Nerol triggers mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS in Candida albicans

Accepted Manuscript Title: Nerol triggers mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS in Candida albicans Authors: Jun Tian, Zhaoqun Lu, Yanzhen Wang, Man Zhang, Xueyan Wang, Xudong Tang, Xue Peng, Hong Zeng PII: DOI: Reference:

S1357-2725(17)30040-7 http://dx.doi.org/doi:10.1016/j.biocel.2017.02.006 BC 5081

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The International Journal of Biochemistry & Cell Biology

Received date: Revised date: Accepted date:

16-12-2016 10-2-2017 11-2-2017

Please cite this article as: Tian, Jun., Lu, Zhaoqun., Wang, Yanzhen., Zhang, Man., Wang, Xueyan., Tang, Xudong., Peng, Xue., & Zeng, Hong., Nerol triggers mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS in Candida albicans.International Journal of Biochemistry and Cell Biology http://dx.doi.org/10.1016/j.biocel.2017.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Article Title: Nerol triggers mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS in Candida albicans

Running Title: Nerol induces apoptosis in Candida albicans

Jun Tian1,2#,*, Zhaoqun Lu1#, Yanzhen Wang1, Man Zhang1, Xueyan Wang2, Xudong Tang2, Xue Peng1, Hong Zeng3** 1

College of Life Science, Jiangsu Normal University, Xuzhou 221116, Jiangsu

Province, PR China 2

Key Lab for New Drug Research of TCM and Shenzhen Branch, State R&D Centre

for Viro-Biotech, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057, Guangdong, People’s Republic of China 3

Key Laboratory of Protection and Utilization of Biological Resources/College of life

science, Tarim University, Alar, 843300, Xinjiang, PR China * Correspondence to: Jun Tian, College of Life Science, Jiangsu Normal University, Xuzhou 221116, Jiangsu Province, PR China. Tel.: +86-516-83403172; Fax: +86-516-83403173. ** Corresponding author: Tel: +86-997-4683860; Fax: +86-997-4682523 E-mail addresses:[email protected] (J. Tian), [email protected] (H. Zeng). #

Jun Tian and Zhaoqun Lu contributed equally to this work 1

ABSTRACT The antifungal activity of Nerol (NEL) against Candida albicans, a pathogenic fungus, has a minimum inhibitory concentration (MIC) of 4.4 mM that causes noteworthy candidacidal activity through an apoptosis-like mechanism. Calcium (Ca2+) levels and reactive oxygen species (ROS) production, which are the major causes of apoptosis, were determined in C. albicans cells treated with NEL and were found to increase, which related to mitochondrial dysfunction and disruption. A series of characteristic changes of apoptosis caused by NEL, including mitochondrial membrane depolarization, cytochrome c (cyt c) release, and metacaspase activation were examined using a flow cytometer and Western blot. The results showed that an increase in intracellular Ca2+ and ROS led to dramatically decreased mitochondrial membrane potential (MMP); cyt c was also released from the mitochondria to the cytosol. Other early apoptotic features were also observed with the metacaspase activation. Finally, the morphological changes of the cells were observed, including phosphatidylserine

(PS)

externalization,

nuclear

condensation,

and

DNA

fragmentation through Annexin V-FITC and PI double staining, TUNEL assay, and DAPI staining. The results supported the hypothesis that NEL was involved in the apoptosis of C. albicans cells not only at the early stages, but also at the late stages. In summary, NEL can trigger mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS leading to apoptosis in C. albicans. This research on the mechanism of cell death triggered by NEL against C. albicans has important significance for providing a novel treatment of C. albicans infections. 2

KEYWORDS: Nerol; Candida albicans; Apoptosis; Antifungal; Calcium; Reactive oxygen species

1. Introduction The incidence of fungal infections has been increasing dramatically, leading to high morbidity and mortality (Sudbery, 2011). Candida albicans is associated with a range of clinical symptoms, and has been prominent among the

systemic fungal pathogen

in individuals who are immunocompromised due to AIDS or cancer chemotherapy (Odds, 1988). Additionally, with the large-scale application of broad-spectrum antifungal agents, such as azoles and polyenes, the prevalence of opportunistic pathogen infections have increased significantly; C. albicans is notable for having an increasing treatment failure because of drug resistance (Cho and Lee, 2011). Because C. albicans is one of the most important opportunistic fungal pathogens, attention is given to how the abuse of antibiotics affects its rate of increase in terms of its invasive infections (Maurya et al., 2013). The development of more effective antifungal therapies is therefore of paramount importance. The discovery of natural compounds with antifungal potential could be promising (Teodoro et al., 2015). Nerol (NEL), a type of monoterpene, is a safe, colorless liquid that is also a volatile compound. Our group recently reported that NEL possesses potent antifungal activity against some Aspergillus spp. (Tian et al., 2013; Wang et al., 2015). Although a previous preliminary investigation reported that NEL has 3

significant activity against C. albicans, the mechanism of how NEL works against C. albicans has not yet been determined (Jirovetz et al., 2007). Therefore, this research on how NEL causes the cell death of C. albicans could be important for providing a novel strategy against C. albicans infections. In this study, we aimed to investigate the mechanism of the antifungal action of NEL and to indicate that NEL has potential as a novel therapeutic antifungal agent. We investigated apoptotic effects caused by NEL, including the production of intracellular Ca2+ and reactive oxygen species (ROS), mitochondrial depolarization, cytochrome c (cyt c) release, and metacaspase activation. We also observed the morphological

changes

in

C.

albicans,

including

phosphatidylserine

(PS)

externalization, nuclear condensation and DNA fragmentation, in order to elucidate its mechanism of action. 2. Materials and methods 2.1. Chemicals The NEL (97.0%; CAS-No. 106-25-2) used in this report was purchased from Aladdin Industrial Corporation (Shanghai, China). It was prepared as a stock solution in 0.1% (v/v) Tween-80. 2.2. Microorganism and media C. albicans (ATCC 64547) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, U.S.A.). The fungal strain cultures were routinely maintained on a Sabouraud dextrose agar (SDA) slant at 4C in a refrigerator. 2.3. Antifungal susceptibility testing 4

To test the susceptibility of NEL-treated cells, the minimum inhibitory concentration (MIC) was determined by the microdilution method (Tian et al., 2014). Tests were performed briefly on sterile U-bottomed 96-well microplates. A single colony of C. albicans from SDA was grown in 50 ml of Sabouraud dextrose broth (SDB) for 18 h in a shaker at 28 ± 2C. The cells were then recovered by centrifugation at 2500 g for 10 min and the pellet resuspended in phosphate buffered saline (PBS) (pH 7.4). The C. albicans cell suspension was adjusted to 1 × 106 CFU/ml with haemocytometer and treated with NEL in the following final concentrations, which ranged from 0.06875 mM to 35.2 mM with a two-fold dilution. The mixture was dispensed into the first ten columns of a 96-well microtiter plate, the eleventh and twelfth columns were used as the blank and negative growth controls wells without inoculation or NEL, respectively. Simultaneously, the Sabouraud dextrose broth (SDB) was distributed into all twelve columns. After 48 h of incubation at 28 ± 2C, the results were noted by visual observation. The concentration of NEL was decided based on the MIC values that prevented the growth of the C. albicans cells. 2.4. Measurement of Ca2+ To evaluate the cytoplasmic mitochondrial Ca2+ levels, the fungal cell suspension was adjusted to 5 × 106 CFU/ml and exposed to NEL with a final concentration of 0, 0.55, 1.1, 2.2, and 4.4 mM (v/v in 0.1% Tween-80, which served as the control treatment). After 2 h, the cells were washed two times with a PBS buffer and resuspended in the buffer. The effect of NEL on the Ca2+ levels was assessed using 5 5

μM Fluo-3/AM or 1 μM Rhod-2/AM, (which was prepared in 2% of Pluronic F-127 solution in dimethyl sulfoxide (DMSO). After incubating the solution at 37C for 50 min, the cells were washed two times and observed using a fluorescence microscope (Tokyo, Japan) (Wang et al., 2011). 2.5. Determination of reactive oxygen species (ROS) production The endogenous ROS levels of C. albicans were measured by fluorometric assay using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Helmerhorst et al., 2001; Kobayashi et al., 2002). Briefly, the C. albicans cell suspension was adjusted to 5 × 106 CFU/ml and exposed to NEL with a final concentration of 0.55, 1.1, 2.2, and 4.4 mM (0.1% Tween-80 control). The controls (without NEL) were handled following the same procedure. After incubating the cells with the NEL at 28 ± 2C for 2 h, DCFH-DA was added to the mixture with a final concentration of 10 μM for 30 min at 30C. The cells were then harvested, washed, and resuspended in 0.5 ml of PBS buffer (pH 7.4). The fluorescence intensities of the suspensions were recorded with an Accuri C6 flow cytometer (BD Biosciences, US) using the excitation and emission wavelengths of 485 and 535 nm, respectively. 2.6. Determination of mitochondrial membrane potential (MMP) The change in the mitochondrial membrane potential (MMP) in C. albicans after treatment with NEL was analyzed using 2-(6-Amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester (Rh-123) through an Accuri C6 flow cytometer (BD Biosciences, US) (Wu et al., 2009). The C. albicans cell suspension (5 × 106 CFU/ml) was treated with 0.55, 1.1, 2.2, and 4.4 mM of NEL and then 100 ng/ml of Rh-123 6

was added to the suspension (0.1% Tween-80 control). The mixture was incubated for 2 h at 28 ± 2C in the dark. The controls (without NEL) were handled following the same procedure. The cells were harvested, washed three times, and resuspended in 0.5 ml of PBS buffer (pH 7.4). The fluorescence intensities of the suspensions were quantitatively analyzed with an Accuri C6 flow cytometer (BD Biosciences, US). 2.7. Analysis of Cyt c release To investigate the cyt c released from the mitochondria, the mitochondria from C. albicans cells were isolated. The C. albicans cells suspension (5 × 106 CFU/ml) was treated with NEL at 0, 2.2, and 4.4 mM (0.1% Tween-80 control) for 2 h at 28 ± 2C. The cultures were then harvested and washed twice with the PBS buffer (pH 7.4). The C. albicans cells were broken using an ultrasonic cell disruptor, and the mitochondria and cytosol were collected in Tris-EDTA buffer (50 mM Tris, pH 7.5, 2 mM EDTA, 1 mM PMSF) and centrifuged at 12000 g for 40 min. The difference in levels of cyt c in the mitochondria and cytosol of C. albicans cells was detected by Western blot analysis (Dumont et al., 1993). The protein content of the mitochondria and cytosol was estimated by a microplate reader with a BCA Protein Assay Kit (Solarbio, Beijing). Equivalent 50 µg of protein each sample was resolved on a 12% SDS– PAGE gel. The separated proteins were transferred onto a nitrocellulose membrane and analyzed by Western blotting using rabbit polyclonal anti-yeast cyt c. Horseradish peroxidase-linked goat antirabbit immunoglobulin G was used as the secondary antibody and the enhanced-chemiluminescence substrate was used for the detection of cyt c. 7

2.8. Detection of metacaspase (caspase-like) activity The activated metacaspases in C. albicans was detected with the CaspACE FITC-VAD-FMK In Situ Marker (Promega; Madison, WI). The C. albicans cell suspension was adjusted to 5 × 106 CFU/ml and exposed to NEL with a final concentration of 0.55, 1.1, 2.2, and 4.4 mM (0.1% Tween-80 control). The controls (without NEL) were handled following the same procedure. After incubating the cells with the NEL at 28 ± 2C for 2 h, the cultures were harvested, washed, and resuspended in 0.5 ml of PBS buffer (pH 7.4). The cells were then stained with 5.0 µg/ml of CaspACE FITC-VADFMK and analyzed with an Accuri C6 flow cytometer (BD Biosciences, US) (Madeo et al., 2002). 2.9. Analysis of phosphatidylserine (PS) For assessing the cellular integrity and externalization of PS, Annexin V-FITC labeling was done using the following modified method (Madeo et al., 1997). C. albicans cells (5 × 106 CFU/ml) were treated with NEL at 0, 0.55, 1.1, 2.2, and 4.4 mM (0.1% Tween-80 control) for 6 h and 12 h. The cultures were then harvested and digested with snailase. After digestion, the cells were stained with 5 µl/ml of propidium iodide (PI) and FITC-labeled Annexin V using the Annexin V-FITC apoptosis detection kit (BD Biosciences, US). After 15 min, the cells were harvested, washed three times, and resuspended in a PBS buffer. PS, which is an apoptosis marker, and the cellular integrity were examined with an Accuri C6 flow cytometer (BD Biosciences, US). 2.10. Measurement of DNA and nuclear damage 8

DNA and nuclear damage is involved in apoptosis. The damage caused by NEL in C. albicans cells was analyzed by fluorescence microscopy using TUNEL and DAPI staining (Madeo et al., 1999; Park and Lee, 2010). The C. albicans cell suspension was adjusted to 5 × 106 CFU/ml and treated for 2 h with 0, 0.55, 1.1, 2.2, and 4.4 mM of NEL (0.1% Tween-80 control). For DNA strand breaks, cells were washed twice with PBS, fixed in 3.6% paraformaldehyde, permeabilized for 2 min on ice, and washed again with PBS. The DNA ends were labeled with an in situ cell death detection kit for 1 h at 37C. The stained cells were observed with a fluorescence microscope (Tokyo, Japan). Nuclear condensation and fragmentation were analyzed by DAPI staining. The cells were harvested, washed, resuspended in 0.5 ml PBS buffer, and incubated with 1 µg/ml DAPI in the dark for 20 min. Cells were then examined with a fluorescence microscope (Tokyo, Japan). 2.11 Statistical analysis For all assays, at least three independent experiments were performed in triplicate. The significant differences between mean values were determined using Duncan’s Multiple Range test (p<0.05) following one-way ANOVA. The statistical analysis was performed using a statistical software (SPSS, 13.0; Chicago, USA). 3 Results 3.1. Antimicrobial activity In this study, we found that the C. albicans cells treated with NEL showed MIC at a concentration of 4.4 mM. Previous research had shown that NEL could be developed into a natural preservative for controlling the infection of table grapes and cherry 9

tomatoes by spoilage fungi, according to our previously published paper (Tian et al., 2013; Wang et al., 2015). The antifungal mechanism of NEL against C. albicans was investigated using subinhibitory concentrations (MIC, MIC/2, MIC/4, and MIC/8) in this study. 3.2. Treatment with NEL increases Ca2+levels In yeast, an increase in intracellular Ca2+ levels leads to cell death, which indicates that free Ca2+ acts as an initiator of apoptosis (Gupta et al., 2003). The intracellular Ca2+ levels were determined after treatment with different concentrations of NEL using two membrane-permeable derivatives of the ratiometric Ca2+ indicator, Fluo-3/AM and Rhod-2/AM. When NEL was not used or was used in low concentrations, the cytosolic and mitochondrial Ca2+ levels in C. albicans were low and nearly undetectable. After treatment with high concentrations of NEL for 2 h, there was an elevation of intracellular Ca2+ levels (Fig. 1). This proved that NEL caused an unexpected movement of Ca2+ and its accumulation in the cytosol and mitochondria. 3.3 Treatment with NEL increases intracellular ROS ROS play important roles as an early initiator of apoptosis in yeasts and other filamentous fungi (Lee et al., 2009; Perrone et al., 2008; Phillips et al., 2003; Shirazi and Kontoyiannis, 2013). To confirm this, the ROS in C. albicans cells was investigated with an Accuri C6 flow cytometer (BD Biosciences, US). Intracellular ROS generation in these cells was monitored using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Compared with the control, which did not have any treatment, at a 10

concentration of 0.55, 1.1, 2.2, and 4.4 mM of NEL, the proportion of fluorescent cells increased to 0.4 ± 0.1%, 0.5 ± 0.1%, 11.5 ± 1.4% and 40.4 ± 1.0%, respectively (Fig. 2). In addition, ROS detection demonstrated that NEL at 0.55 mM and 1.1 mM had a less obvious effect on intracellular ROS levels, but NEL at 2.2 mM and 4.4 mM resulted in a significant increase in ROS (Fig. 2G). Therefore, C. albicans cells treated with NEL at high concentrations had higher ROS levels than untreated controls. 3.4 Treatment with NEL exacerbates the depolarization of mitochondrial membrane potential (MMP) The depolarization of the MMP has been considered to be a characteristic feature in the early stages of apoptosis (Hwang et al., 2012). The dysfunction of mitochondria might be caused by stimulus of ROS. To investigate the change in MMP caused by NEL, the MMP was examined by Rh-123 staining. As shown in Fig. 3, under normal (control) or low concentrations of NEL, just a few of the cells displayed fluorescence, which indicated that the MMP was hyperpolarized in these cells. However, under high concentrations of NEL, C. albicans cells had a significant loss in MMP (Fig. 3G). NEL caused a depolarization in the MMP in a dose-dependent manner. 3.5 Treatment with NEL induces the release of Cyt c The cyt c release from the mitochondria to the cytosol is a representative hallmark of an apoptotic cell (Wang and Youle, 2009), as well as a crucial event in apoptosis, resulting from ROS generation and metacaspase activation. It was assumed that the NEL might induce a release of cyt c from the mitochondria to the cytosol. The Western blot analysis was used to investigate the levels of cyt c in the mitochondria 11

and cytosol of C. albicans cells. The results showed that the cytosolic cyt c levels increased, while the mitochondrial cyt c levels decreased compared with the controls, which were not treated with NEL (Fig. 4). These results indicated that NEL induced the release of cyt c from the mitochondria in C. albicans. 3.6 Treatment with NEL induces metacaspase activation Metacaspases, which are caspase-like cysteine proteases in yeast, are significantly associated with the generation of ROS and mitochondrial dysfunction (Mazzoni and Falcone, 2008). Caspases play a central role in the early stages of the apoptosis and can be detected by FITC-VAD-FMK staining. In our study, cells with activated intracellular metacaspases have fluorescence, whereas control cells appear unstained (Fig. 5). The increasing presence of metacaspase activity in C. albicans cells was observed from 1.9 ± 0.3% to 19.9 ± 1.0% as the concentration of NEL increased in contrast to 0.8 ± 0.1% of the control cells (Fig. 5G). These results demonstrated that NEL induced metacaspase activation. 3.7. Treatment with NEL leads to phosphatidylserine (PS) externalization In yeast, most membrane PS exist in the inner leaflet of the cytoplasmic membrane. During apoptosis and necrosis, PS is translocated to the outer leaflet from the inner surface, which is considered to be an early marker of apoptosis in fungi (Madeo et al., 1997). In yeast, the exposure of PS can be observed by double staining with Annexin V-FITC and PI. In this assay, the apoptotic cells were detected using Annexin V-FITC staining (green fluorescence), whereas necrotic cells were detected using PI staining (red fluorescence). After treatment with different concentrations of NEL for different 12

amounts of time, the cells increased in the Annexin V+/PI– quadrant. In the Annexin V+/PI+ quadrant, the cell population also increased (Fig. 6). This result indicated that NEL leads to both apoptosis and necrosis by PS externalization and membrane disruption. 3.8. Treatment with NEL produces morphological change in DNA and the nuclei DNA fragmentation is one of the late stages in apoptosis (Wadskog et al., 2004). DNA fragmentation can be analyzed by fluorescence microscopy using TUNEL assay, which is a common method for detecting apoptotic DNA cleavage in individual nuclei by labeling fluorescent dUTP at the 3’-OH ends of DNA (Madeo et al., 1997). It was found that cells exposed to NEL showed a TUNEL positive phenotype, while the controls that were not exposed to NEL showed a TUNEL negative phenotype (Fig. 7A). These results indicated that the treatment of C. albicans cells with NEL caused DNA fragmentation and margination. Nuclear

morphologic

changes, including

chromosome

condensation

and

fragmentation, occur during late-stage apoptosis (Kapuscinski, 1995). Nuclear condensation and fragmentation were analyzed using a DAPI stain, which is a cell-permeable fluorescent dye. C. albicans cells that were treated with different concentrations of NEL were stained with DAPI and showed a more concentrated fluorescence intensity in single cells compared with untreated cells (Fig. 7B). These results showed that the treatment of C. albicans cells with NEL caused nuclear morphologic changes. 4. Discussion 13

New-generation antifungal drugs are urgently required to treat fungal infections because the currently available clinically prescribed drugs are associated with low efficacy, high toxicity, and drug resistance (Khan et al., 2014). Therefore, it is necessary to investigate the fungal cell death processes to identify the manner of cell death. Apoptosis is a major form of programmed cell death (PCD), as well as a highly regulated cellular suicide program that is essential for metazoan development (Khan et al., 2014). Damaged, infected, and superfluous cells can be homeostatically removed through apoptosis, while necrotic cell death is characterized by cell lysis (Kroemer et al., 2007). Apoptosis is usually mediated by two key molecular signals: Ca2+ and ROS (Brookes et al., 2004). In yeast, intracellular Ca2+ leads to cell death; an increase in Ca2+ levels indicates that free Ca2+ acts as an initiator of apoptosis (Gupta et al., 2003; Mallick et al., 2015). During apoptosis, the depolarization of the plasma membrane potential causes an imbalance between the Ca2+ influx and the Ca2+ export, which results in a progressive increase in mitochondrial Ca2+ uptake (Hajnoczky et al., 2006). For this study, intracellular Ca2+ levels were determined after NEL treatment of C. albicans cells using two fluorescent dyes, Fura-2AM and Rhod-2AM, which are cell permeable and selective for free cytosolic and mitochondrial Ca2+, respectively. The results demonstrated that NEL can cause Ca2+ accumulation in the cytosol and mitochondria. In recent years, several studies have reported that ROS are sufficient as a primary cell death regulator of the apoptotic pathways in yeast (Price et al., 2009). The 14

production of ROS is one of the early changes that are implicated in apoptosis (Benaroudj et al., 2001). Mitochondria represent the major source of intracellular ROS production (Tian et al., 2012). The accumulation of ROS can cause oxidative damage to the macromolecules, which results in DNA damage, DNA strand breaks, and mitochondrial damage in cells (Helmerhorst et al., 2001; Park et al., 2004). On the other hand, ROS that are generated as metabolic byproducts can induce apoptosis in cells in a metacaspase-dependent manner (Wu et al., 2010). In this sense, we analyzed the ROS-stimulated apoptosis in C. albicans cells that were treated with NEL. A DCFH-DA assay was performed to investigate the intracellular ROS levels; results indicated that NEL triggered a noticeable generation of ROS in C. albicans cells. Excessive ROS is a major cause of apoptosis because it disrupts the balance of intracellular ROS, which leads to oxidative stress (Scandalios, 2002; Waris and Ahsan, 2006). The generation of ROS that was induced by NEL might be connected with metacaspase activity (Kajiwara et al., 2001). It was also reported that ROS causes damage to chromosomal DNA and a very rapid decline in MMP with some enzymatic activity (Fleury et al., 2002). During the early stages of apoptosis, the depolarization of the MMP is followed by ROS generation (Kobayashi et al., 2002). The change in MMP was analyzed with Rh-123 dye through an Accuri C6 flow cytometer. As shown in Fig. 3, NEL can induce the depolarization of the mitochondrial membrane, which is a hallmark of apoptosis that is caused by the opening of the membrane pores that are located in the outer membrane of the mitochondria (Chen et al., 1998). In addition, the collapse of 15

the MMP has been considered to be a characteristic feature of the early stages of apoptosis and might initiate a series of events, such as the release of cyt c, metacaspase activation, the disorganization DNA, and cell demise (Hwang et al., 2012). Cyt c, a soluble protein that is electrostatically bound to the outer face of the inner mitochondrial membrane, is a typical feature of apoptotic cell death (Adrain and Martin, 2001; Ludovico et al., 2002). Thus, the levels of cyt c in the mitochondria and in the cytosol of C. albicans cells that were treated with NEL was investigated by Western blot analysis. During apoptosis, the mitochondrial depolarization and disruption resulted in the exposure of cyt c to the intermembrane space from the mitochondria (Gottlieb et al., 2003). Our results indicated that NEL induced the release of cyt c from the mitochondria in C. albicans cells (Fig. 4). After its release into the cytosol, cyt c binds the activation site of the caspase cascade, resulting in apoptosis (Buttner et al., 2007). Caspases are members of a family of cysteine proteases and are recognized as one of the key processes linked to apoptosis in mammalian cells (Madeo et al., 2002). In C. albicans, a putative caspase is encoded by metacaspase 1 (CaMCA1) (orf19.5995) that is involved in the oxidative stress-induced cell death by hydrogen peroxide (H2O2) (Cao et al., 2009). Metacaspase activation plays a central role in the early stages of apoptosis (Zivna et al., 2010). The activated metacaspases in C. albicans that was treated with NEL were detected using the CaspACE FITC-VAD-FMK In Situ Marker. The results significantly demonstrated that NEL induced metacaspase activation. 16

To further confirm that NEL had an effect on general apoptotic features, the morphological changes seen in early and late apoptosis were investigated. In yeast, most membrane PS exists in the inner leaflet of the cytoplasmic membrane (Elmore, 2007). During apoptosis and necrosis, the PS component of the phospholipid bilayer is translocated to the outer leaflet from the inner surface, which is considered to be an early marker of apoptosis in fungi (Elmore, 2007). The exposure of PS can be observed by double staining with Annexin V-FITC and PI. The result of the double staining assay verified that NEL led to both apoptosis and necrosis by PS externalization and membrane disruption. Nuclear condensation and fragmentation are also considered to be late apoptotic phenotypes that result from ROS accumulation, which could lead to apoptosis through the shrinkage of the cell (Cho and Lee, 2011). In this study, nuclear condensation and DNA fragmentation were analyzed by fluorescence microscopy using DAPI staining and TUNEL assay. As shown in Fig. 7, the treatment of C. albicans cells with NEL caused nuclear condensation and fragmentation, which are markers of apoptosis. The results of this study demonstrate the potential mechanism of apoptosis in C. albicans exposed to NEL in vitro (Fig. 8). NEL induced elevations of Ca2+ and ROS production in the cells, which further resulted in mitochondrial dysfunction and disruption. Then, it appeared that the MMP obviously decreased and that cyt c was exposed to the intermembrane space from the mitochondria. Subsequently, both ROS and cyt c induced metacaspase activation, which led to the apoptosis of C. albicans. Meanwhile, the typical morphological characteristics, including PS externalization 17

and DNA fragmentation, were observed at the early and late phases of apoptosis. In short, NEL can trigger mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS leading to apoptosis in C. albicans. The results of this investigation indicate that NEL is a potent candidacidal candidate for the treatment of C. albicans infection.

Conflict of interest The authors declare no competing financial interest.

Acknowledgements This work was supported by National Natural Science Foundation of China (31671944, 31301585), Qing Lan Project of Jiangsu Province, Xinjiang Production & Construction Crops, Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin (BRZD1502), China Postdoctoral Science Foundation (2016M600678), Shenzhen basic research project (JCYJ20140414145007216, JCYJ20160301100720906),

National

Undergraduate

Training

Programs

for

Innovation and Entrepreneurship (201610320027), the Industry-University-Academy Prospective Joint Research Project of Jiangsu Province (BY2016028-01) and the PAPD of Jiangsu Higher Education Institutions.

18

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Figure Captions Fig. 1. Assay for Ca2+ activation was visualized with fluorescence microscopy using Fluo-3/AM and Rhod-2/AM staining. C. albicans cells were incubated with 0, 0.55, 1.1, 2.2, and 4.4 mM of NEL for 2 h at 28C. Scale bars = 5 µm. Fig. 2. The change of intracellular ROS (A–F) was detected by a flow cytometry using DCFH-DA. (A) Autofluorescence of cells without any treatment; (B) fluorescence of cells without NEL treatment for DCFH-DA staining; (C – F) fluorescence of cells exposed to 0.55, 1.1, 2.2, and 4.4 mM NEL. (G) histogram of percentage of stained cells. Values are mean (n = 3) ± SD (*p<0.05). Fig. 3. The depolarization of MMP (A–F) was detected with a flow cytometry by Rh-123. (A) Autofluorescence of cells without any treatment; (B) fluorescence of cells without NEL treatment for Rh-123 staining; (C–F) fluorescence of cells exposed to 0.55, 1.1, 2.2, and 4.4 mM NEL. (G) histogram of percentage of stained cells. Values are mean (n = 3) ± SD (*p<0.05). Fig. 4. The release of cyt c from the mitochondria in C. albicans was measured using the Western blotting method. GAPDH expression is shown as a loading control. Cells were incubated with 0, 2.2, and 4.4 mM NEL for 2 h at 28C. The C. albicans cells were broken using an ultrasonic cell disruptor, and the mitochondria and cytosol were collected in Tris-EDTA buffer. Fig. 5. The effect of NEL on metacaspase activity in C. albicans was confirmed using FITC-VAD-FMK staining by a flow cytometry. (A) Autofluorescence of cells without any treatment; (B) fluorescence of cells without NEL treatment for FITC-VAD-FMK 26

staining; (C–F) fluorescence of cells exposed to 0.55, 1.1, 2.2, and 4.4 mM NEL. (G) histogram of percentage of stained cells. Values are mean (n = 3) ± SD (*p<0.05). Fig. 6. Density plots showing the fluorescence patterns of C. albicans cells exposed to 0, 0.55, 1.1, 2.2, and 4.4 mM NEL. Region Q1-UL shows dead debris (Annexin V– /PI+), region Q1-LL shows viable cells (Annexin V–/PI–), region Q1-LR shows apoptotic cells (Annexin V+/PI–), and Q1-UR shows necrotic cells (Annexin V+/PI+). Fig. 7. DNA and nuclear damage by 0, 0.55, 1.1, 2.2, and 4.4 mM NEL was visualized with fluorescence microscopy using TUNEL and DAPI staining. (A) DNA fragmentation by NEL was determined by TUNEL assay using green fluorescence, which was especially concentrated in margins of cells. (B) Using DAPI staining, the morphological changes in individual nuclei were observed. Scale bars = 5 µm. Fig. 8. The potential mechanism of NEL in C. albicans. NEL induces an elevation of Ca2+ and ROS production, and results in mitochondrial dysfunction and disruption. Metacaspases are activated by ROS and cyt c is exposed to the cytosol, which leads to apoptosis in C. albicans.

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