoSOX Red (20 μM) and visualized by CLSM. The bars indicate 20 μm. (E) Effect of thiourea on the fungicidal activity of FC. Candida albicans cells were treated with FC (8 μg/ml) for 3 h after pre-incubation with 5 mM thiourea for 2 h. The number of viable cells was determined by a colony-counting method. Reactive oxygen species accumulation The accumulation of intracellular reactive oxygen species (ROS) in C. albicans was determined using 2΄,7΄-dichlorodihydro fluorescein diacetate (Sigma-Aldrich) (Li et al.2015) or MitoSOX Red (Invitrogen). 2΄,7΄-Dichlorodihydro fluorescein diacetate is a fluorogenic dye that moves freely across the cell membrane and reacts with ROS inside the cell to form the highly fluorescent compound dichlorofluorescein. Briefly, yeast cells were incubated with 2΄,7΄-dichlorodihydrofluorescein diacetate (40 μg/ml) in the dark for 30 min after incubated with FC for 6 h. The cells were then washed twice with PBS and the fluorescence intensity of stained cells was measured by a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA, USA) and visualized by confocal laser scanning microscopy (CLSM). MitoSOX Red accumulates in the mitochondrial matrix and is oxidized to a fluorescent product by superoxide. Cells within FC-treated mature biofilm were stained with MitoSOX Red (20 μM) for 30 min, followed by visualization by CLSM to assess the oxidative state of cells within biofilms. Mitochondrial isolation and ROS assay Mitochondria were isolated from C. albicans spheroplasts, essentially as previously described (Yu et al.2016). Briefly, overnight cultured C. albicans were washed and re-suspended with buffer A (50 mM K2HPO4, 50 mM KH2PO4, 5 mM EDTA, 50 mM dithiothreitol, 40 mM β-mercaptoethanol, 2 M sorbitol, pH 7.2) containing 5 μl of zymolyase (2000 U/mL, Zymo Research, USA). The mixture was incubated at 30°C for 90 min. Cells were collected by centrifugation at 1000 g and washed twice with buffer B (0.6 M mannitol, 0.2% BSA, 10 mM imidazole) to obtain the yeast protoplasts. The obtained protoplasts were then homogenized for 20–50 strokes with a Dounce homogenizer. Cell lysates were centrifuged at 4°C at 1000 g to remove the nuclei and intact cells. The supernatant was then centrifuged at 10 000 g, obtaining the yeast mitochondria. Mitochondria were diluted to an OD620 of 1.0 in buffer B containing 0.2 M succinate. A dilution series of FC was added to the suspensions to a final concentration of 0, 4, 8, 16 and 32 μg/ml and MitoSOX was added at 20 μM. After incubation of 6 h, the fluorescence intensity was determined by a spectrofluorophotometer (excitation wavelength 520 nm, emission wavelength 600 nm). Analysis of mitochondrial membrane potential Changes in mitochondrial membrane potential (mtΔψ) were detected with rhodamine 123 (Rh123) (Chang et al.2011). SC5314 cells (1 × 106 cells/ml in SD medium) were incubated with various doses of FC at 30°C for 6 h. Rh123 was added at a final concentration of 5 μM, and the mixture was incubated for 30 min in the dark. Fluorescence intensities were recorded with a flow cytometer. ATP luciferase assay Intracellular ATP was measured according to a previously described method (Chang et al.2011). FC-treated C. albicans SC5314 cells were disrupted by vortexing with glass beads and centrifuged at 13523 g for 10 min. The ATP content in the cell lysis was determined using an ATP analysis kit (Beyotime, Haimen, China). Cytochrome c release from mitochondria The measurement of cytochrome c (Cyt c) release from mitochondria was performed as previously described (Wu et al.2010). Specifically, C. albicans cell suspension (5 × 106 cells/ml) was incubated with 16 μg/ml of FC for 12 h, and collected at 5000 g for 5 min. The cell pellets were resuspended in homogenization medium (50 mM Tris (pH 7.5), 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) and disrupted by vortexing with glass beads. The supernatants were collected at 2348 g for 10 min to remove the cell debris and unbroken cells. To estimate the levels of cytosolic and mitochondrial Cyt c, the supernatant was centrifuged at 30 000 g for 45 min. The supernatants were collected for assay of Cyt c released from mitochondria into the cytoplasm. The pellet was suspended in buffer (50 mM Tris, pH 5.0, 2 mM EDTA), incubated for 5 min, and centrifuged at 5000 g for 30 s. The pellet was collected as mitochondria for determination of Cyt c remaining in mitochondria. After being reduced by ascorbic acid at room temperature for 5 min, the relative quantities of the reduced Cyt c were assessed using a spectrophotometer at 550 nm. The protein content was quantified using BSA as a standard. Synthesis of FC–N-acetylcysteine and FC–reduced glutathione A 0.5 mg quantity of N-acetylcysteine (NAC) or reduced glutathione (GSH) in 0.5 ml of water was added to a solution of FC (0.5 mg) in 0.5 ml of methanol. The mixture was stirred for 0.5 h at room temperature to give the target compounds, which were determined by liquid chromatography–mass spectrometry (LC-MS). Effect of thiourea or N-acetylcysteine on the antifungal activity of FC Candida albicans cells (1 × 105 cells/ml) were pre-incubated with 5 mM thiourea or NAC for 2 h before treatment with FC (8 μg/ml) for 3 h. The number of viable cells was determined by a colony-counting method. Intracellular glutathione analysis Intracellular glutathione was monitored by the production of the fluorescent adduct glutathione-bimane from monochlorobimane and GSH catalyzed by glutathione- S-transferase (Katey et al.2005). Candida albicans cells were stained with 50 μM monochlorobimane after exposure to different concentrations of FC for 3 h. After incubation for 30 min, the stained cells were quantified in a spectrofluorophotometer at 385 nm excitation and 478 nm emission. Nuclear morphology assay Nuclear damage was analyzed by 4΄,6-diamidino-2-phenylindole staining. Candida albicans cells were treated with FC or vehicle control. After 12 h, cells were collected, washed twice with PBS, and resuspended in 70% ethanol for brief fixation and permeability (Wu et al.2010). After 10 min fixation, cells were washed with PBS and incubated with 10 μg/ml of 4΄,6-diamidino-2-phenylindole in the dark for 20 min. The stained cells were then observed with CLSM. The apoptosis and necrosis of C. albicans induced by FC Overnight-incubated cells were treated by FC (0, 16 and 32 μg/ml) for 12 h and the following treatments were performed as previously described in our lab (Chang et al.2011). Protoplasts were washed with PBS three times and incubated with 5 μl of Annexin V–fluorescein isothiocyanate (FITC) and 5 μl of propidium iodide (PI) for 20 min in the dark. Cells were then observed by CLSM using a ×63 objective. Effects of FC on biofilm formation Candida biofilms were developed on the surface of 96-well polystyrene plates and quantified using a 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay as described previously (Zhang et al.2017). Briefly, standard cell suspensions (100 μl) of Candida cells (1 × 106 cells/ml) containing different concentrations of FC (ranging from 0 to 32 μg/ml) were seeded in selected wells. After 24 h of incubation at 37°C, the biofilms were washed twice with PBS, and the metabolic activity of the biofilm cells was semi-quantified using a standard XTT reduction assay, which measures the activity of mitochondrial dehydrogenase (Chandra, Mukherjee and Ghannoum 2008). In addition, the biofilms were qualitatively inspected using an Olympus microscope. Effects of FC on pre-formed Candida biofilms Candida cells were seeded in 96-well polystyrene plates at an initial concentration of 1 × 106 cells/ml and grown in RPMI 1640 medium at 37°C for 48 h to form mature biofilms (Ramage et al.2002). The supernatants were discarded, and wells were washed with PBS to remove unbound cells. Fresh medium containing different concentrations of FC (ranging from 0 to 64 μg/ml) was then added to the wells, and the plates were incubated for another 24 h at 37°C. The effects of FC on the pre-formed biofilms were assessed using the XTT reduction assay as described previously. Candida albicans TDH3-GFP-CAI4 was used to visualize live/dead cells in pre-formed biofilms. The mature biofilms were treated with FC, followed by PI (5 μg/ml) staining for 45 min. The stained biofilms were imaged by CLSM. In this assay, dead cells displayed red fluorescence due to intracellular accumulation of PI. Antifungal activity of FC in Caenorhabditis elegans–C. albicans infection model A Caenorhabditis elegans–Candida albicans infection assay was performed as previously described (Sun, Liao and Wang 2015). Caenorhabditis elegans was exposed to C. albicans for 2 h and washed three times with sterile M9 buffer. Then the infected worms were challenged with different concentrations of FC. The infection was started by adding 20–25 animals to each well of a 96-well plate at 25°C, and scoring for dead or alive every 24 h. After 6 days of incubation, the wells were washed and visualized by an Olympus microscope with a ×4 objective lens. Statistical analysis In the Caenorhabditis elegans–Candida albicans infection assay, data were statistically analyzed using the log rank test. The other experimental data were statistically analyzed using Student's t-test. P < 0.05 was considered significant. RESULTS AND DISCUSSION Effect of FC on intracellular ROS generation In our previous study, FC was observed to disrupt the cytoplasmic membrane and exert a fungicidal action against C. albicans (Li et al.2016). In this study, we found FC also induced ROS accumulation in C. albicans cells. Flow cytometry analysis revealed that FC induced ROS generation in a dose-dependent manner in planktonic cells (Fig. 1B). This finding was further corroborated by CLSM observation (Fig. 1C). In addition, ROS accumulation, as indicated by MitoSOX Red fluorescence, was observed in FC-treated C. albicans mature biofilms (Fig. 1D). To determine whether increased ROS contributes to FC-induced cellular death, we explored the scavenging effect of the antioxidant thiourea. Addition of thiourea increased the survival percentage compared with the group treated with FC alone (Fig. 1E), suggesting that ROS accumulation is an important factor in FC-mediated cell death. Effect of FC on ROS accumulation in isolated mitochondria ROS are byproducts of cellular metabolism primarily generated in the mitochondria (Kowaltowski et al.2009). Thus, we speculated that FC might cause damage to mitochondria to increase ROS generation. We assessed the ROS levels in isolated mitochondria of C. albicans under FC treatment using MitoSOX Red as an indicator. The fluorescence intensity increased in FC-treated mitochondria compared with vehicle-treated mitochondria, suggesting that FC could directly cause ROS accumulation in isolated mitochondria (Fig. 2). Figure 2. View largeDownload slide The effect of FC on ROS generation in isolated mitochondria. The isolated mitochondria from SC5314 cells were treated with various doses of FC and stained with MitoSOX. The fluorescence intensity was determined by a spectrofluorophotometer (excitation wavelength 520 nm, emission wavelength 600 nm). **P < 0.01, ***P < 0.001. Figure 2. View largeDownload slide The effect of FC on ROS generation in isolated mitochondria. The isolated mitochondria from SC5314 cells were treated with various doses of FC and stained with MitoSOX. The fluorescence intensity was determined by a spectrofluorophotometer (excitation wavelength 520 nm, emission wavelength 600 nm). **P < 0.01, ***P < 0.001. Effect of FC on mitochondrial function in C. albicans cells Given the disruptive effect of FC on isolated mitochondria, we next assessed the effects of FC on mitochondria in C. albicans cells. The activity of mitochondrial proton pumps and electrogenic transport systems and the activation of the mitochondrial permeability transition can be assessed using mtΔψ, an indicator of the energetic state of the mitochondria (Li et al.2015). A low dose of FC significantly increased the fraction of cells with high fluorescence intensity (Fig. 3A). However, the fluorescence decreased when cells were treated with high dose of FC. The geometric mean (GMean) value was utilized to reflect the change of fluorescence intensity. As illustrated in Fig. 3B, the GMean value increased from 35.07 (control) to 98.36 and 172.72 at 4 and 8 μg/ml, respectively when exposed to FC. However, when the dose of FC was increased to 32 and 64 μg/ml, the GMean value decreased to 17.48 and 7.03, respectively. Intracellular ATP content, as another representative of mitochondrial function (Yu et al.2016), was measured in the presence of FC. The levels of ATP varied according to the change in mtΔψ (Fig. 3C). This suggested that C. albicans cells tried to increase mtΔψ and ATP generation to counteract the stress from the low dose of FC. However, a high dose of FC fatally hit the cells and disrupted the mitochondria. Figure 3. View largeDownload slide The dysfunction of mitochondria when C. albicans cells were treated with FC. (A, B) Candida albicans cells were cultured in SD medium with FC for 6 h. Cells were then stained with Rh123, and the fluorescence intensity was detected by flow cytometry. (C) Candida albicans was treated with FC at the indicated concentrations, followed by cell lysis and measurement of the intracellular ATP content. The bars represent the means ± SD of three independent experiments. ***P < 0.001. (D) Release of Cyt c from mitochondria to cytosol. The FC-treated cells were lysed to extract Cyt c, and the Cyt c contents of mitochondria and cytosol were analyzed by measuring absorbance at 550 nm with a spectrophotometer. Figure 3. View largeDownload slide The dysfunction of mitochondria when C. albicans cells were treated with FC. (A, B) Candida albicans cells were cultured in SD medium with FC for 6 h. Cells were then stained with Rh123, and the fluorescence intensity was detected by flow cytometry. (C) Candida albicans was treated with FC at the indicated concentrations, followed by cell lysis and measurement of the intracellular ATP content. The bars represent the means ± SD of three independent experiments. ***P < 0.001. (D) Release of Cyt c from mitochondria to cytosol. The FC-treated cells were lysed to extract Cyt c, and the Cyt c contents of mitochondria and cytosol were analyzed by measuring absorbance at 550 nm with a spectrophotometer. Cyt c is tightly bound to the inner mitochondrial membrane by its electrostatic interactions with acidic phospholipids, but it can be released to the cytosol when the mitochondria are damaged. (Yun et al.2016). Compared with control cells, the relative level of Cyt c in the mitochondria was decreased under FC treatment, and that in the cytosol was significantly increased (Fig. 3D), indicating that FC induced the release of Cyt c from mitochondria in C. albicans. Based on above results, we concluded that FC could directly disrupt the mitochondria and result in ROS accumulation when entering the cells. FC can chelate thiol-based agents through its α,β-unsaturated carbonyl moiety We analyzed the chemical structure of FC, and noticed the presence of an α,β-unsaturated carbonyl in the structure, which is considered to react with a mercapto moiety via the Michael reaction. To confirm this conjugate formation, FC was mixed with NAC or GSH, which both possess a mercapto moiety. The generated products were then analyzed by LC-MS. FC levels decreased when NAC or GSH was added, concomitant with the formation of a new product with a relative molecular mass of 469 (FC–NAC) or 613 (FC–GSH) (Fig. 4A and B). At the cellular level, NAC dramatically diminished the activity of FC against C. albicans (data not shown), which is further evidence for the occurrence of Michael reaction between FC and NAC. Figure 4. View largeDownload slide FC chelated thiol-based agents through its α,β-unsaturated carbonyl moiety. (A, B) The chemical reaction between FC and NAC (A) or GSH (B). (C) FC resulted in decreased intracellular GSH content. FC-treated C. albicans cells were stained with 50 μM monochlorobimane. The resultant fluorescent adduct, glutathione-bimane, was detected by a spectrofluorophotometer to indicate the decrease of intracellular GSH content. (D) The outlined reaction between FC and thiol-based agents. Figure 4. View largeDownload slide FC chelated thiol-based agents through its α,β-unsaturated carbonyl moiety. (A, B) The chemical reaction between FC and NAC (A) or GSH (B). (C) FC resulted in decreased intracellular GSH content. FC-treated C. albicans cells were stained with 50 μM monochlorobimane. The resultant fluorescent adduct, glutathione-bimane, was detected by a spectrofluorophotometer to indicate the decrease of intracellular GSH content. (D) The outlined reaction between FC and thiol-based agents. GSH, a thiol-based intracellular antioxidant, is a major ROS scavenging agent in cells (González-Párraga et al.2005). To determine if the Michael reaction occurs in C. albicans cells, the intracellular GSH content was measured in the presence of FC. FC at a dose of 8 μg/ml resulted in a decrease in GSH content, although the decrease was not as obvious as expected (Fig. 4C). Moreover, we speculate FC could also react with other thiol-based molecules or proteins (Fig. 4D), and these chelating effects altogether might damage redox homeostasis and result in the dysfunction of key proteins. The apoptosis and necrosis of C. albicans induced by FC If the production of ROS overwhelms the antioxidant capacity of the cell, oxidative stress occurs and causes cell damage (Perrone, Tan and Dawes 2008). Nuclear morphological changes, including chromosome condensation and fragmentation, indicate cell damage (Kapuscinski 1995). We next utilized 4΄,6-diamidino-2-phenylindole staining to visualize the nuclear morphology when C. albicans cells were treated with FC. In non-treated cells, chromatin appeared as a single round spot in the cells, the normal appearance (Fig. 5A). In contrast, 4΄,6-diamidino-2-phenylindole dye was dispersed in the whole cell in FC-treated cells (Fig. 5A), indicating the disruptive effect of FC on nuclear membrane or nuclear structure. The Annexin V–FITC Kit was applied to determine the apoptosis and necrosis induced by FC. The results showed that the majority of stained cells (under treatment with 32 μg/ml FC) were penetrated by PI rather than annexin V–FITC, suggesting that the 32 μg/ml FC-treated C. albicans cells mainly undergo necrosis rather than apoptosis (Fig. 5B). However, the low dose of FC induced more apoptotic cells than necrotic ones, suggesting that FC causes both apoptosis and necrosis of C. albicans cells. Figure 5. View largeDownload slide Candida albicans cell death induced by FC. (A) The nuclear damage of C. albicans cells in response to FC treatment. FC-treated cells were fixed and stained with 10 μg/ml of 4΄,6-diamidino-2-phenylindole. The stained cells were then observed with CLSM. The bars indicate 5 μm. (B) The apoptosis and necrosis of C. albicans induced by FC. Cells were stained by Annexin V–FITC or PI to indicate the characteristics of apoptosis or necrosis under treatment with FC. The stained cells were observed with CLSM. BF, bright field. The bars indicate 10 μm. Figure 5. View largeDownload slide Candida albicans cell death induced by FC. (A) The nuclear damage of C. albicans cells in response to FC treatment. FC-treated cells were fixed and stained with 10 μg/ml of 4΄,6-diamidino-2-phenylindole. The stained cells were then observed with CLSM. The bars indicate 5 μm. (B) The apoptosis and necrosis of C. albicans induced by FC. Cells were stained by Annexin V–FITC or PI to indicate the characteristics of apoptosis or necrosis under treatment with FC. The stained cells were observed with CLSM. BF, bright field. The bars indicate 10 μm. FC inhibits C. albicans biofilm formation in vitro Candida albicans biofilm formation on implanted medical devices is increasingly recognized as a key mediator of fungal infections in the host. The formation of biofilms is associated with antifungal resistance and increased pathogenicity (Rosseti, Chagas and Costa 2014). To investigate whether FC has an effect on C. albicans biofilm formation, XTT reduction assays were performed. The results demonstrated that FC reduced biofilm formation in a dose-dependent manner (Fig. 6A). FC at its minimal inhibitory concentration (MIC) reduced the metabolic activity of C. albicans biofilm formation by greater than 50%. Amphotericin B at the dose of 2 μg/ml was used as a positive control. Microscopy revealed that FC treatment resulted in a defect of biofilm formation, with only a few filamentous cells on the substratum at a dose of 8 μg/ml or greater (Fig. 6B). From the inspection of cell morphology, we noticed that FC had little effect on inhibiting the yeast-to-hyphae transition. The inhibition of biofilm formation was attributable to its fungicidal activity. Figure 6. View largeDownload slide The activity of FC against C. albicans biofilms. (A, B) The inhibitory effect of FC on biofilm formation was measured using XTT reduction assay and observed by microscopy. The bars indicate 50 μm. (C) The eradicating effect of FC on mature biofilms of C. albicans was assessed by XTT reduction assay. (D) Candida albicans TDH3-GFP-CAI4 mature biofilms were treated with FC for 24 h and stained with PI for CLSM observation. The bars indicate 50 μm. **P < 0.01, ***P < 0.001. Figure 6. View largeDownload slide The activity of FC against C. albicans biofilms. (A, B) The inhibitory effect of FC on biofilm formation was measured using XTT reduction assay and observed by microscopy. The bars indicate 50 μm. (C) The eradicating effect of FC on mature biofilms of C. albicans was assessed by XTT reduction assay. (D) Candida albicans TDH3-GFP-CAI4 mature biofilms were treated with FC for 24 h and stained with PI for CLSM observation. The bars indicate 50 μm. **P < 0.01, ***P < 0.001. FC exhibits fungicidal activity against mature biofilms Mature biofilms are much more resistance to antimicrobial agents and host immune factors than planktonic cells (Finkel and Mitchell 2011). Notably, FC also potently eradicated pre-formed biofilms in a dose-dependent manner, as detected by the XTT reduction assay (Fig. 6C). Compared with the drug-free group, 16 μg/ml FC significantly decreased cell survival in mature biofilms (P < 0.05). When the dose was increased to 32 or 64 μg/ml, FC treatment resulted in greater than 60% killing in mature biofilms. This effect was confirmed by CLSM (Fig. 6D). Collectively, FC exhibited a significant anti-biofilm effect against both developing biofilms and mature biofilms. Antifungal effect of FC on fluconazole-resistant C. albicans and other non-albicans Candida species We examined the susceptibility of four clinical fluconazole-resistant C. albicans strains to FC using the broth microdilution method following the Clinical and Laboratory Standards Institute's M27-A3 guidelines (CLSI 2008). The MICs against these four fluconazole-resistant strains were similar to that against wild type strain SC5314 (Table 1) (Li et al.2016), suggesting the application of FC in treating azole-resistant C. albicans caused infections. In addition to C. albicans, other clinically derived Candida species such as C. krusei (CK1), C. tropicalis (CT2) and C. glabrata (CG1) were also tested for evidence of antifungal activity of FC. FC exhibited potent antifungal activity against these three tested Candida species with its MICs ranging from 8 to 16 μg/ml (Table 1). We further evaluated the activity of FC against the biofilms of other Candida species. The data showed that FC could prevent biofilm formation as well as eradicate pre-formed biofilms of other Candida species at a concentration of 32 μg/ml (Fig. 7). Figure 7. View largeDownload slide The antifungal effect of FC on three non-albicans Candida species. (A) The inhibitory effect of FC on biofilm formation of tested Candida strains. After incubation with 32 μg/ml of FC for 24 h, the biofilms were washed twice with PBS, and assessed by XTT reduction assay. (B) The eradicating activity of FC on tested Candida mature biofilms. Candida cells were incubated at 37°C for 48 h to form mature biofilms. Then the mature biofilms were challenged with 32 μg/ml of FC for another 24 h, and assessed by XTT reduction assay. Figure 7. View largeDownload slide The antifungal effect of FC on three non-albicans Candida species. (A) The inhibitory effect of FC on biofilm formation of tested Candida strains. After incubation with 32 μg/ml of FC for 24 h, the biofilms were washed twice with PBS, and assessed by XTT reduction assay. (B) The eradicating activity of FC on tested Candida mature biofilms. Candida cells were incubated at 37°C for 48 h to form mature biofilms. Then the mature biofilms were challenged with 32 μg/ml of FC for another 24 h, and assessed by XTT reduction assay. FC improves the survival of C. albicans-infected nematodes We further investigated the in vivo antifungal activity of FC using the Caenorhabditis elegans–Candida albicans infection model. Results showed that FC could significantly prolong the survival of C. albicans-infected Caenorhabditis elegans at 8 μg/ml (P = 0.008), 16 μg/ml (P < 0.001), and 32 μg/ml (P = 0.002) compared with the control group (Fig. 8A). The toxicity test demonstrated that 64 μg/ml FC had little effect on the survival of non-infected Caenorhabditis elegans within 6 days (Fig. 8B). These data suggested a potential application of FC in treating C. albicans infection in vivo. Figure 8. View largeDownload slide FC improves the survival of Candida albicans-infected Caenorhabditis elegans. (A) Worms infected by C. albicans SC5314 were treated with indicated concentrations of FC. The survival curves were plotted based on the survival state of each worm, which was monitored each day. The data were statistically analyzed using Log Rank tests. **P < 0.01, ***P < 0.001. (B) Healthy worms were incubated with or without 64 μg/ml of FC for 6 days and imaged using an Olympus microscope to show the toxicity of FC on worms. Figure 8. View largeDownload slide FC improves the survival of Candida albicans-infected Caenorhabditis elegans. (A) Worms infected by C. albicans SC5314 were treated with indicated concentrations of FC. The survival curves were plotted based on the survival state of each worm, which was monitored each day. The data were statistically analyzed using Log Rank tests. **P < 0.01, ***P < 0.001. (B) Healthy worms were incubated with or without 64 μg/ml of FC for 6 days and imaged using an Olympus microscope to show the toxicity of FC on worms. Taken together, we have demonstrated that FC has promising fungicidal activity against pathogenic Candida species both in the planktonic state and in biofilms, thus expanding current potential antifungal agents for combatting fungal infections. Further research demonstrated that FC could directly damage mitochondria to increase ROS accumulation. In addition, the α,β-unsaturated carbonyl of FC can react with GSH or thiol-based proteins through the Michael reaction, resulting in an imbalance of intracellular redox homeostasis and ROS accumulation. The accumulated ROS ultimately induce mitochondria dysfunction, nuclear fragmentation and, consequently, cell death. Acknowledgements This work was financially supported by the State Key Program of the National Natural Science Foundation (no. 81630093) and the National Natural Science Foundation (nos. 81402804 and 81273383). AUTHOR CONTRIBUTIONS Conceived and designed the experiments: HXL, MZ and WQC. Performed the experiments: MZ, HZS, SZ, YL and WL. Analyzed the data: MZ. Contributed reagents/materials/analysis tools: HXL. Wrote the paper: MZ, WQC and HXL. Conflict of interest. None declared. REFERENCES Braga PC, Culici M, Alfieri M et al.   Thymol inhibits Candida albicans biofilm formation and mature biofilm. Int J Antimicrob Agents  2008; 31: 472– 7. Google Scholar CrossRef Search ADS PubMed  Chandra J, Mukherjee PK, Ghannoum MA. In vitro growth and analysis of Candida biofilms. Nat Protoc  2008; 3: 1909– 24. Google Scholar CrossRef Search ADS PubMed  Chang WQ, Wu XZ, Cheng AX et al.   Retigeric acid B exerts antifungal effect through enhanced reactive oxygen species and decreased cAMP. 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Silibinin triggers yeast apoptosis related to mitochondrial Ca2+ influx in Candida albicans. Int J Biochem Cell Biol  2016; 80: 1– 9. Google Scholar CrossRef Search ADS PubMed  Zhang M, Chang W, Shi H et al.   Biatriosporin D displays anti-virulence activity through decreasing the intracellular cAMP levels. Toxicol Appl Pharmacol  2017; 322: 104– 12. Google Scholar CrossRef Search ADS PubMed  © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Yeast Research Oxford University Press

Floricolin C elicits intracellular reactive oxygen species accumulation and disrupts mitochondria to exert fungicidal action

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Abstract

Abstract Candida albicans, one of the most prevalent fungal pathogens, causes severe mucosal and invasive infections in predisposed individuals. The rise of fungal infection and drug resistance demands the development of novel antifungal agents. In this study, we observed that floricolin C (FC), a p-terphenyl pigment from an endolichenic fungus, killed C. albicans cells in the planktonic state or within biofilms through reactive oxygen species (ROS) accumulation. Further tests revealed that FC could directly damage the mitochondria to cause ROS accumulation. In addition, FC can quench thiol-based agents through a Michael reaction involving the α,β-unsaturated carbonyl group, whose effect may chelate intracellular thiol-based molecules or proteins in C. albicans, resulting in an imbalance in redox homeostasis. Increased ROS generation led to mitochondrial dysfunction, nuclear dispersion and consequently cell death. We further demonstrated that FC could prevent biofilm formation of other Candida species and eradicate their pre-formed biofilms. An in vivo study demonstrated that FC prolonged the survival of C. albicans-infected Caenorhabditis elegans. Taken together, our study provides the basis for the application of FC to combat Candida infections. Candida albicans, floricolin C, mitochondria, ROS, biofilms INTRODUCTION Candida albicans, a commensal fungus that inhabits the human gastrointestinal tract and skin, can cause severe mucosal infections as well as fatal invasive infections in predisposed individuals, including those suffering from immune deficiencies due to cancer chemotherapy or immunosuppression due to transplantation of solid organs or stem cells (Ganguly and Mitchell 2011, Rajendran et al.2015). During infection, C. albicans has a propensity to form biofilms on a variety of surfaces ranging from human tissues to implanted medical devices, such as intravascular catheters, urinary catheters, acrylic surfaces, heart valves and orthopaedic implants (Kojic and Darouiche 2004, Ramage, Martinez and Lopez-Ribot 2006; Ramage, Wickes and Lopez-Ribot 2007). Unfortunately, clinically important antifungal agents such as fluconazole, flucytosine, itraconazole, ketoconazole and miconazole are either ineffective in eradicating C. albicans biofilms or must be used in very high concentrations (Braga et al.2008; Monteiro et al.2011). Moreover, as the result of overuse, even abuse, of antifungal agents in the clinical setting, resistance has emerged (Müller et al.2000). The continued emergence of fungal infections and drug resistance requires the development of new effective antifungal agents. Natural products are a source of bioactive molecules that have been explored for novel antimicrobial/antifungal properties (Newman 2008). The chemical investigation of p-terphenyl pigments dates to 1877 (Liu 2006), and these compounds have been reported to possess a range of biological properties, including immunosuppressive, antioxidative, neuroprotective, antimicrobial and cytotoxic activities (Holzapfel, Kilpert and Steglich 1989; Yonezawa et al.1998; Kuhnert et al.2015). We previously reported that floricolin C (FC), a p-terphenyl pigment, exhibited potent antifungal activity against C. albicans via disruption of the cytoplasmic membrane (Li et al.2016). In this study, we observed that FC could directly damage the mitochondria to cause reactive oxygen species accumulation, which in turn led to mitochondrial dysfunction, nuclear dispersion and consequently cell death. MATERIAL AND METHODS Strains and cultures Candida albicans strains SC5314 and TDH3-GFP-CAI4 (Chang et al.2015), clinical isolates of C. albicans (CA10, CA137, 24D, 28I), C. krusei (CK1), C. tropicalis (CT2) and C. glabrata (CG1) were used in this study (Table 1). Candida albicans (24D, 28I) were kindly donated by Dr Q. Qingguo from the School of Stomatology in Shandong University, and other clinical isolates were kindly provided by the Shandong Provincial Qianfoshan Hospital. Cells from stocks stored at −80°C were routinely propagated on YPD agar plates (yeast extract 1%, peptone 2%, dextrose 2% and agar 2%) and incubated overnight at 30°C in YPD broth (yeast extract 1%, peptone 2% and dextrose 2%) before each experiment. The overnight fungal cells were harvested by centrifugation and washed twice in phosphate-buffered saline (PBS) before use. FC used in this study was previously isolated in our lab from the endolichenic fungus Floricola striata, and its chemical structure is shown in Fig. 1A. Table 1. The MIC80 values of FC against fluconazole-resistant C. albicans strains and other Candida species. Strain  MIC80 (μg/ml)  Wild type C. albicans  SC5314  8a    CA10  8  Fluconazole-resistant C. albicans  CA137  16    24D  8    28I  8    CT2  8  Other Candida species  CG1  16    CK1  16  Strain  MIC80 (μg/ml)  Wild type C. albicans  SC5314  8a    CA10  8  Fluconazole-resistant C. albicans  CA137  16    24D  8    28I  8    CT2  8  Other Candida species  CG1  16    CK1  16  aThe MIC80 was defined as the first well with an approximate 80% reduction in growth compared to the growth of the vehicle well. The MIC value has previously been reported in our publication (Li et al.2016). View Large Table 1. The MIC80 values of FC against fluconazole-resistant C. albicans strains and other Candida species. Strain  MIC80 (μg/ml)  Wild type C. albicans  SC5314  8a    CA10  8  Fluconazole-resistant C. albicans  CA137  16    24D  8    28I  8    CT2  8  Other Candida species  CG1  16    CK1  16  Strain  MIC80 (μg/ml)  Wild type C. albicans  SC5314  8a    CA10  8  Fluconazole-resistant C. albicans  CA137  16    24D  8    28I  8    CT2  8  Other Candida species  CG1  16    CK1  16  aThe MIC80 was defined as the first well with an approximate 80% reduction in growth compared to the growth of the vehicle well. The MIC value has previously been reported in our publication (Li et al.2016). View Large Figure 1. View largeDownload slide The effect of FC on intracellular ROS generation. (A) The structure of floricolin C (FC). (B, C) Yeast cells were exposed to increasing concentrations of FC for 6 h. After staining with 40 μg/ml 2΄,7΄-dichlorodihydrofluorescein diacetate (DCFHDA), the samples were detected by flow cytometry and visualized by CLSM. BF, bright field. The bars indicate 10 μm. (D) FC-treated C. albicans mature biofilms were stained with MitoSOX Red (20 μM) and visualized by CLSM. The bars indicate 20 μm. (E) Effect of thiourea on the fungicidal activity of FC. Candida albicans cells were treated with FC (8 μg/ml) for 3 h after pre-incubation with 5 mM thiourea for 2 h. The number of viable cells was determined by a colony-counting method. Figure 1. View largeDownload slide The effect of FC on intracellular ROS generation. (A) The structure of floricolin C (FC). (B, C) Yeast cells were exposed to increasing concentrations of FC for 6 h. After staining with 40 μg/ml 2΄,7΄-dichlorodihydrofluorescein diacetate (DCFHDA), the samples were detected by flow cytometry and visualized by CLSM. BF, bright field. The bars indicate 10 μm. (D) FC-treated C. albicans mature biofilms were stained with MitoSOX Red (20 μM) and visualized by CLSM. The bars indicate 20 μm. (E) Effect of thiourea on the fungicidal activity of FC. Candida albicans cells were treated with FC (8 μg/ml) for 3 h after pre-incubation with 5 mM thiourea for 2 h. The number of viable cells was determined by a colony-counting method. Reactive oxygen species accumulation The accumulation of intracellular reactive oxygen species (ROS) in C. albicans was determined using 2΄,7΄-dichlorodihydro fluorescein diacetate (Sigma-Aldrich) (Li et al.2015) or MitoSOX Red (Invitrogen). 2΄,7΄-Dichlorodihydro fluorescein diacetate is a fluorogenic dye that moves freely across the cell membrane and reacts with ROS inside the cell to form the highly fluorescent compound dichlorofluorescein. Briefly, yeast cells were incubated with 2΄,7΄-dichlorodihydrofluorescein diacetate (40 μg/ml) in the dark for 30 min after incubated with FC for 6 h. The cells were then washed twice with PBS and the fluorescence intensity of stained cells was measured by a FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA, USA) and visualized by confocal laser scanning microscopy (CLSM). MitoSOX Red accumulates in the mitochondrial matrix and is oxidized to a fluorescent product by superoxide. Cells within FC-treated mature biofilm were stained with MitoSOX Red (20 μM) for 30 min, followed by visualization by CLSM to assess the oxidative state of cells within biofilms. Mitochondrial isolation and ROS assay Mitochondria were isolated from C. albicans spheroplasts, essentially as previously described (Yu et al.2016). Briefly, overnight cultured C. albicans were washed and re-suspended with buffer A (50 mM K2HPO4, 50 mM KH2PO4, 5 mM EDTA, 50 mM dithiothreitol, 40 mM β-mercaptoethanol, 2 M sorbitol, pH 7.2) containing 5 μl of zymolyase (2000 U/mL, Zymo Research, USA). The mixture was incubated at 30°C for 90 min. Cells were collected by centrifugation at 1000 g and washed twice with buffer B (0.6 M mannitol, 0.2% BSA, 10 mM imidazole) to obtain the yeast protoplasts. The obtained protoplasts were then homogenized for 20–50 strokes with a Dounce homogenizer. Cell lysates were centrifuged at 4°C at 1000 g to remove the nuclei and intact cells. The supernatant was then centrifuged at 10 000 g, obtaining the yeast mitochondria. Mitochondria were diluted to an OD620 of 1.0 in buffer B containing 0.2 M succinate. A dilution series of FC was added to the suspensions to a final concentration of 0, 4, 8, 16 and 32 μg/ml and MitoSOX was added at 20 μM. After incubation of 6 h, the fluorescence intensity was determined by a spectrofluorophotometer (excitation wavelength 520 nm, emission wavelength 600 nm). Analysis of mitochondrial membrane potential Changes in mitochondrial membrane potential (mtΔψ) were detected with rhodamine 123 (Rh123) (Chang et al.2011). SC5314 cells (1 × 106 cells/ml in SD medium) were incubated with various doses of FC at 30°C for 6 h. Rh123 was added at a final concentration of 5 μM, and the mixture was incubated for 30 min in the dark. Fluorescence intensities were recorded with a flow cytometer. ATP luciferase assay Intracellular ATP was measured according to a previously described method (Chang et al.2011). FC-treated C. albicans SC5314 cells were disrupted by vortexing with glass beads and centrifuged at 13523 g for 10 min. The ATP content in the cell lysis was determined using an ATP analysis kit (Beyotime, Haimen, China). Cytochrome c release from mitochondria The measurement of cytochrome c (Cyt c) release from mitochondria was performed as previously described (Wu et al.2010). Specifically, C. albicans cell suspension (5 × 106 cells/ml) was incubated with 16 μg/ml of FC for 12 h, and collected at 5000 g for 5 min. The cell pellets were resuspended in homogenization medium (50 mM Tris (pH 7.5), 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) and disrupted by vortexing with glass beads. The supernatants were collected at 2348 g for 10 min to remove the cell debris and unbroken cells. To estimate the levels of cytosolic and mitochondrial Cyt c, the supernatant was centrifuged at 30 000 g for 45 min. The supernatants were collected for assay of Cyt c released from mitochondria into the cytoplasm. The pellet was suspended in buffer (50 mM Tris, pH 5.0, 2 mM EDTA), incubated for 5 min, and centrifuged at 5000 g for 30 s. The pellet was collected as mitochondria for determination of Cyt c remaining in mitochondria. After being reduced by ascorbic acid at room temperature for 5 min, the relative quantities of the reduced Cyt c were assessed using a spectrophotometer at 550 nm. The protein content was quantified using BSA as a standard. Synthesis of FC–N-acetylcysteine and FC–reduced glutathione A 0.5 mg quantity of N-acetylcysteine (NAC) or reduced glutathione (GSH) in 0.5 ml of water was added to a solution of FC (0.5 mg) in 0.5 ml of methanol. The mixture was stirred for 0.5 h at room temperature to give the target compounds, which were determined by liquid chromatography–mass spectrometry (LC-MS). Effect of thiourea or N-acetylcysteine on the antifungal activity of FC Candida albicans cells (1 × 105 cells/ml) were pre-incubated with 5 mM thiourea or NAC for 2 h before treatment with FC (8 μg/ml) for 3 h. The number of viable cells was determined by a colony-counting method. Intracellular glutathione analysis Intracellular glutathione was monitored by the production of the fluorescent adduct glutathione-bimane from monochlorobimane and GSH catalyzed by glutathione- S-transferase (Katey et al.2005). Candida albicans cells were stained with 50 μM monochlorobimane after exposure to different concentrations of FC for 3 h. After incubation for 30 min, the stained cells were quantified in a spectrofluorophotometer at 385 nm excitation and 478 nm emission. Nuclear morphology assay Nuclear damage was analyzed by 4΄,6-diamidino-2-phenylindole staining. Candida albicans cells were treated with FC or vehicle control. After 12 h, cells were collected, washed twice with PBS, and resuspended in 70% ethanol for brief fixation and permeability (Wu et al.2010). After 10 min fixation, cells were washed with PBS and incubated with 10 μg/ml of 4΄,6-diamidino-2-phenylindole in the dark for 20 min. The stained cells were then observed with CLSM. The apoptosis and necrosis of C. albicans induced by FC Overnight-incubated cells were treated by FC (0, 16 and 32 μg/ml) for 12 h and the following treatments were performed as previously described in our lab (Chang et al.2011). Protoplasts were washed with PBS three times and incubated with 5 μl of Annexin V–fluorescein isothiocyanate (FITC) and 5 μl of propidium iodide (PI) for 20 min in the dark. Cells were then observed by CLSM using a ×63 objective. Effects of FC on biofilm formation Candida biofilms were developed on the surface of 96-well polystyrene plates and quantified using a 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay as described previously (Zhang et al.2017). Briefly, standard cell suspensions (100 μl) of Candida cells (1 × 106 cells/ml) containing different concentrations of FC (ranging from 0 to 32 μg/ml) were seeded in selected wells. After 24 h of incubation at 37°C, the biofilms were washed twice with PBS, and the metabolic activity of the biofilm cells was semi-quantified using a standard XTT reduction assay, which measures the activity of mitochondrial dehydrogenase (Chandra, Mukherjee and Ghannoum 2008). In addition, the biofilms were qualitatively inspected using an Olympus microscope. Effects of FC on pre-formed Candida biofilms Candida cells were seeded in 96-well polystyrene plates at an initial concentration of 1 × 106 cells/ml and grown in RPMI 1640 medium at 37°C for 48 h to form mature biofilms (Ramage et al.2002). The supernatants were discarded, and wells were washed with PBS to remove unbound cells. Fresh medium containing different concentrations of FC (ranging from 0 to 64 μg/ml) was then added to the wells, and the plates were incubated for another 24 h at 37°C. The effects of FC on the pre-formed biofilms were assessed using the XTT reduction assay as described previously. Candida albicans TDH3-GFP-CAI4 was used to visualize live/dead cells in pre-formed biofilms. The mature biofilms were treated with FC, followed by PI (5 μg/ml) staining for 45 min. The stained biofilms were imaged by CLSM. In this assay, dead cells displayed red fluorescence due to intracellular accumulation of PI. Antifungal activity of FC in Caenorhabditis elegans–C. albicans infection model A Caenorhabditis elegans–Candida albicans infection assay was performed as previously described (Sun, Liao and Wang 2015). Caenorhabditis elegans was exposed to C. albicans for 2 h and washed three times with sterile M9 buffer. Then the infected worms were challenged with different concentrations of FC. The infection was started by adding 20–25 animals to each well of a 96-well plate at 25°C, and scoring for dead or alive every 24 h. After 6 days of incubation, the wells were washed and visualized by an Olympus microscope with a ×4 objective lens. Statistical analysis In the Caenorhabditis elegans–Candida albicans infection assay, data were statistically analyzed using the log rank test. The other experimental data were statistically analyzed using Student's t-test. P < 0.05 was considered significant. RESULTS AND DISCUSSION Effect of FC on intracellular ROS generation In our previous study, FC was observed to disrupt the cytoplasmic membrane and exert a fungicidal action against C. albicans (Li et al.2016). In this study, we found FC also induced ROS accumulation in C. albicans cells. Flow cytometry analysis revealed that FC induced ROS generation in a dose-dependent manner in planktonic cells (Fig. 1B). This finding was further corroborated by CLSM observation (Fig. 1C). In addition, ROS accumulation, as indicated by MitoSOX Red fluorescence, was observed in FC-treated C. albicans mature biofilms (Fig. 1D). To determine whether increased ROS contributes to FC-induced cellular death, we explored the scavenging effect of the antioxidant thiourea. Addition of thiourea increased the survival percentage compared with the group treated with FC alone (Fig. 1E), suggesting that ROS accumulation is an important factor in FC-mediated cell death. Effect of FC on ROS accumulation in isolated mitochondria ROS are byproducts of cellular metabolism primarily generated in the mitochondria (Kowaltowski et al.2009). Thus, we speculated that FC might cause damage to mitochondria to increase ROS generation. We assessed the ROS levels in isolated mitochondria of C. albicans under FC treatment using MitoSOX Red as an indicator. The fluorescence intensity increased in FC-treated mitochondria compared with vehicle-treated mitochondria, suggesting that FC could directly cause ROS accumulation in isolated mitochondria (Fig. 2). Figure 2. View largeDownload slide The effect of FC on ROS generation in isolated mitochondria. The isolated mitochondria from SC5314 cells were treated with various doses of FC and stained with MitoSOX. The fluorescence intensity was determined by a spectrofluorophotometer (excitation wavelength 520 nm, emission wavelength 600 nm). **P < 0.01, ***P < 0.001. Figure 2. View largeDownload slide The effect of FC on ROS generation in isolated mitochondria. The isolated mitochondria from SC5314 cells were treated with various doses of FC and stained with MitoSOX. The fluorescence intensity was determined by a spectrofluorophotometer (excitation wavelength 520 nm, emission wavelength 600 nm). **P < 0.01, ***P < 0.001. Effect of FC on mitochondrial function in C. albicans cells Given the disruptive effect of FC on isolated mitochondria, we next assessed the effects of FC on mitochondria in C. albicans cells. The activity of mitochondrial proton pumps and electrogenic transport systems and the activation of the mitochondrial permeability transition can be assessed using mtΔψ, an indicator of the energetic state of the mitochondria (Li et al.2015). A low dose of FC significantly increased the fraction of cells with high fluorescence intensity (Fig. 3A). However, the fluorescence decreased when cells were treated with high dose of FC. The geometric mean (GMean) value was utilized to reflect the change of fluorescence intensity. As illustrated in Fig. 3B, the GMean value increased from 35.07 (control) to 98.36 and 172.72 at 4 and 8 μg/ml, respectively when exposed to FC. However, when the dose of FC was increased to 32 and 64 μg/ml, the GMean value decreased to 17.48 and 7.03, respectively. Intracellular ATP content, as another representative of mitochondrial function (Yu et al.2016), was measured in the presence of FC. The levels of ATP varied according to the change in mtΔψ (Fig. 3C). This suggested that C. albicans cells tried to increase mtΔψ and ATP generation to counteract the stress from the low dose of FC. However, a high dose of FC fatally hit the cells and disrupted the mitochondria. Figure 3. View largeDownload slide The dysfunction of mitochondria when C. albicans cells were treated with FC. (A, B) Candida albicans cells were cultured in SD medium with FC for 6 h. Cells were then stained with Rh123, and the fluorescence intensity was detected by flow cytometry. (C) Candida albicans was treated with FC at the indicated concentrations, followed by cell lysis and measurement of the intracellular ATP content. The bars represent the means ± SD of three independent experiments. ***P < 0.001. (D) Release of Cyt c from mitochondria to cytosol. The FC-treated cells were lysed to extract Cyt c, and the Cyt c contents of mitochondria and cytosol were analyzed by measuring absorbance at 550 nm with a spectrophotometer. Figure 3. View largeDownload slide The dysfunction of mitochondria when C. albicans cells were treated with FC. (A, B) Candida albicans cells were cultured in SD medium with FC for 6 h. Cells were then stained with Rh123, and the fluorescence intensity was detected by flow cytometry. (C) Candida albicans was treated with FC at the indicated concentrations, followed by cell lysis and measurement of the intracellular ATP content. The bars represent the means ± SD of three independent experiments. ***P < 0.001. (D) Release of Cyt c from mitochondria to cytosol. The FC-treated cells were lysed to extract Cyt c, and the Cyt c contents of mitochondria and cytosol were analyzed by measuring absorbance at 550 nm with a spectrophotometer. Cyt c is tightly bound to the inner mitochondrial membrane by its electrostatic interactions with acidic phospholipids, but it can be released to the cytosol when the mitochondria are damaged. (Yun et al.2016). Compared with control cells, the relative level of Cyt c in the mitochondria was decreased under FC treatment, and that in the cytosol was significantly increased (Fig. 3D), indicating that FC induced the release of Cyt c from mitochondria in C. albicans. Based on above results, we concluded that FC could directly disrupt the mitochondria and result in ROS accumulation when entering the cells. FC can chelate thiol-based agents through its α,β-unsaturated carbonyl moiety We analyzed the chemical structure of FC, and noticed the presence of an α,β-unsaturated carbonyl in the structure, which is considered to react with a mercapto moiety via the Michael reaction. To confirm this conjugate formation, FC was mixed with NAC or GSH, which both possess a mercapto moiety. The generated products were then analyzed by LC-MS. FC levels decreased when NAC or GSH was added, concomitant with the formation of a new product with a relative molecular mass of 469 (FC–NAC) or 613 (FC–GSH) (Fig. 4A and B). At the cellular level, NAC dramatically diminished the activity of FC against C. albicans (data not shown), which is further evidence for the occurrence of Michael reaction between FC and NAC. Figure 4. View largeDownload slide FC chelated thiol-based agents through its α,β-unsaturated carbonyl moiety. (A, B) The chemical reaction between FC and NAC (A) or GSH (B). (C) FC resulted in decreased intracellular GSH content. FC-treated C. albicans cells were stained with 50 μM monochlorobimane. The resultant fluorescent adduct, glutathione-bimane, was detected by a spectrofluorophotometer to indicate the decrease of intracellular GSH content. (D) The outlined reaction between FC and thiol-based agents. Figure 4. View largeDownload slide FC chelated thiol-based agents through its α,β-unsaturated carbonyl moiety. (A, B) The chemical reaction between FC and NAC (A) or GSH (B). (C) FC resulted in decreased intracellular GSH content. FC-treated C. albicans cells were stained with 50 μM monochlorobimane. The resultant fluorescent adduct, glutathione-bimane, was detected by a spectrofluorophotometer to indicate the decrease of intracellular GSH content. (D) The outlined reaction between FC and thiol-based agents. GSH, a thiol-based intracellular antioxidant, is a major ROS scavenging agent in cells (González-Párraga et al.2005). To determine if the Michael reaction occurs in C. albicans cells, the intracellular GSH content was measured in the presence of FC. FC at a dose of 8 μg/ml resulted in a decrease in GSH content, although the decrease was not as obvious as expected (Fig. 4C). Moreover, we speculate FC could also react with other thiol-based molecules or proteins (Fig. 4D), and these chelating effects altogether might damage redox homeostasis and result in the dysfunction of key proteins. The apoptosis and necrosis of C. albicans induced by FC If the production of ROS overwhelms the antioxidant capacity of the cell, oxidative stress occurs and causes cell damage (Perrone, Tan and Dawes 2008). Nuclear morphological changes, including chromosome condensation and fragmentation, indicate cell damage (Kapuscinski 1995). We next utilized 4΄,6-diamidino-2-phenylindole staining to visualize the nuclear morphology when C. albicans cells were treated with FC. In non-treated cells, chromatin appeared as a single round spot in the cells, the normal appearance (Fig. 5A). In contrast, 4΄,6-diamidino-2-phenylindole dye was dispersed in the whole cell in FC-treated cells (Fig. 5A), indicating the disruptive effect of FC on nuclear membrane or nuclear structure. The Annexin V–FITC Kit was applied to determine the apoptosis and necrosis induced by FC. The results showed that the majority of stained cells (under treatment with 32 μg/ml FC) were penetrated by PI rather than annexin V–FITC, suggesting that the 32 μg/ml FC-treated C. albicans cells mainly undergo necrosis rather than apoptosis (Fig. 5B). However, the low dose of FC induced more apoptotic cells than necrotic ones, suggesting that FC causes both apoptosis and necrosis of C. albicans cells. Figure 5. View largeDownload slide Candida albicans cell death induced by FC. (A) The nuclear damage of C. albicans cells in response to FC treatment. FC-treated cells were fixed and stained with 10 μg/ml of 4΄,6-diamidino-2-phenylindole. The stained cells were then observed with CLSM. The bars indicate 5 μm. (B) The apoptosis and necrosis of C. albicans induced by FC. Cells were stained by Annexin V–FITC or PI to indicate the characteristics of apoptosis or necrosis under treatment with FC. The stained cells were observed with CLSM. BF, bright field. The bars indicate 10 μm. Figure 5. View largeDownload slide Candida albicans cell death induced by FC. (A) The nuclear damage of C. albicans cells in response to FC treatment. FC-treated cells were fixed and stained with 10 μg/ml of 4΄,6-diamidino-2-phenylindole. The stained cells were then observed with CLSM. The bars indicate 5 μm. (B) The apoptosis and necrosis of C. albicans induced by FC. Cells were stained by Annexin V–FITC or PI to indicate the characteristics of apoptosis or necrosis under treatment with FC. The stained cells were observed with CLSM. BF, bright field. The bars indicate 10 μm. FC inhibits C. albicans biofilm formation in vitro Candida albicans biofilm formation on implanted medical devices is increasingly recognized as a key mediator of fungal infections in the host. The formation of biofilms is associated with antifungal resistance and increased pathogenicity (Rosseti, Chagas and Costa 2014). To investigate whether FC has an effect on C. albicans biofilm formation, XTT reduction assays were performed. The results demonstrated that FC reduced biofilm formation in a dose-dependent manner (Fig. 6A). FC at its minimal inhibitory concentration (MIC) reduced the metabolic activity of C. albicans biofilm formation by greater than 50%. Amphotericin B at the dose of 2 μg/ml was used as a positive control. Microscopy revealed that FC treatment resulted in a defect of biofilm formation, with only a few filamentous cells on the substratum at a dose of 8 μg/ml or greater (Fig. 6B). From the inspection of cell morphology, we noticed that FC had little effect on inhibiting the yeast-to-hyphae transition. The inhibition of biofilm formation was attributable to its fungicidal activity. Figure 6. View largeDownload slide The activity of FC against C. albicans biofilms. (A, B) The inhibitory effect of FC on biofilm formation was measured using XTT reduction assay and observed by microscopy. The bars indicate 50 μm. (C) The eradicating effect of FC on mature biofilms of C. albicans was assessed by XTT reduction assay. (D) Candida albicans TDH3-GFP-CAI4 mature biofilms were treated with FC for 24 h and stained with PI for CLSM observation. The bars indicate 50 μm. **P < 0.01, ***P < 0.001. Figure 6. View largeDownload slide The activity of FC against C. albicans biofilms. (A, B) The inhibitory effect of FC on biofilm formation was measured using XTT reduction assay and observed by microscopy. The bars indicate 50 μm. (C) The eradicating effect of FC on mature biofilms of C. albicans was assessed by XTT reduction assay. (D) Candida albicans TDH3-GFP-CAI4 mature biofilms were treated with FC for 24 h and stained with PI for CLSM observation. The bars indicate 50 μm. **P < 0.01, ***P < 0.001. FC exhibits fungicidal activity against mature biofilms Mature biofilms are much more resistance to antimicrobial agents and host immune factors than planktonic cells (Finkel and Mitchell 2011). Notably, FC also potently eradicated pre-formed biofilms in a dose-dependent manner, as detected by the XTT reduction assay (Fig. 6C). Compared with the drug-free group, 16 μg/ml FC significantly decreased cell survival in mature biofilms (P < 0.05). When the dose was increased to 32 or 64 μg/ml, FC treatment resulted in greater than 60% killing in mature biofilms. This effect was confirmed by CLSM (Fig. 6D). Collectively, FC exhibited a significant anti-biofilm effect against both developing biofilms and mature biofilms. Antifungal effect of FC on fluconazole-resistant C. albicans and other non-albicans Candida species We examined the susceptibility of four clinical fluconazole-resistant C. albicans strains to FC using the broth microdilution method following the Clinical and Laboratory Standards Institute's M27-A3 guidelines (CLSI 2008). The MICs against these four fluconazole-resistant strains were similar to that against wild type strain SC5314 (Table 1) (Li et al.2016), suggesting the application of FC in treating azole-resistant C. albicans caused infections. In addition to C. albicans, other clinically derived Candida species such as C. krusei (CK1), C. tropicalis (CT2) and C. glabrata (CG1) were also tested for evidence of antifungal activity of FC. FC exhibited potent antifungal activity against these three tested Candida species with its MICs ranging from 8 to 16 μg/ml (Table 1). We further evaluated the activity of FC against the biofilms of other Candida species. The data showed that FC could prevent biofilm formation as well as eradicate pre-formed biofilms of other Candida species at a concentration of 32 μg/ml (Fig. 7). Figure 7. View largeDownload slide The antifungal effect of FC on three non-albicans Candida species. (A) The inhibitory effect of FC on biofilm formation of tested Candida strains. After incubation with 32 μg/ml of FC for 24 h, the biofilms were washed twice with PBS, and assessed by XTT reduction assay. (B) The eradicating activity of FC on tested Candida mature biofilms. Candida cells were incubated at 37°C for 48 h to form mature biofilms. Then the mature biofilms were challenged with 32 μg/ml of FC for another 24 h, and assessed by XTT reduction assay. Figure 7. View largeDownload slide The antifungal effect of FC on three non-albicans Candida species. (A) The inhibitory effect of FC on biofilm formation of tested Candida strains. After incubation with 32 μg/ml of FC for 24 h, the biofilms were washed twice with PBS, and assessed by XTT reduction assay. (B) The eradicating activity of FC on tested Candida mature biofilms. Candida cells were incubated at 37°C for 48 h to form mature biofilms. Then the mature biofilms were challenged with 32 μg/ml of FC for another 24 h, and assessed by XTT reduction assay. FC improves the survival of C. albicans-infected nematodes We further investigated the in vivo antifungal activity of FC using the Caenorhabditis elegans–Candida albicans infection model. Results showed that FC could significantly prolong the survival of C. albicans-infected Caenorhabditis elegans at 8 μg/ml (P = 0.008), 16 μg/ml (P < 0.001), and 32 μg/ml (P = 0.002) compared with the control group (Fig. 8A). The toxicity test demonstrated that 64 μg/ml FC had little effect on the survival of non-infected Caenorhabditis elegans within 6 days (Fig. 8B). These data suggested a potential application of FC in treating C. albicans infection in vivo. Figure 8. View largeDownload slide FC improves the survival of Candida albicans-infected Caenorhabditis elegans. (A) Worms infected by C. albicans SC5314 were treated with indicated concentrations of FC. The survival curves were plotted based on the survival state of each worm, which was monitored each day. The data were statistically analyzed using Log Rank tests. **P < 0.01, ***P < 0.001. (B) Healthy worms were incubated with or without 64 μg/ml of FC for 6 days and imaged using an Olympus microscope to show the toxicity of FC on worms. Figure 8. View largeDownload slide FC improves the survival of Candida albicans-infected Caenorhabditis elegans. (A) Worms infected by C. albicans SC5314 were treated with indicated concentrations of FC. The survival curves were plotted based on the survival state of each worm, which was monitored each day. The data were statistically analyzed using Log Rank tests. **P < 0.01, ***P < 0.001. (B) Healthy worms were incubated with or without 64 μg/ml of FC for 6 days and imaged using an Olympus microscope to show the toxicity of FC on worms. Taken together, we have demonstrated that FC has promising fungicidal activity against pathogenic Candida species both in the planktonic state and in biofilms, thus expanding current potential antifungal agents for combatting fungal infections. Further research demonstrated that FC could directly damage mitochondria to increase ROS accumulation. In addition, the α,β-unsaturated carbonyl of FC can react with GSH or thiol-based proteins through the Michael reaction, resulting in an imbalance of intracellular redox homeostasis and ROS accumulation. The accumulated ROS ultimately induce mitochondria dysfunction, nuclear fragmentation and, consequently, cell death. Acknowledgements This work was financially supported by the State Key Program of the National Natural Science Foundation (no. 81630093) and the National Natural Science Foundation (nos. 81402804 and 81273383). AUTHOR CONTRIBUTIONS Conceived and designed the experiments: HXL, MZ and WQC. Performed the experiments: MZ, HZS, SZ, YL and WL. Analyzed the data: MZ. Contributed reagents/materials/analysis tools: HXL. Wrote the paper: MZ, WQC and HXL. Conflict of interest. None declared. REFERENCES Braga PC, Culici M, Alfieri M et al.   Thymol inhibits Candida albicans biofilm formation and mature biofilm. Int J Antimicrob Agents  2008; 31: 472– 7. Google Scholar CrossRef Search ADS PubMed  Chandra J, Mukherjee PK, Ghannoum MA. In vitro growth and analysis of Candida biofilms. Nat Protoc  2008; 3: 1909– 24. Google Scholar CrossRef Search ADS PubMed  Chang WQ, Wu XZ, Cheng AX et al.   Retigeric acid B exerts antifungal effect through enhanced reactive oxygen species and decreased cAMP. 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Published: Feb 1, 2018

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