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Reactive oxygen species-independent apoptotic pathway by gold nanoparticles in Candida albicans
Microbiological Research 207 (2018) 33–40
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Reactive oxygen species-independent apoptotic pathway by gold nanoparticles in Candida albicans
MARK
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Minju Seong, Dong Gun Lee
School of Life Sciences, KNU Creative BioResearch Group (BK21 Plus Program), College of Natural Sciences, Kyungpook National University, Daehak-ro 80, Buk-gu, Daegu 41566, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Gold nanoparticles Genomic DNA interactions Mitochondrial dysfunction ROS-independent apoptosis Candida albicans
Candida albicans is the most common pathogenic fungus in humans, causing cutaneous and life-threatening systemic infections. In this study, we confirmed using propidium iodide influx that gold nanoparticles (AuNPs), which are promising materials for use as antimicrobial agents, did not affect the membrane permeability of C. albicans. Thus, the fungal cell death mechanisms induced by AuNPs were assessed at intracellular levels including DNA damage, mitochondrial dysfunction, and reactive oxygen species (ROS) overproduction. AuNPs interacted with C. albicans DNA leading to increased nuclear condensation and DNA fragmentation. Changes in the mitochondria induced by AuNPs involving mass, Ca2+ concentrations, and membrane potential indicated dysfunction, though the level of intracellular and mitochondrial ROS were maintained. Although ROS signaling was not disrupted, DNA damage and mitochondrial dysfunction triggered the release of mitochondrial cytochrome c into the cytosol, metacaspase activation, and phosphatidylserine externalization. Additionally, the AuNPs-induced apoptotic pathway was not influenced by N-acetylcysteine, an ROS scavenger. This indicates that ROS signaling is not linked with the apoptosis. In conclusion, AuNPs induce ROS-independent apoptosis in C. albicans by causing DNA damage and mitochondria dysfunction.
1. Introduction The common pathogenic fungi, Candida albicans resides as a commensal organism in the mucocutaneous cavities of the skin, vagina and intestine of humans. It can cause infections under certain pathological and physiological conditions such as infancy, diabetes, pregnancy, steroidal chemotherapy, and prolonged broad spectrum antibiotic administration as well as acquired immunodeficiency syndrome (AIDS) (Manohar et al., 2001). Resistance to conventional drugs is rapidly emerging, and the decreased activity of these drugs against C. albicans has been observed on some level for every currently used drug class (Sanglard, 2017). Thus, development of novel and effective antifungal agents against C. albicans has gained major interest. Nanoparticles, defined as being between 10 and 100 nanometers in size, are promising materials owing to their wide variety of potential biological, biomedical, catalytic, optoelectronic, and pharmaceutical applications (Mohanraj and Chen, 2006). Previous studies have shown that nanoparticles can act as antimicrobial agents because of their ability to interact with microorganisms (Albanese et al., 2012). Because of these characteristics, various metal nanoparticles have been studied to determine their unique antimicrobial properties and their potential
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usage in a wide range of applications such as medical instruments, textiles, and purifications (Sondi and Salopek-Sondi, 2004; Espitia et al., 2012). Gold nanoparticles (AuNPs) are a well-studied material and their unique chemical and physical properties make them promising therapeutic agents without inherently toxic to human cells (Wang et al., 2008; Alkilany et al., 2012; Conde et al., 2014). AuNPs have been widely used in cancer treatments as a drug delivery system and thermal therapy (Huang et al., 2008; Brown et al., 2010). Furthermore, in previous studies, AuNPs showed that antimicrobial activity against various pathogens including Escherichia coli, Streptococcus mutans, and Candida species (Hernandez-Sierra et al., 2008; Lima et al., 2013; Wani et al., 2013). However, while their antimicrobial activity has been the focus of numerous studies, and there has yet been no study demonstrating their mechanism against C. albicans. Therefore, in this context, this study aims to confirm the mode of fungicidal action of AuNPs on C. albicans.
Corresponding author. E-mail address: [email protected] (D.G. Lee).
https://doi.org/10.1016/j.micres.2017.11.003 Received 12 August 2017; Received in revised form 28 October 2017; Accepted 4 November 2017 Available online 06 November 2017 0944-5013/ © 2017 Elsevier GmbH. All rights reserved.
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genomic DNA that was extracted from C. albicans cells (Hanna and Xiao, 2006). Fluorescence changes in the AuNPs after the addition of genomic DNA were recorded using a RF-5301PC spectrofluorometer. An excitation wavelength of 527 nm (approximate absorption maximum) was used for the AuNPs. The emission spectra were recorded in the wavelength range from 460 nm to 600 nm. The fluorescence spectra of AuNPs were determined at room temperature in the presence of 1 μg of genomic DNA (An and Jin, 2012).
Table 1 The antifungal effect of AuNPs and H2O2. Fungal strains
C. albicans ATCC 90028
MIC (μg/ml) AuNPs
H2O2
32
20
2. Materials and methods
2.6. Mitochondrial mass detection
2.1. Nanoparticle preparation and antifungal activity of AuNPs
To evaluate mitochondrial mass, Mitotracker Green FM fluorescent probe (Invitrogen, Carlsbad, CA) was used. Harvested C. albicans cells were washed with PBS and incubated with AuNPs for 4 h at 28 °C. Then, AuNP-treated cells were resuspended in PBS and incubated with 100 nM Mitotracker Green FM for 45 min. Next, the cells were washed twice, and the fluorescence was measured using a spectrofluorophotometer (Blanquer-Rossello et al., 2017).
C. albicans (ATCC 90028) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in aerated YPD (yeast extract-peptone-dextrose) broth (BD Bioscience, Franklin Lakes, NJ, USA) at 28 °C. Gold nanoparticles (AuNPs) were purchased from Sigma-Aldrich. The particles were 30 nm in diameter and a stabilized suspension in phosphate-buffered saline (PBS). H2O2 (SigmaAldrich) was used as a positive control for comparisons of the physiological responses caused by AuNPs. C. albicans cells were inoculated into YPD and then dispensed into microtiter plates. The minimum inhibitory concentration (MIC) was determined by a standard microdilution method. After incubation with AuNPs for 12 h, the growth was measured using a microtiter ELISA Reader (Molecular Devices Emax, CA; Table 1).
2.7. Mitochondrial Ca2+ concentration detection To assess mitochondrial Ca2+ levels, a Rhod-2AM (Molecular Probes) assay was used. Harvested C. albicans cells were washed in PBS. Next, the cells were incubated with AuNPs or H2O2 for 4 h at 28 °C. After incubation, the cells were centrifuged twice at 12,000 rpm (Sorvall Biofuge Fresco) for 5 min with Krebs buffer (pH 7.4; 4 mM KCl, 132 mM NaCl, 1.4 mM MgCl2, 10 mM HEPES, 6 mM glucose, 10 mM NaHCO3, and 1 mM CaCl2) and treated with 1% bovine serum albumin and 0.01% pluronic acid F-127. The cells were then incubated with a Ca2+-sensitive fluorescent dyes (Rhod-2AM; Molecular Probes) for 30 min and washed three times with Ca2+-free Krebs buffer. The fluorescence intensity of Rhod-2AM was examined using a spectrofluorophotometer (excitation/emission = 552 nm/581 nm) (Shimadzu RF-5301PC, Shimadzu) (Valipour et al., 2015).
2.2. Membrane permeabilization detection Membrane permeabilization was detected using propidium iodide (PI). The cells were washed with PBS (2.7 mM KCl, 137 mM NaCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) and incubated with 32 μg/mL AuNPs for 4 h at 28 °C. The fluorescent intensity was measured using a FACSverse flow cytometer (BD Biosciences) (Lecoeur et al., 2001).
2.8. Mitochondrial membrane potential detection
2.3. DNA condensation assay
The collapse of the electrochemical gradient across the mitochondrial membrane was measured using a JC-1 dye (Molecular Probes). The fungal cells were washed with PBS and the cells were then incubated with AuNPs for 4 h at 28 °C. After centrifugation, the cells were resuspended in warm PBS and incubated with JC-1 for 10 min. The fluorescent intensity of the JC-1 monomer and aggregate (FL1 and FL2, green and red fluorescence, respectively) was measured using a FACSverse flow cytometer (Alonso-Monge et al., 2009).
A DNA-specific fluorescent dye, 4′-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was used to analyze nuclear condensation. C. albicans cells were washed with PBS buffer and incubated with AuNPs for 4 h at 28 °C. After incubation, the cells were resuspended in PBS and treated with DAPI for 20 min. Next, the cells were washed, and the fluorescence was analyzed using a spectrofluorophotometer (Shimadzu RF-5301PC; Shimadzu, Kyoto, Japan) and visualized using a fluorescence microscope (Nikon Eclipse Ti-S; Nikon, Japan) (Madeo et al., 1997).
2.9. Intracellular ROS formation assay To detect intracellular ROS levels, an oxidation-sensitive fluorescent dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes) was used. The cells were washed with PBS buffer and incubated with AuNPs for 4 h at 28 °C. After centrifugation, the cells were incubated with H2DCFDA for 1 h in the dark and washed twice. The fluorescent intensity was measured using a FACSverse flow cytometer (Echave et al., 2003). Mitochondrial ROS were measured using MitoSOX Red (Molecular Probes). C. albicans cells were incubated with AuNPs for 4 h at 28 °C and centrifuged at 12,000 rpm (Sorvall Biofuge Fresco). The cells were suspended in 5 μM MitoSOX Red reagent at 28 °C for 30 min and then the stained cells were washed with PBS. The fluorescent cells were analyzed with a FACSverse flow cytometer (Bankapalli et al., 2015).
2.4. DNA fragmentation assay For the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, a method for assessing DNA damage, the cells were washed with PBS and incubated with AuNPs for 4 h at 28 °C. Next, the cells were fixed with 2% paraformaldehyde for 1 h and incubated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) on ice for 2 min. Then, the cells were incubated with the TUNEL reaction mixture from an in situ cell death detection kit (Roche Diagnostics, Mannheim, Germany) for 1 h at 28 °C. Finally, the cells were washed, and the fluorescence was assayed using a spectrofluorophotometer (Madeo et al., 1997; Valipour, 2012). 2.5. Interactions of AuNPs with C. albicans DNA
2.10. Detection of cytochrome c concentration To investigate interactions of DNA with AuNPs, fluorescence measurements were evaluated by the process described by Atay et al. (2009) and Sung and Lee (2007), with slight modification. we prepared
To confirm the effect of AuNPs on cytochrome c release, the levels of cytochrome c in the cytosol and mitochondria were measured as 34
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directly injure the membrane of C. albicans. Because the antifungal activity of AuNPs is not related to membrane disruption, the effect of AuNPs on several intracellular responses was tested.
previously described (Wu et al., 2010; Barbu et al., 2013; Choi and Lee, 2015). C. albicans cells were washed with PBS and pretreated with 1 mM N-acetylcysteine (NAC; Sigma-Aldrich) at 28 °C for 10 min. Next, the C. albicans cells were incubated with AuNPs for 4 h at 28 °C. After centrifugation, the cells were homogenized in a homogenization medium [50 mM Tris (pH 7.5), 2 mM EDTA, and 1 mM phenylmethylsulphonyl fluoride] supplemented with 2% glucose. After centrifugation for 45 min, the cytosolic cytochrome c content of the supernatant was measured, and the pellet was suspended in 50 mM Tris (pH 5.0) with 2 mM EDTA to measure the mitochondrial cytochrome c content. After reduction via the addition of 500 mg/mL ascorbic acid, the cytosolic and mitochondrial cytochrome c levels were measured at 550 nm using a spectrophotometer (GE Healthcare, Little Chalfont, Buckinghamshire, UK) (Wu et al., 2010; Dwyer et al., 2012).
3.2. AuNPs induce nucleic acid damage through physical interactions in C. albicans We next investigated DNA damage because cells with damaged DNA fail to undergo productive DNA repair, and this trigger the cell death response (Roos and Kaina, 2006). DNA damage was indicated by morphologic changes including nuclear condensation and fragmentation (Hong et al., 2004). DAPI was used to monitor morphological changes in the nucleus, based on the roundness and size through binding to the minor groove of A-T rich regions of DNA (Kapuscinski, 1995; Daniel and DeCoster, 2004). Spectrofluorophotometric and fluorescence microscopic analyses revealed that cells treated with AuNPs or H2O2 displayed more concentrated fluorescence in comparison with untreated cells, indicating nuclear condensation (Fig. 2a). The TUNEL assay has been designed to detect the extensive DNA degradation that is present in apoptotic cells (Negoescu et al., 1998). This assay revealed that C. albicans cells exposed to AuNPs or H2O2 displayed increases in fluorescence indicating DNA fragmentation (Fig. 2b). AuNPstreated cells undergo physical changes such as nucleus condensation and DNA fragmentation. Physical changes in C. albicans DNA may induce a failure to complete cell division that leads to apoptosis (Henry et al., 2013). We thus suggest that AuNPs induce physical changes leading to a failure in the normal function of DNA in C. albicans. The potential impact of nanoparticles binding directly to DNA to create cellular damage has been discussed previously (Lecoeur et al., 2001). The nanoparticles containing AuNPs have native fluorescence, and the fluorescence intensity was reduced by binding DNA (He et al., 2008; Atay et al., 2009). To investigate the direct action of AuNPs on the DNA of C. albicans, we measured the AuNPs fluorescence spectrum, which was used to elucidate the interactions between AuNPs and C. albicans DNA. The emission maximum and intensity depend upon the surroundings of the AuNPs (Sung and Lee, 2007). We assessed the changes in AuNPs fluorescence in the presence and absence of C. albicans DNA. The results showed that the fluorescence intensity decreased in the presence of C. albicans DNA (Fig. 2c). Based on these experiments showing fluorescence changes, we suggest that AuNPs directly interact with the DNA and lead to nucleus condensation and DNA fragmentation. These changes would support to the breakdown of intracellular maintenance caused by AuNPs.
2.11. Metacaspase activation Activation of metacaspases was detected with the CaspACE FITCVAD-FMK In Situ Marker (Promega, Madison, WI, USA). The cells were washed with PBS and pretreated with 1 mM NAC at 28 °C for 10 min. After 10 min, the cells were incubated with AuNPs for 4 h at 28 °C. Then, the cells were washed twice and incubated with CaspACE fluorescein isothiocyanate (FITC)-VAD-FMK for 20 min. After centrifugation, the cells were resuspended in PBS, and the fluorescence was measured using a FACSverse flow cytometer (Madeo et al., 2002). 2.12. Phosphatidylserine (PS) externalization PS external exposure was assessed after binding of FITC-conjugated annexin V and the loss of membrane integrity after PI uptake. For preparation of protoplasts, the cells were digested with 0.1 M potassium phosphate buffer (PPB, pH 6.0) including 1 M sorbitol and 15 mg/mL lysing enzyme (Sigma-Aldrich) for 4 h at 28 °C to remove the cell wall. The protoplasts were then suspended in 0.1 M PPB (pH 6.0) containing 1 M sorbitol at 28 °C. After centrifugation, the cells were pretreated with 1 mM NAC at 28 °C for 10 min, incubated with AuNPs for 4 h at 28 °C, and washed twice with 0.1 M PPB (pH 6.0) with 1 M sorbitol. Then, the cells were stained with FITC-labeled annexin V and PI (BD Pharmingen, San Diego, CA). PS externalization was analyzed using a FACSverse flow cytometer (Madeo et al., 1997). 2.13. Statistical analysis The results are expressed as mean ± standard deviation of at least three independent experiments. Analysis of variance (ANOVA) calculated using SPSS Software was used to determine any significant differences among the samples. For pairwise comparisons, Tukey’s Honestly Significant Difference test (HSD) was used. Statistically significant results had P-values < 0.05.
3.3. Mitochondrial dysfunction is involved in AuNP-induced cell death Mitochondria play important roles in cell survival, providing cellular energy as adenosine triphosphate (ATP), and the apoptotic pathway (Akbar et al., 2016). Therefore, we assessed whether AuNPs caused changes in mitochondrial mass, Ca2+ concentration and membrane potential. The fluorescent MitoTracker Green FM, a dye which binds covalently to the inner mitochondrial membrane and fluoresces independent of membrane potential, was used to investigate mitochondrial mass (Blanquer-Rossello et al., 2017). In both AuNP- and H2O2-treated cells, the mitochondrial mass was significantly increased compared to control cells (Fig. 3a). These results indicate that AuNPs cause alterations in mitochondrial mass. Increase in mitochondrial mass appeared when mitochondrial proliferation and fusion (MahyarRoemer et al., 2001; Arakaki et al., 2006; Peng et al., 2016; Ampawong et al., 2017), and the increase were these results of a cellular response to compensate for reduced mitochondrial function (Nugent et al., 2007). For the reason, increased mitochondrial mass indicates mitochondrial dysfunction. Therefore, we conclude that mitochondria in AuNPs-treated C. albicans cells are functioning abnormally. Imbalances of the mitochondrial ion homeostasis have been shown to indicate an abnormal status of the mitochondria. Mitochondrial Ca2+
3. Results and discussion 3.1. AuNPs do not have an effect on membranes The membrane of a living cell plays a vital role in regulating activities within and around the cell (Madeo et al., 1997). Cell death is precipitated by a sudden breakdown of the plasma membrane permeability barrier (Lemasters et al., 1987). To clarify whether AuNPs cause membrane disruption, membrane permeability was investigated. We measured membrane permeability using PI, a membrane-impermeable dye that only enters cells that have damaged membranes (Pina-Vaz et al., 2001). For flow cytometric analysis (Fig. 1), the cells treated with AuNPs showed similar PI fluorescence intensities (0.73%) as untreated cells (0.61%). When H2O2 was applied, the fluorescence increased to 97.67%. These results suggest that AuNPs may not have an effect on membrane permeability in C. albicans. Therefore, the particles did not 35
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Fig. 1. Flow cytometry analysis of membrane permeabilization by AuNPs using propidium iodide (PI) staining in C. albicans. The x-axis shows the PI fluorescence and the y-axis shows the SSC.
Fig. 2. DNA and nuclear damage during late stage apoptosis by AuNPs (a) Concentrated or split fluorescence of DAPI indicates nuclear condensation by a spectroflourophotometer and visualized by fluorescence microscopy. 1: Control, 2: AuNPs treatment, 3: H2O2 treatment. (b) DNA fragmentation was observed using a TUNEL assay. Data were analyzed using one-way ANOVA followed by a post hoc Tukey’s test, *P < 0.05 versus control. (c) Quenching of the fluorescence of AuNPs, both in the absence and presence of DNA. The x-axis shows the excitation wavelength and the y-axis shows the emission wavelength.
(Fig. 3b). Increased Rhod-2AM fluorescence indicates a transient elevation in mitochondrial Ca2+ levels (Duchen, 2000). When C. albicans cells were exposed to AuNPs or H2O2, mitochondrial Ca2+ levels
affects several steps of energy metabolism such as synchronize ATP generation (Hajnóczky et al., 2006). Therefore, mitochondrial Ca2+ was detected using the mitochondrial Ca2+-sensitive dye Rhod-2AM
Fig. 3. Analysis of mitochondrial dysfunction (a) Mitochondrial mass was assessed using Mitotracker Green FM and analyzed by a spectrofluorophotometer. The y-axis shows the relative Mitotracker Green fluorescence. These data were analyzed using one-way ANOVA followed by a post hoc Tukey’s test, *P < 0.05 versus control. (b) Influx of mitochondrial Ca2+ was assessed using Rhod-2AM and analyzed by spectrofluorophotometer. The y-axis shows the relative Rhod-2 AM fluorescence. Data were analyzed using one-way ANOVA followed by a post-hoc Tukey’s test, *P < 0.05 versus control. (c) Mitochondrial membrane depolarization was detected using JC-1 and analyzed by flow cytometry. The x-axis shows the FL1 fluorescence and the y-axis shows the FL2 fluorescence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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(Fig. 4b). Overproduction of ROS was not detected in AuNP-treated cells. Numerous studies have suggested that ROS are generated by the cellular disruption that occurs during apoptosis and that ROS overproduction triggers cell death (Carmona-Gutierrez et al., 2010). However, a pathway directly linking ROS to yeast cell death has yet to be elucidated (Wysocki and Kron, 2004). ROS-independent cell apoptosis has rarely been reported in cells and very little is known about this pathway (Wysocki and Kron, 2004). Thus, we expect that ROS signaling does not influence the mechanisms of AuNP-induced cell damage.
increased significantly compared to control cells. These results showed that AuNPs trigger an increase in mitochondrial Ca2+ levels. Mitochondrial Ca2+ uptake can induce the opening of the mitochondrial permeability transition pore leading to changes in the mitochondrial mass and cell death by apoptosis (Rizzuto et al., 2012). AuNPs cause collapse of mitochondrial Ca2+ homeostasis via overload of mitochondrial Ca2+. Thus, disruption of mitochondrial Ca2+ homeostasis could play a role in initiating AuNPs-induced cell death. The disruption of mitochondrial Ca2+ homeostasis is typically coupled with a reduction in mitochondrial membrane potential, and it is known that the reduction of mitochondrial membrane potential constitutes an early, irreversible step of programmed cell death (Carraro and Bernardi, 2016). JC-1, a lipophilic cationic dye that interacts with the mitochondrial membrane, was used to estimate mitochondrial membrane depolarization (Sifaoui et al., 2014). As expected, flow cytometry analysis showed that AuNP-treated cells showed decreased emitted fluorescence (Y mean/X mean = 7.53) in comparison with untreated cells (Y mean/X mean = 44.39). H2O2-treated cells also showed fluorescence change (Y mean/X mean = 0.85) (Fig. 3c). Thus, depolarization of the mitochondrial membrane occurred after AuNP treatment. Measurement of membrane potential provides information about energy metabolism, matrix configuration, and cytochrome c release during apoptosis (Gottlieb et al., 2003). Mitochondria membrane depolarization in AuNPs-treated C. albicans can allude changes in intracellular networks. To summarize, these results shows that physical and physiological changes occurred such as increased mass, mitochondrial calcium disruption, and loss of mitochondrial membrane potential, and it supports mitochondrial dysfunction in the presence of AuNPs in C. albicans.
3.5. AuNPs trigger an apoptotic pathway unrelated to ROS signaling Cytochrome c is a component of the electron transport chain in mitochondria. It transfers electrons from complex III to IV. Thus, the release of cytochrome c is an indicator of electron transport chain failures (Huttemann et al., 2011). To investigate the translocation of cytochrome c to the cytoplasm from the mitochondria, we detected cytochrome c levels using spectrophotometric analysis (Wu et al., 2010; Barbu et al., 2013; Choi and Lee, 2015). Additionally, NAC pretreated groups were employed to ensure that ROS signaling is not involved in the apoptotic pathway induced by AuNPs. NAC is a source of sulfhydryl groups in cells and a scavenger of free radicals as it interacts with ROS (Bavarsad Shahripour et al., 2014). Cytosolic cytochrome c levels increased in AuNPs treated cells compared to untreated cells. NAC-pretreated cells with AuNPs also showed increased cytochrome c levels in the cytosol (Fig. 5a). In the mitochondria, cytochrome c levels decreased in both AuNP-treated and NAC-pretreated cells in comparison with untreated cells (Fig. 5b). The H2O2-treated cells showed also significantly increased levels of release. These results suggest that cytochrome c is released from the mitochondria into the cytosol after treatment with AuNPs and thus loses its function as an electron transmitter. Additionally, we propose that ROS do not have an influence on cytochrome c release based on the results from NAC-pretreated cells with AuNPs. Cytochrome c has been found to have dual functions in controlling both cellular energetic metabolism and apoptosis. Opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential and the release of apoptogenic factors (Di Lisa et al., 2001). AuNPs-induced mitochondrial membrane depolarization can lead to the release of cytochrome c into the cytosol. Mitochondria play key roles through the release of cytochrome c into cytosol in AuNPs-mediated apoptosis. The release of cytochrome c into the cytosol from the mitochondria can lead to the activation of metacaspase. The metacaspase of yeast is a functional homologue of the mammalian caspases that cleave specific substrates and trigger apoptotic cell death. Metacaspase is always present within the cell in an inactive form and can be activated by cleavage (Barbu et al., 2013). FITC-VAD-FMK is a fluorescent analogue of the cell permeable pan-caspase inhibitor Z-VAD-FMK [carbobenzoxy-valyl-alanyel-aspartyl-(O-methyl)-fluoromethylketone] and is a green fluorescent marker that penetrates intact living cells where it irreversibly
3.4. AuNPs did not disrupt ROS signaling ROS are generated as a consequence of or as part of cellular disruption in many different cellular processes including proliferation, inflammation, aging, and death (Li et al., 2011; Schieber and Chandel, 2014). Moderate concentration of ROS are also involved in physiological responses as part of defense mechanisms and signaling processes (Espinosa-Diez et al., 2015). We assessed the levels of intracellular and mitochondrial ROS using the fluorescent ROS indicators H2DCFDA and MitoSOX Red, respectively. In the presence of ROS, H2DCF is rapidly oxidized into the highly fluorescent DCF (Jin et al., 2005). In the flow cytometry analysis, the cells treated with AuNPs showed minimal increases in the H2DCFDA fluorescent intensity (3.79%) in comparison with untreated cells. When H2O2 was applied, the fluorescence intensity increased to 49.14% (Fig. 4a). Mitochondrial ROS were detected using MitoSOX Red, a fluorogenic dye that selectively detects mitochondrial superoxide (Bankapalli et al., 2015). Similar to the intracellular ROS results, the cells treated with AuNPs showed minimal increases in MitoSOX Red fluorescence with an intensity of 7.54% while H2O2 treated cells had notable fluorescence increases with an intensity of 79.48%
Fig. 4. Assessment of ROS accumulation after AuNPs treatment (a) ROS accumulation in C. albicans was evaluated by H2DCFDA and analyzed by flow cytometry. The x-axis shows the H2DCFDA fluorescence and the y-axis shows the cell counts. (b) Mitochondrial-specific ROS was evaluated using MitoSOX Red and analyzed by flow cytometry. The x-axis shows the MitoSOX Red fluorescence and the y-axis shows the cell counts. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Measurment of intracellular cytochrome c levels. The release of cytochrome c to the cytoplasm (a) from the mitochondria (b) in C. albicans was analyzed by measuring the absorbance at 550 nm with a spectrophotometer, and the y-axis shows the relative cytochrome c level. Data were analyzed using one-way ANOVA followed by a post-hoc Tukey’s test, *P < 0.05 versus control.
Fig. 6. Apoptosis induced by AuNPs (a) Caspase activation in C. albicans was evaluated using a FITC-VAD-FMK assay, and was analyzed by flow cytometry. The x-axis shows the FITCVAD-FMK fluorescence and the y-axis shows the SSC. (b) Phosphatidylserine (PS) externalization during early apoptosis was assessed by FITC-annexin V and PI double staining, and was analyzed by flow cytometry. The x-axis shows the Annexin V FITC fluorescence and the y-axis shows the propidium iodide fluorescence.
shown in Fig. 6b. The annexin V-stained apoptotic cell population increased from 11.82% to 25.64% after AuNP treatment, and NAC-pretreated cells showed similar results. Additionally, the apoptotic cell population increased to 37.03% after H2O2 treatment. This result shows that AuNPs can cause PS external exposure in C. albicans indicating that AuNPs-induced early apoptosis. In addition, PS externalization mediated by AuNPs is unrelated to ROS in C. albicans. These apoptotic features support that the cells under investigation underwent ROS-independent apoptosis. In all of the NAC-pretreated experiments, the cells had similar results as AuNP-treated cells. Generally, apoptotic responses are blocked by ROS scavengers (Simon et al., 2000), whereas, antioxidant have no protective effects against apoptotic responses in ROS-independent apoptosis (Ko et al., 2005). The AuNPs-induced apoptotic pathway functioned regardless of NAC-pretreatment in C. albicans cells. Thus, ROS do not affect the AuNP-mediated apoptotic process.
binds to activated caspases (Lee and Lee, 2017). The CaspACE FITCVAD-FMK In Situ Marker was used to examine metacaspase activation. We also preincubated the cells with NAC to further confirm that ROS were not associated with the AuNP-induced activation of metacaspase. As shown in Fig. 6a, increase of metacaspase activation was detected in cells treated with AuNPs (35.83%), NAC-pretreated AuNPs (31.82%), and H2O2 (51.87%) compared to untreated cells (16.65%). This result shows that AuNPs induce metacaspase activation. Since NAC-pretreated cells have similar results as cells treated with only AuNPs, ROS does not appear to have an effect on metacaspase activation. In yeast, apoptosis is governed chiefly by metacaspases, which are responsible for destroying the cell from within (Carmona-Gutierrez et al., 2010). Thus, we suggest that C. albicans undergoes apoptosis via metacaspase activation induced by AuNPs. PS is the most abundant negatively charged phospholipid in eukaryotic membranes. In the early stages of apoptosis, PS become exposed on the external surface of the cell (Tian et al., 2017). PS externalization was detected using annexin V-FITC and PI double staining. Annexin V has a high affinity for PS after it has been externalized from the inner to the outer plasma membrane of apoptotic cells (Liu et al., 2009). Staining cells with a combination of annexin V and PI allows the distinction between living cells (annexin V-/PI-), apoptotic cells (annexin V+/PI-), and late apoptotic/necrotic cells (annexin V+/PI+) (Lecoeur et al., 2001). The results of bivariate FITC-Annexin V/PI analysis of C. albicans cells after treatment with AuNPs and H2O2 are
4. Conclusions In this study, the mode of fungicidal action of AuNPs was confirmed on C. albicans. AuNPs induce destruction of the nucleus, nucleic acids and attenuation of mitochondrial homeostasis, leading to apoptosis (Fig. 7). The apoptotic responses are not associated with ROS, different from the conventional apoptotic mechanism. This ROS-independent apoptosis in yeast is significant because it has not been previously 38
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Fig. 7. AuNPs exert ROS-independent apoptosis effects against C. albicans through intracellular disruption, including destruction of nucleus and nucleic acid and attenuation of mitochondrial homeostasis.
reported. The present study provides a new insight of apoptotic mechanisms by AuNP in C. albicans. In addition, since AuNPs has no toxicity in human cells (Wang et al., 2008; Conde et al., 2014), this study could help the improved understanding of AuNPs as antifungal agents, and suggests the potential for broad applications in the treatment of human pathogens.
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Reactive oxygen species-independent apoptotic pathway by gold nanoparticles in Candida albicans
MARK
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Minju Seong, Dong Gun Lee
School of Life Sciences, KNU Creative BioResearch Group (BK21 Plus Program), College of Natural Sciences, Kyungpook National University, Daehak-ro 80, Buk-gu, Daegu 41566, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Gold nanoparticles Genomic DNA interactions Mitochondrial dysfunction ROS-independent apoptosis Candida albicans
Candida albicans is the most common pathogenic fungus in humans, causing cutaneous and life-threatening systemic infections. In this study, we confirmed using propidium iodide influx that gold nanoparticles (AuNPs), which are promising materials for use as antimicrobial agents, did not affect the membrane permeability of C. albicans. Thus, the fungal cell death mechanisms induced by AuNPs were assessed at intracellular levels including DNA damage, mitochondrial dysfunction, and reactive oxygen species (ROS) overproduction. AuNPs interacted with C. albicans DNA leading to increased nuclear condensation and DNA fragmentation. Changes in the mitochondria induced by AuNPs involving mass, Ca2+ concentrations, and membrane potential indicated dysfunction, though the level of intracellular and mitochondrial ROS were maintained. Although ROS signaling was not disrupted, DNA damage and mitochondrial dysfunction triggered the release of mitochondrial cytochrome c into the cytosol, metacaspase activation, and phosphatidylserine externalization. Additionally, the AuNPs-induced apoptotic pathway was not influenced by N-acetylcysteine, an ROS scavenger. This indicates that ROS signaling is not linked with the apoptosis. In conclusion, AuNPs induce ROS-independent apoptosis in C. albicans by causing DNA damage and mitochondria dysfunction.
1. Introduction The common pathogenic fungi, Candida albicans resides as a commensal organism in the mucocutaneous cavities of the skin, vagina and intestine of humans. It can cause infections under certain pathological and physiological conditions such as infancy, diabetes, pregnancy, steroidal chemotherapy, and prolonged broad spectrum antibiotic administration as well as acquired immunodeficiency syndrome (AIDS) (Manohar et al., 2001). Resistance to conventional drugs is rapidly emerging, and the decreased activity of these drugs against C. albicans has been observed on some level for every currently used drug class (Sanglard, 2017). Thus, development of novel and effective antifungal agents against C. albicans has gained major interest. Nanoparticles, defined as being between 10 and 100 nanometers in size, are promising materials owing to their wide variety of potential biological, biomedical, catalytic, optoelectronic, and pharmaceutical applications (Mohanraj and Chen, 2006). Previous studies have shown that nanoparticles can act as antimicrobial agents because of their ability to interact with microorganisms (Albanese et al., 2012). Because of these characteristics, various metal nanoparticles have been studied to determine their unique antimicrobial properties and their potential
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usage in a wide range of applications such as medical instruments, textiles, and purifications (Sondi and Salopek-Sondi, 2004; Espitia et al., 2012). Gold nanoparticles (AuNPs) are a well-studied material and their unique chemical and physical properties make them promising therapeutic agents without inherently toxic to human cells (Wang et al., 2008; Alkilany et al., 2012; Conde et al., 2014). AuNPs have been widely used in cancer treatments as a drug delivery system and thermal therapy (Huang et al., 2008; Brown et al., 2010). Furthermore, in previous studies, AuNPs showed that antimicrobial activity against various pathogens including Escherichia coli, Streptococcus mutans, and Candida species (Hernandez-Sierra et al., 2008; Lima et al., 2013; Wani et al., 2013). However, while their antimicrobial activity has been the focus of numerous studies, and there has yet been no study demonstrating their mechanism against C. albicans. Therefore, in this context, this study aims to confirm the mode of fungicidal action of AuNPs on C. albicans.
Corresponding author. E-mail address: [email protected] (D.G. Lee).
https://doi.org/10.1016/j.micres.2017.11.003 Received 12 August 2017; Received in revised form 28 October 2017; Accepted 4 November 2017 Available online 06 November 2017 0944-5013/ © 2017 Elsevier GmbH. All rights reserved.
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genomic DNA that was extracted from C. albicans cells (Hanna and Xiao, 2006). Fluorescence changes in the AuNPs after the addition of genomic DNA were recorded using a RF-5301PC spectrofluorometer. An excitation wavelength of 527 nm (approximate absorption maximum) was used for the AuNPs. The emission spectra were recorded in the wavelength range from 460 nm to 600 nm. The fluorescence spectra of AuNPs were determined at room temperature in the presence of 1 μg of genomic DNA (An and Jin, 2012).
Table 1 The antifungal effect of AuNPs and H2O2. Fungal strains
C. albicans ATCC 90028
MIC (μg/ml) AuNPs
H2O2
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20
2. Materials and methods
2.6. Mitochondrial mass detection
2.1. Nanoparticle preparation and antifungal activity of AuNPs
To evaluate mitochondrial mass, Mitotracker Green FM fluorescent probe (Invitrogen, Carlsbad, CA) was used. Harvested C. albicans cells were washed with PBS and incubated with AuNPs for 4 h at 28 °C. Then, AuNP-treated cells were resuspended in PBS and incubated with 100 nM Mitotracker Green FM for 45 min. Next, the cells were washed twice, and the fluorescence was measured using a spectrofluorophotometer (Blanquer-Rossello et al., 2017).
C. albicans (ATCC 90028) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in aerated YPD (yeast extract-peptone-dextrose) broth (BD Bioscience, Franklin Lakes, NJ, USA) at 28 °C. Gold nanoparticles (AuNPs) were purchased from Sigma-Aldrich. The particles were 30 nm in diameter and a stabilized suspension in phosphate-buffered saline (PBS). H2O2 (SigmaAldrich) was used as a positive control for comparisons of the physiological responses caused by AuNPs. C. albicans cells were inoculated into YPD and then dispensed into microtiter plates. The minimum inhibitory concentration (MIC) was determined by a standard microdilution method. After incubation with AuNPs for 12 h, the growth was measured using a microtiter ELISA Reader (Molecular Devices Emax, CA; Table 1).
2.7. Mitochondrial Ca2+ concentration detection To assess mitochondrial Ca2+ levels, a Rhod-2AM (Molecular Probes) assay was used. Harvested C. albicans cells were washed in PBS. Next, the cells were incubated with AuNPs or H2O2 for 4 h at 28 °C. After incubation, the cells were centrifuged twice at 12,000 rpm (Sorvall Biofuge Fresco) for 5 min with Krebs buffer (pH 7.4; 4 mM KCl, 132 mM NaCl, 1.4 mM MgCl2, 10 mM HEPES, 6 mM glucose, 10 mM NaHCO3, and 1 mM CaCl2) and treated with 1% bovine serum albumin and 0.01% pluronic acid F-127. The cells were then incubated with a Ca2+-sensitive fluorescent dyes (Rhod-2AM; Molecular Probes) for 30 min and washed three times with Ca2+-free Krebs buffer. The fluorescence intensity of Rhod-2AM was examined using a spectrofluorophotometer (excitation/emission = 552 nm/581 nm) (Shimadzu RF-5301PC, Shimadzu) (Valipour et al., 2015).
2.2. Membrane permeabilization detection Membrane permeabilization was detected using propidium iodide (PI). The cells were washed with PBS (2.7 mM KCl, 137 mM NaCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) and incubated with 32 μg/mL AuNPs for 4 h at 28 °C. The fluorescent intensity was measured using a FACSverse flow cytometer (BD Biosciences) (Lecoeur et al., 2001).
2.8. Mitochondrial membrane potential detection
2.3. DNA condensation assay
The collapse of the electrochemical gradient across the mitochondrial membrane was measured using a JC-1 dye (Molecular Probes). The fungal cells were washed with PBS and the cells were then incubated with AuNPs for 4 h at 28 °C. After centrifugation, the cells were resuspended in warm PBS and incubated with JC-1 for 10 min. The fluorescent intensity of the JC-1 monomer and aggregate (FL1 and FL2, green and red fluorescence, respectively) was measured using a FACSverse flow cytometer (Alonso-Monge et al., 2009).
A DNA-specific fluorescent dye, 4′-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was used to analyze nuclear condensation. C. albicans cells were washed with PBS buffer and incubated with AuNPs for 4 h at 28 °C. After incubation, the cells were resuspended in PBS and treated with DAPI for 20 min. Next, the cells were washed, and the fluorescence was analyzed using a spectrofluorophotometer (Shimadzu RF-5301PC; Shimadzu, Kyoto, Japan) and visualized using a fluorescence microscope (Nikon Eclipse Ti-S; Nikon, Japan) (Madeo et al., 1997).
2.9. Intracellular ROS formation assay To detect intracellular ROS levels, an oxidation-sensitive fluorescent dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes) was used. The cells were washed with PBS buffer and incubated with AuNPs for 4 h at 28 °C. After centrifugation, the cells were incubated with H2DCFDA for 1 h in the dark and washed twice. The fluorescent intensity was measured using a FACSverse flow cytometer (Echave et al., 2003). Mitochondrial ROS were measured using MitoSOX Red (Molecular Probes). C. albicans cells were incubated with AuNPs for 4 h at 28 °C and centrifuged at 12,000 rpm (Sorvall Biofuge Fresco). The cells were suspended in 5 μM MitoSOX Red reagent at 28 °C for 30 min and then the stained cells were washed with PBS. The fluorescent cells were analyzed with a FACSverse flow cytometer (Bankapalli et al., 2015).
2.4. DNA fragmentation assay For the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, a method for assessing DNA damage, the cells were washed with PBS and incubated with AuNPs for 4 h at 28 °C. Next, the cells were fixed with 2% paraformaldehyde for 1 h and incubated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) on ice for 2 min. Then, the cells were incubated with the TUNEL reaction mixture from an in situ cell death detection kit (Roche Diagnostics, Mannheim, Germany) for 1 h at 28 °C. Finally, the cells were washed, and the fluorescence was assayed using a spectrofluorophotometer (Madeo et al., 1997; Valipour, 2012). 2.5. Interactions of AuNPs with C. albicans DNA
2.10. Detection of cytochrome c concentration To investigate interactions of DNA with AuNPs, fluorescence measurements were evaluated by the process described by Atay et al. (2009) and Sung and Lee (2007), with slight modification. we prepared
To confirm the effect of AuNPs on cytochrome c release, the levels of cytochrome c in the cytosol and mitochondria were measured as 34
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directly injure the membrane of C. albicans. Because the antifungal activity of AuNPs is not related to membrane disruption, the effect of AuNPs on several intracellular responses was tested.
previously described (Wu et al., 2010; Barbu et al., 2013; Choi and Lee, 2015). C. albicans cells were washed with PBS and pretreated with 1 mM N-acetylcysteine (NAC; Sigma-Aldrich) at 28 °C for 10 min. Next, the C. albicans cells were incubated with AuNPs for 4 h at 28 °C. After centrifugation, the cells were homogenized in a homogenization medium [50 mM Tris (pH 7.5), 2 mM EDTA, and 1 mM phenylmethylsulphonyl fluoride] supplemented with 2% glucose. After centrifugation for 45 min, the cytosolic cytochrome c content of the supernatant was measured, and the pellet was suspended in 50 mM Tris (pH 5.0) with 2 mM EDTA to measure the mitochondrial cytochrome c content. After reduction via the addition of 500 mg/mL ascorbic acid, the cytosolic and mitochondrial cytochrome c levels were measured at 550 nm using a spectrophotometer (GE Healthcare, Little Chalfont, Buckinghamshire, UK) (Wu et al., 2010; Dwyer et al., 2012).
3.2. AuNPs induce nucleic acid damage through physical interactions in C. albicans We next investigated DNA damage because cells with damaged DNA fail to undergo productive DNA repair, and this trigger the cell death response (Roos and Kaina, 2006). DNA damage was indicated by morphologic changes including nuclear condensation and fragmentation (Hong et al., 2004). DAPI was used to monitor morphological changes in the nucleus, based on the roundness and size through binding to the minor groove of A-T rich regions of DNA (Kapuscinski, 1995; Daniel and DeCoster, 2004). Spectrofluorophotometric and fluorescence microscopic analyses revealed that cells treated with AuNPs or H2O2 displayed more concentrated fluorescence in comparison with untreated cells, indicating nuclear condensation (Fig. 2a). The TUNEL assay has been designed to detect the extensive DNA degradation that is present in apoptotic cells (Negoescu et al., 1998). This assay revealed that C. albicans cells exposed to AuNPs or H2O2 displayed increases in fluorescence indicating DNA fragmentation (Fig. 2b). AuNPstreated cells undergo physical changes such as nucleus condensation and DNA fragmentation. Physical changes in C. albicans DNA may induce a failure to complete cell division that leads to apoptosis (Henry et al., 2013). We thus suggest that AuNPs induce physical changes leading to a failure in the normal function of DNA in C. albicans. The potential impact of nanoparticles binding directly to DNA to create cellular damage has been discussed previously (Lecoeur et al., 2001). The nanoparticles containing AuNPs have native fluorescence, and the fluorescence intensity was reduced by binding DNA (He et al., 2008; Atay et al., 2009). To investigate the direct action of AuNPs on the DNA of C. albicans, we measured the AuNPs fluorescence spectrum, which was used to elucidate the interactions between AuNPs and C. albicans DNA. The emission maximum and intensity depend upon the surroundings of the AuNPs (Sung and Lee, 2007). We assessed the changes in AuNPs fluorescence in the presence and absence of C. albicans DNA. The results showed that the fluorescence intensity decreased in the presence of C. albicans DNA (Fig. 2c). Based on these experiments showing fluorescence changes, we suggest that AuNPs directly interact with the DNA and lead to nucleus condensation and DNA fragmentation. These changes would support to the breakdown of intracellular maintenance caused by AuNPs.
2.11. Metacaspase activation Activation of metacaspases was detected with the CaspACE FITCVAD-FMK In Situ Marker (Promega, Madison, WI, USA). The cells were washed with PBS and pretreated with 1 mM NAC at 28 °C for 10 min. After 10 min, the cells were incubated with AuNPs for 4 h at 28 °C. Then, the cells were washed twice and incubated with CaspACE fluorescein isothiocyanate (FITC)-VAD-FMK for 20 min. After centrifugation, the cells were resuspended in PBS, and the fluorescence was measured using a FACSverse flow cytometer (Madeo et al., 2002). 2.12. Phosphatidylserine (PS) externalization PS external exposure was assessed after binding of FITC-conjugated annexin V and the loss of membrane integrity after PI uptake. For preparation of protoplasts, the cells were digested with 0.1 M potassium phosphate buffer (PPB, pH 6.0) including 1 M sorbitol and 15 mg/mL lysing enzyme (Sigma-Aldrich) for 4 h at 28 °C to remove the cell wall. The protoplasts were then suspended in 0.1 M PPB (pH 6.0) containing 1 M sorbitol at 28 °C. After centrifugation, the cells were pretreated with 1 mM NAC at 28 °C for 10 min, incubated with AuNPs for 4 h at 28 °C, and washed twice with 0.1 M PPB (pH 6.0) with 1 M sorbitol. Then, the cells were stained with FITC-labeled annexin V and PI (BD Pharmingen, San Diego, CA). PS externalization was analyzed using a FACSverse flow cytometer (Madeo et al., 1997). 2.13. Statistical analysis The results are expressed as mean ± standard deviation of at least three independent experiments. Analysis of variance (ANOVA) calculated using SPSS Software was used to determine any significant differences among the samples. For pairwise comparisons, Tukey’s Honestly Significant Difference test (HSD) was used. Statistically significant results had P-values < 0.05.
3.3. Mitochondrial dysfunction is involved in AuNP-induced cell death Mitochondria play important roles in cell survival, providing cellular energy as adenosine triphosphate (ATP), and the apoptotic pathway (Akbar et al., 2016). Therefore, we assessed whether AuNPs caused changes in mitochondrial mass, Ca2+ concentration and membrane potential. The fluorescent MitoTracker Green FM, a dye which binds covalently to the inner mitochondrial membrane and fluoresces independent of membrane potential, was used to investigate mitochondrial mass (Blanquer-Rossello et al., 2017). In both AuNP- and H2O2-treated cells, the mitochondrial mass was significantly increased compared to control cells (Fig. 3a). These results indicate that AuNPs cause alterations in mitochondrial mass. Increase in mitochondrial mass appeared when mitochondrial proliferation and fusion (MahyarRoemer et al., 2001; Arakaki et al., 2006; Peng et al., 2016; Ampawong et al., 2017), and the increase were these results of a cellular response to compensate for reduced mitochondrial function (Nugent et al., 2007). For the reason, increased mitochondrial mass indicates mitochondrial dysfunction. Therefore, we conclude that mitochondria in AuNPs-treated C. albicans cells are functioning abnormally. Imbalances of the mitochondrial ion homeostasis have been shown to indicate an abnormal status of the mitochondria. Mitochondrial Ca2+
3. Results and discussion 3.1. AuNPs do not have an effect on membranes The membrane of a living cell plays a vital role in regulating activities within and around the cell (Madeo et al., 1997). Cell death is precipitated by a sudden breakdown of the plasma membrane permeability barrier (Lemasters et al., 1987). To clarify whether AuNPs cause membrane disruption, membrane permeability was investigated. We measured membrane permeability using PI, a membrane-impermeable dye that only enters cells that have damaged membranes (Pina-Vaz et al., 2001). For flow cytometric analysis (Fig. 1), the cells treated with AuNPs showed similar PI fluorescence intensities (0.73%) as untreated cells (0.61%). When H2O2 was applied, the fluorescence increased to 97.67%. These results suggest that AuNPs may not have an effect on membrane permeability in C. albicans. Therefore, the particles did not 35
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Fig. 1. Flow cytometry analysis of membrane permeabilization by AuNPs using propidium iodide (PI) staining in C. albicans. The x-axis shows the PI fluorescence and the y-axis shows the SSC.
Fig. 2. DNA and nuclear damage during late stage apoptosis by AuNPs (a) Concentrated or split fluorescence of DAPI indicates nuclear condensation by a spectroflourophotometer and visualized by fluorescence microscopy. 1: Control, 2: AuNPs treatment, 3: H2O2 treatment. (b) DNA fragmentation was observed using a TUNEL assay. Data were analyzed using one-way ANOVA followed by a post hoc Tukey’s test, *P < 0.05 versus control. (c) Quenching of the fluorescence of AuNPs, both in the absence and presence of DNA. The x-axis shows the excitation wavelength and the y-axis shows the emission wavelength.
(Fig. 3b). Increased Rhod-2AM fluorescence indicates a transient elevation in mitochondrial Ca2+ levels (Duchen, 2000). When C. albicans cells were exposed to AuNPs or H2O2, mitochondrial Ca2+ levels
affects several steps of energy metabolism such as synchronize ATP generation (Hajnóczky et al., 2006). Therefore, mitochondrial Ca2+ was detected using the mitochondrial Ca2+-sensitive dye Rhod-2AM
Fig. 3. Analysis of mitochondrial dysfunction (a) Mitochondrial mass was assessed using Mitotracker Green FM and analyzed by a spectrofluorophotometer. The y-axis shows the relative Mitotracker Green fluorescence. These data were analyzed using one-way ANOVA followed by a post hoc Tukey’s test, *P < 0.05 versus control. (b) Influx of mitochondrial Ca2+ was assessed using Rhod-2AM and analyzed by spectrofluorophotometer. The y-axis shows the relative Rhod-2 AM fluorescence. Data were analyzed using one-way ANOVA followed by a post-hoc Tukey’s test, *P < 0.05 versus control. (c) Mitochondrial membrane depolarization was detected using JC-1 and analyzed by flow cytometry. The x-axis shows the FL1 fluorescence and the y-axis shows the FL2 fluorescence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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(Fig. 4b). Overproduction of ROS was not detected in AuNP-treated cells. Numerous studies have suggested that ROS are generated by the cellular disruption that occurs during apoptosis and that ROS overproduction triggers cell death (Carmona-Gutierrez et al., 2010). However, a pathway directly linking ROS to yeast cell death has yet to be elucidated (Wysocki and Kron, 2004). ROS-independent cell apoptosis has rarely been reported in cells and very little is known about this pathway (Wysocki and Kron, 2004). Thus, we expect that ROS signaling does not influence the mechanisms of AuNP-induced cell damage.
increased significantly compared to control cells. These results showed that AuNPs trigger an increase in mitochondrial Ca2+ levels. Mitochondrial Ca2+ uptake can induce the opening of the mitochondrial permeability transition pore leading to changes in the mitochondrial mass and cell death by apoptosis (Rizzuto et al., 2012). AuNPs cause collapse of mitochondrial Ca2+ homeostasis via overload of mitochondrial Ca2+. Thus, disruption of mitochondrial Ca2+ homeostasis could play a role in initiating AuNPs-induced cell death. The disruption of mitochondrial Ca2+ homeostasis is typically coupled with a reduction in mitochondrial membrane potential, and it is known that the reduction of mitochondrial membrane potential constitutes an early, irreversible step of programmed cell death (Carraro and Bernardi, 2016). JC-1, a lipophilic cationic dye that interacts with the mitochondrial membrane, was used to estimate mitochondrial membrane depolarization (Sifaoui et al., 2014). As expected, flow cytometry analysis showed that AuNP-treated cells showed decreased emitted fluorescence (Y mean/X mean = 7.53) in comparison with untreated cells (Y mean/X mean = 44.39). H2O2-treated cells also showed fluorescence change (Y mean/X mean = 0.85) (Fig. 3c). Thus, depolarization of the mitochondrial membrane occurred after AuNP treatment. Measurement of membrane potential provides information about energy metabolism, matrix configuration, and cytochrome c release during apoptosis (Gottlieb et al., 2003). Mitochondria membrane depolarization in AuNPs-treated C. albicans can allude changes in intracellular networks. To summarize, these results shows that physical and physiological changes occurred such as increased mass, mitochondrial calcium disruption, and loss of mitochondrial membrane potential, and it supports mitochondrial dysfunction in the presence of AuNPs in C. albicans.
3.5. AuNPs trigger an apoptotic pathway unrelated to ROS signaling Cytochrome c is a component of the electron transport chain in mitochondria. It transfers electrons from complex III to IV. Thus, the release of cytochrome c is an indicator of electron transport chain failures (Huttemann et al., 2011). To investigate the translocation of cytochrome c to the cytoplasm from the mitochondria, we detected cytochrome c levels using spectrophotometric analysis (Wu et al., 2010; Barbu et al., 2013; Choi and Lee, 2015). Additionally, NAC pretreated groups were employed to ensure that ROS signaling is not involved in the apoptotic pathway induced by AuNPs. NAC is a source of sulfhydryl groups in cells and a scavenger of free radicals as it interacts with ROS (Bavarsad Shahripour et al., 2014). Cytosolic cytochrome c levels increased in AuNPs treated cells compared to untreated cells. NAC-pretreated cells with AuNPs also showed increased cytochrome c levels in the cytosol (Fig. 5a). In the mitochondria, cytochrome c levels decreased in both AuNP-treated and NAC-pretreated cells in comparison with untreated cells (Fig. 5b). The H2O2-treated cells showed also significantly increased levels of release. These results suggest that cytochrome c is released from the mitochondria into the cytosol after treatment with AuNPs and thus loses its function as an electron transmitter. Additionally, we propose that ROS do not have an influence on cytochrome c release based on the results from NAC-pretreated cells with AuNPs. Cytochrome c has been found to have dual functions in controlling both cellular energetic metabolism and apoptosis. Opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential and the release of apoptogenic factors (Di Lisa et al., 2001). AuNPs-induced mitochondrial membrane depolarization can lead to the release of cytochrome c into the cytosol. Mitochondria play key roles through the release of cytochrome c into cytosol in AuNPs-mediated apoptosis. The release of cytochrome c into the cytosol from the mitochondria can lead to the activation of metacaspase. The metacaspase of yeast is a functional homologue of the mammalian caspases that cleave specific substrates and trigger apoptotic cell death. Metacaspase is always present within the cell in an inactive form and can be activated by cleavage (Barbu et al., 2013). FITC-VAD-FMK is a fluorescent analogue of the cell permeable pan-caspase inhibitor Z-VAD-FMK [carbobenzoxy-valyl-alanyel-aspartyl-(O-methyl)-fluoromethylketone] and is a green fluorescent marker that penetrates intact living cells where it irreversibly
3.4. AuNPs did not disrupt ROS signaling ROS are generated as a consequence of or as part of cellular disruption in many different cellular processes including proliferation, inflammation, aging, and death (Li et al., 2011; Schieber and Chandel, 2014). Moderate concentration of ROS are also involved in physiological responses as part of defense mechanisms and signaling processes (Espinosa-Diez et al., 2015). We assessed the levels of intracellular and mitochondrial ROS using the fluorescent ROS indicators H2DCFDA and MitoSOX Red, respectively. In the presence of ROS, H2DCF is rapidly oxidized into the highly fluorescent DCF (Jin et al., 2005). In the flow cytometry analysis, the cells treated with AuNPs showed minimal increases in the H2DCFDA fluorescent intensity (3.79%) in comparison with untreated cells. When H2O2 was applied, the fluorescence intensity increased to 49.14% (Fig. 4a). Mitochondrial ROS were detected using MitoSOX Red, a fluorogenic dye that selectively detects mitochondrial superoxide (Bankapalli et al., 2015). Similar to the intracellular ROS results, the cells treated with AuNPs showed minimal increases in MitoSOX Red fluorescence with an intensity of 7.54% while H2O2 treated cells had notable fluorescence increases with an intensity of 79.48%
Fig. 4. Assessment of ROS accumulation after AuNPs treatment (a) ROS accumulation in C. albicans was evaluated by H2DCFDA and analyzed by flow cytometry. The x-axis shows the H2DCFDA fluorescence and the y-axis shows the cell counts. (b) Mitochondrial-specific ROS was evaluated using MitoSOX Red and analyzed by flow cytometry. The x-axis shows the MitoSOX Red fluorescence and the y-axis shows the cell counts. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Measurment of intracellular cytochrome c levels. The release of cytochrome c to the cytoplasm (a) from the mitochondria (b) in C. albicans was analyzed by measuring the absorbance at 550 nm with a spectrophotometer, and the y-axis shows the relative cytochrome c level. Data were analyzed using one-way ANOVA followed by a post-hoc Tukey’s test, *P < 0.05 versus control.
Fig. 6. Apoptosis induced by AuNPs (a) Caspase activation in C. albicans was evaluated using a FITC-VAD-FMK assay, and was analyzed by flow cytometry. The x-axis shows the FITCVAD-FMK fluorescence and the y-axis shows the SSC. (b) Phosphatidylserine (PS) externalization during early apoptosis was assessed by FITC-annexin V and PI double staining, and was analyzed by flow cytometry. The x-axis shows the Annexin V FITC fluorescence and the y-axis shows the propidium iodide fluorescence.
shown in Fig. 6b. The annexin V-stained apoptotic cell population increased from 11.82% to 25.64% after AuNP treatment, and NAC-pretreated cells showed similar results. Additionally, the apoptotic cell population increased to 37.03% after H2O2 treatment. This result shows that AuNPs can cause PS external exposure in C. albicans indicating that AuNPs-induced early apoptosis. In addition, PS externalization mediated by AuNPs is unrelated to ROS in C. albicans. These apoptotic features support that the cells under investigation underwent ROS-independent apoptosis. In all of the NAC-pretreated experiments, the cells had similar results as AuNP-treated cells. Generally, apoptotic responses are blocked by ROS scavengers (Simon et al., 2000), whereas, antioxidant have no protective effects against apoptotic responses in ROS-independent apoptosis (Ko et al., 2005). The AuNPs-induced apoptotic pathway functioned regardless of NAC-pretreatment in C. albicans cells. Thus, ROS do not affect the AuNP-mediated apoptotic process.
binds to activated caspases (Lee and Lee, 2017). The CaspACE FITCVAD-FMK In Situ Marker was used to examine metacaspase activation. We also preincubated the cells with NAC to further confirm that ROS were not associated with the AuNP-induced activation of metacaspase. As shown in Fig. 6a, increase of metacaspase activation was detected in cells treated with AuNPs (35.83%), NAC-pretreated AuNPs (31.82%), and H2O2 (51.87%) compared to untreated cells (16.65%). This result shows that AuNPs induce metacaspase activation. Since NAC-pretreated cells have similar results as cells treated with only AuNPs, ROS does not appear to have an effect on metacaspase activation. In yeast, apoptosis is governed chiefly by metacaspases, which are responsible for destroying the cell from within (Carmona-Gutierrez et al., 2010). Thus, we suggest that C. albicans undergoes apoptosis via metacaspase activation induced by AuNPs. PS is the most abundant negatively charged phospholipid in eukaryotic membranes. In the early stages of apoptosis, PS become exposed on the external surface of the cell (Tian et al., 2017). PS externalization was detected using annexin V-FITC and PI double staining. Annexin V has a high affinity for PS after it has been externalized from the inner to the outer plasma membrane of apoptotic cells (Liu et al., 2009). Staining cells with a combination of annexin V and PI allows the distinction between living cells (annexin V-/PI-), apoptotic cells (annexin V+/PI-), and late apoptotic/necrotic cells (annexin V+/PI+) (Lecoeur et al., 2001). The results of bivariate FITC-Annexin V/PI analysis of C. albicans cells after treatment with AuNPs and H2O2 are
4. Conclusions In this study, the mode of fungicidal action of AuNPs was confirmed on C. albicans. AuNPs induce destruction of the nucleus, nucleic acids and attenuation of mitochondrial homeostasis, leading to apoptosis (Fig. 7). The apoptotic responses are not associated with ROS, different from the conventional apoptotic mechanism. This ROS-independent apoptosis in yeast is significant because it has not been previously 38
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Fig. 7. AuNPs exert ROS-independent apoptosis effects against C. albicans through intracellular disruption, including destruction of nucleus and nucleic acid and attenuation of mitochondrial homeostasis.
reported. The present study provides a new insight of apoptotic mechanisms by AuNP in C. albicans. In addition, since AuNPs has no toxicity in human cells (Wang et al., 2008; Conde et al., 2014), this study could help the improved understanding of AuNPs as antifungal agents, and suggests the potential for broad applications in the treatment of human pathogens.
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