RESEARCH LETTER
Melittin triggers apoptosis in Candida albicans through the reactive oxygen species-mediated mitochondria/caspasedependent pathway Juneyoung Lee* & Dong Gun Lee School of Life Sciences, KNU Creative BioResearch Group (BK21 Plus Program), College of Natural Sciences, Kyungpook National University, Daegu, Korea
Correspondence: 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 702701, Korea. Tel.: +82 53 9505373; fax: +82 53 9555522; e-mail: [email protected] *Present address: Juneyoung Lee, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Japan
MICROBIOLOGY LETTERS
Received 16 February 2014; revised 5 April 2014; accepted 23 April 2014. Final version published online 13 May 2014. DOI: 10.1111/1574-6968.12450
Abstract Melittin is one of the best-studied antimicrobial peptides, and many studies have focused on the membrane underlying its membrane-disruptive activity. We previously showed that melittin could cause some hallmarks of apoptosis in Candida albicans. Here, we first report the exact mechanism of melittin-induced fungal apoptosis. We first characterized the reactive oxygen species generated by melittin. The results showed that melittin strongly produced highly reactive hydroxyl radicals (˙OH), which contribute to cell death. Next, we showed that melittin also disrupted the mitochondrial membrane potential (DΨm) and induced the Ca2+ release from the endoplasmic reticulum and its remarkable accumulation in mitochondria. Finally, we investigated the role of caspase in the apoptotic pathway. The results showed that melittin activated metacaspase, which was mediated by cytochrome c release. To summarize, melittin is involved in the mitochondria- and caspase-dependent apoptotic pathway in C. albicans. Our findings suggest that melittin possesses a dual antimicrobial mechanism, including membrane-disruptive and apoptotic actions.
Editor: Stefan Olsson Keywords antimicrobial peptide; fungal; programmed cell death.
Introduction Antimicrobial peptides (AMPs) are a major component of the innate immune systems in numerous organisms. AMP deficiency may lead to infection by pathogenic microorganisms or even death (Lehrer & Ganz, 1999). The classic mechanism of AMP is disruption of the microbial plasma membrane (Reddy et al., 2004). Briefly, AMPs interact directly with the bacterial membrane through electrostatic forces between positively charged amino acids, specifically arginine (R) and lysine (K), and negatively charged phospholipids in the bacterial membrane, such as phosphatidylglycerol (PG) and zwitterionic phosphatidylethanolamine (Lohner, 2009). Because the extracellular side of the mammalian cell membrane bilayer contains abundant neutral phospholipids, such as phosphatidylcholine (PC) and ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
sphingomyelin, the antimicrobial activity of most AMPs is microbial cell-specific (Guilhelmelli et al., 2013). Most AMPs exert their activity by disrupting target microorganism membranes via the barrel-stave model or the toroidal model. In the toroidal model, peptides insert into the membrane by forming a bundle, resulting in pore formation (Chen et al., 2013). Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2), the principal active component in the venom of the honeybee, Apis mellifera (Habermann, 1972), is a prototype toroidal poreforming peptide (Guilhelmelli et al., 2013), and its membranolytic mechanism has been well-established in many studies. At nanomolar concentrations, melittin induces the formation of transient pores, which causes leakage of atomic ions and, at micromolar concentrations, melittin forms larger pores that allow the leakage of kilodalton-
FEMS Microbiol Lett 355 (2014) 36–42
37
Novel mechanism of melittin as an apoptosis inducer
sized molecules (Lee et al., 2013). The molecular mechanism of melittin-induced pore formation has been further characterized employing an aspirated giant unilamellar vesicle experiment, fully hydrated multi-layers of peptidelipid (PC/PG) mixtures, and grazing-angle X-ray anomalous diffraction. Specifically, the procedure of stable pore formation, above a critical P/L (peptide-to-lipid molar ratio), the changes in the mechanism that occur as a result of differences in peptide concentration, and the lipid structure of the pore have been thoroughly investigated (Lee et al., 2013). Owing to its potent activity and welldefined effect against the plasma membrane, melittin is always used as a positive control peptide in AMP studies. Previously, we focused on the distinctive effects of melittin in addition to its membrane-disruptive action, and suggested its potential as an apoptosis inducer in the human pathogenic fungus, Candida albicans. After treatment of 2.5 lM melittin, we observed some apparent morphologic changes during apoptosis, such as the appearance of phosphatidylserine on the outer leaflet of cell membranes and both DNA degradation and nuclear fragmentation (Park & Lee, 2010). However, the exact apoptotic pathway remained elusive. Therefore, in this study, we investigated the pathway of melittin-induced fungal apoptosis. In line with our previous study, we used the same concentration (2.5 lM) of melittin in this study. Finally, we demonstrated the potential of AMPs with dual mechanisms.
buffered saline (PBS) with 5 lM 2-[6-(40 -hydroxy)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (HPF). The cells were washed in PBS and analyzed using an FACSCalibur flow cytometer (Becton-Dickinson). In this study, thiourea was used in millimolar levels, as ˙OH scavenger (Whiteman & Halliwell, 1997). For that, 150 mM thiourea was treated simultaneously with 2.5 lM melittin or 10 mM H2O2. The cells were treated with 2.5 lM melittin or 10 mM H2O2 with or without thiourea and cultured in yeast extract peptone dextrose (YPD) media plates for 4 h at 28 °C. Finally, the colony was counted. Data are presented from three independent experiments using the means SD. JC-1 staining for the detection of mitochondrial membrane potential (DΨm)
JC-1 dye (Molecular Probes) was used to detect the change of mitochondrial membrane potential. Candida albicans (ATCC 90028) cells (2 9 104 cells mL1) were treated with 2.5 lM melittin or 10 mM H2O2 for 2 h at 28 °C. The treated cells were washed in PBS and then suspended in 200 lL staining solution containing 2 lg mL1 JC-1 for 20 min at 37 °C. The cells were centrifuged at 500 g for 5 min and the pellet was re-suspended in 1 mL PBS. The cells were analyzed using an FACSCalibur flow cytometer (Becton-Dickinson) (Lee et al., 2012).
Materials and methods
Fura-2, AM and Rhod-2, AM staining for the detection of cytosolic and mitochondrial Ca2+
Solid-phase peptide synthesis
Candida albicans (ATCC 90028) cells (2 9 104 cells mL1) were treated with 2.5 lM melittin or 10 mM H2O2, for 2 h at 28 °C. The cells were washed twice with Krebs buffer (pH 7.2) and treated with 1 mL Krebs buffer, including both 0.01% Pluronic F127 (Molecular Probes) and 1% bovine serum albumin. Thereafter, the cells were treated with 5 lM Fura-2, AM (Molecular Probes) or 10 lM Rhod-2, AM (Molecular Probes) and incubated for 30 min at 28 °C. The cells treated with dyes were then washed with calcium-free Krebs buffer (pH 7.2). Finally, the stained cells were observed with a fluorescence microscope (Nikon Eclipse Ti-S, Japan) and the fluorescence intensity was detected with a spectrofluorophotometer (Shimadzu, RF-5301PC; Arduino et al., 2009). Data are presented from three independent experiments using the means SD.
Melittin was chemically synthesized by Anygen Co. Ltd (Gwangju, Korea). Briefly, the assembly of peptides consisted of a 60-min cycle for each residue at ambient temperature as follows: (1) the 2-chlorotrityl (or 4-methylbenzhydrylamine amide) resin was charged in a reactor and then washed with dichloromethane and N,N-dimethylformamide (DMF), respectively, and (2) a coupling step with vigorous shaking using a 0.14 mM solution of FmocL-amino acids and Fmoc-L-amino acids pre-activated for c. 60 min with a 0.1 mM solution of 0.5 M HOBt/DIC in DMF. Finally, the peptide was cleaved from the resin using a trifluoroacetic acid cocktail solution at ambient temperature (Merrifield, 1986; Sheppard, 2003). HPF staining for the detection of hydroxyl radical generation
The intracellular hydroxyl radical (˙OH) levels were measured by incubating the C. albicans (ATCC 90028) cells (2 9 104 cells mL1) with 2.5 lM melittin (Park & Lee, 2010) or 10 mM H2O2 for 2 h at 28 °C, in phosphateFEMS Microbiol Lett 355 (2014) 36–42
Metacaspase activation and cytochrome c release detection
Activated metacaspases in C. albicans (ATCC 90028) cells were measured using the CaspACETM FITC-VAD-FMK In ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
38
Situ Marker (Promega). The cells (2 9 104 cells mL1) were treated with 2.5 lM melittin or 10 mM H2O2 for 2 h at 28 °C. The treated cells were washed in PBS, suspended in 200 lL of staining solution containing 10 lM CaspACETM FITC-VAD-FMK In Situ Marker, and incubated for 30 min at room temperature in the dark. The cells were re-suspended in PBS and metacaspase activation was analyzed with an FACSCalibur flow cytometer (Becton-Dickinson; Lee et al., 2012). To investigate cytochrome c release after the treatment of melittin, C. albicans (ATCC 90028) cells were treated with 2.5 lM melittin or 10 mM H2O2 for 2 h at 28 °C. The cells were harvested and homogenized in buffer A [50 mM Tris, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.5]. To remove the impurities, the mixture was supplemented with 2% glucose and centrifuged. The supernatant was collected, centrifuged at 30 000 g for 45 min and then used to examine cytoplasmic cytochrome c. To obtain pure mitochondria, the pel-
J. Lee & D.G. Lee
let was washed in buffer B (50 mM Tris, 2 mM EDTA, pH 5.0). Mitochondria was suspended in 2 mg mL1 of Tris-EDTA buffer. After the treatment of 500 mg mL1 ascorbic acid for 5 min, the cytochrome c levels in the cytosolic or mitochondrial fractions were determined at 550 nm with spectrophotometer (Beckman, DU530; Barbu et al., 2013). Data are presented from three independent experiments using the means SD.
Results and discussion Hydroxyl radical-mediated oxidative stress caused by melittin
AMPs are thought to be a novel candidate as a substitute for conventional antibiotics. The general mechanism of AMPs is a potent membrane-disruptive action; however, we hypothesize that AMPs can induce other additional effects after the cell entry. In this study, for the first time,
(a)
(b)
Fig. 1. Hydroxyl radical (˙OH) generation by melittin in Candida albicans. (a) Candida albicans cells were treated with 2.5 lM melittin or 10 mM H2O2 for 2 h at 28 °C, with 5 lM HPF dye, to specifically detect ˙OH. The cells stained were analyzed by FACSCalibur flow cytometer. (b) The survival rates of C. albicans after melittin treatment for 4 h at 28 °C, in the presence of 150 mM thiourea as a scavenger of ˙OH, were examined. The black bars indicate no treatment of thiourea, whereas the white bars indicate the treatment of thiourea. Data are presented from three independent experiments using the means SD. ***P < 0.001 (Student’s t-test).
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Lett 355 (2014) 36–42
39
Novel mechanism of melittin as an apoptosis inducer
Fig. 2. JC-1 staining assay for detecting the change of mitochondrial membrane potential (DΨm) after melittin treatment.
(a)
(b)
Fig. 3. Melittin significantly increased mitochondrial Ca2+ levels in Candida albicans, compared to cytosolic Ca2+. (a) Fluorescence microscopy of Fura-2AM and Rhod-2AM for detection of cytosolic Ca2+ and mitochondrial Ca2+, respectively. The gray images indicate the DIC (differential interference contrast) images. (b) Percentages of Fura-2AM-positive cells or Rhod-2AM-positive cells were assessed. Data are presented from three independent experiments using the means SD. **P < 0.01, ***P < 0.001 (Student’s t-test).
FEMS Microbiol Lett 355 (2014) 36–42
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
40
(a)
(d)
J. Lee & D.G. Lee
(b)
(c)
(e)
Fig. 4. Metacaspase activation and cytochrome c release by melittin in Candida albicans. (a–d) Candida albicans cells were treated with 2.5 lM melittin or 10 mM H2O2 for 2 h at 28 °C, with 10 lM CaspACETM FITC-VAD-FMK In Situ Marker, to detect metacaspase. The cells stained were analyzed by an FACSCalibur flow cytometer. (e) Cytochrome c release from cytosol to mitochondria was examined and the patterns showed mutual release patterns between cytosol and mitochondria. Data are presented from three independent experiments using mean SD. *P < 0.05, **P < 0.01 (Student’s t-test).
we propose a melittin-induced apoptotic mechanism in C. albicans. Reactive oxygen species (ROS) has been focused on, due to their pivotal role in the apoptosis of many different cell types. For example, hydrogen peroxide (H2O2), an ROS, induced CD95 (Fas/APO-1)-mediated neutrophil apoptosis (Kasahara et al., 1997). Nitric oxide (NO˙), another component of ROS, also induced apoptosis in various cells, including mesangial cells, epithelial cells, endothelial cells (M€ uhl et al., 1996) and, by extension, cancer cells (Hajri et al., 1998; Shami et al., 1998). Previously, we suggested that endogenous ROS generation was involved in the antifungal activity of melittin against C. albicans. In this study, among the ROS, we focused on hydroxyl radical (˙OH), as it is a highly reactive molecule and a crucial mediator of apoptosis (Lee et al., 2012). We used HPF staining to detect melittin-induced ˙OH and investigated the survival rates in the presence of thiourea, which mimics the role of an ˙OH scavenger. The ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
results showed that melittin induced higher ˙OH generation (76.12%) than induced by the negative control (4.51%). H2O2 (97.27%) showed more potent inductive activity than melittin; however, the activity of melittin was also remarkable considering the established effect of H2O2 (Fig. 1a). To further investigate the ˙OH generation, we investigated the survival rates after treatment with melittin and H2O2 in the presence of thiourea, as a scavenger of ˙OH. Although there are some discrepancies between H2O2 and melittin, the increased survival rates after treatment with both melittin and thiourea compared with those after treatment with melittin alone, demonstrated that hydroxyl radicals constitute a significant portion of melittin-induced ROS (Fig. 1b). Melittin-induced mitochondrial dysfunction
We subsequently investigated mitochondrial involvement in melittin-induced apoptosis, using JC-1 dye. MitoFEMS Microbiol Lett 355 (2014) 36–42
41
Novel mechanism of melittin as an apoptosis inducer
chondria is the most important site regarding the occurrence of apoptotic key events. In particular, the loss of mitochondrial membrane potential (DΨm) and mitochondrial permeability transition are distinctive features of early apoptosis. In normal cells, JC-1 containing delocalized positive charges can be observed as red fluorescence in the mitochondrial matrix. In contrast, in apoptotic cells, the dye remains in the cytoplasm as fluorescent green (Smiley et al., 1991). The Y mean/X mean ratio of the control was 5.1; however, the ratios in melittin-treated cells (0.6) and H2O2-treated cells (0.4) were remarkably decreased (Fig. 2). These decreases indicated that melittin greatly reduced the mitochondrial membrane potential, triggering the mitochondrial dysfunction of C. albicans. Mitochondrial Ca2+ and melittin-induced fungal apoptosis
In the regulation of apoptosis, Ca2+ homeostasis in both the endoplasmic reticulum (ER) and mitochondria is involved in the localization of regulator proteins, Bcl-2 (Pinton & Rizzuto, 2006). For this reason, investigations of Ca2+ homeostasis or communication between the ER and mitochondria through Ca2+, are critical in apoptosis studies. In this study, to investigate the change in Ca2+ as a mediator of ER-mitochondrial communication and its essential role in apoptosis, we used two dyes, Fura-2AM and Rhod-2AM, to assess the cytosol and mitochondria, respectively. The results showed that melittin caused a slight increase in intracellular Ca2+ levels (Fura-2AM levels), whereas melittin remarkably increased mitochondrial Ca2+ levels (Rhod-2AM levels), even compared with H2O2 (Fig. 3a and b). This mitochondrial Ca2+ overload would be one of the main factors to induce mitochondrial damage through perturbation or rupture by the decrease of DΨm. Namely, we confirm that the mitochondria are primarily involved in the apoptotic pathway of melittin in C. albicans. Effect of melittin on intracellular cytochrome c release and caspase activation
Caspase, an intracellular protease, is activated when apoptosis is induced; specifically, metacaspase 1 (CaMCA1), which encodes a putative caspase in C. albicans, is activated (Shirtliff et al., 2009). We examined the metacaspase dependency of melittin-induced apoptosis. The results showed that the melittin-exposed cells exhibited increased fluorescence intensity of metacaspase (34.17%) similar to that of H2O2 (39.08%) (Fig. 4a–d). This indicates that as a central regulator of apoptosis, metacaspase is associated with melittin-induced fungal FEMS Microbiol Lett 355 (2014) 36–42
cell death. We subsequently investigated the release of cytochrome c, an important stimulus for caspase activation. Cytochrome c is released from the mitochondria into the cytosol before caspase activation during apoptosis (Bossy-Wetzel et al., 1998). Therefore, we investigated the translocation of cytochrome c after peptide treatment. The results showed that after melittin exposure, the cytochrome c concentration in the mitochondria decreased. Conversely, the concentration of cytochrome c in the cytosol increased (Fig. 4e). This could demonstrate that metacaspase activation is induced by cytochrome c leakage. Moreover, the results also suggested that these activities were correlated with mitochondrial membrane potential disruption, as noted previously. In the present study we suggested that melittin-induced apoptosis in C. albicans is triggered by hydroxyl radial generation, mitochondrial membrane permeabilization, and metacaspase activation. It is well-established that melittin disrupts the fungal cell membrane of C. albicans, with potent activity (Lee et al., 2010). Here, we showed a novel mechanism of melittin through the apoptotic pathway. Although the relationship between these two mechanisms remains to be investigated, this study suggests that melittin could trigger fungal apoptosis after membrane damage utilizing a dual mechanism against pathogenic microorganisms.
Acknowledgements This work was supported by a grant from the Next-Generation BioGreen 21 Program (no. PJ008158), Rural Development Administration, Republic of Korea.
References Arduino DM, Esteves AR, Domingues AF, Pereira CM, Cardoso SM & Oliveira CR (2009) ER-mediated stress induces mitochondrial-dependent caspases activation in NT2 neuron-like cells. BMB Rep 42: 719–724. Barbu EM, Shirazi F, McGrath DM, Albert N, Sidman RL, Pasqualini R, Arap W & Kontoyiannis DP (2013) An antimicrobial peptidomimetic induces mucorales cell death through mitochondria-mediated apoptosis. PLoS ONE 8: e76981. Bossy-Wetzel E, Newmeyer DD & Green DR (1998) Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J 17: 37–49. Chen L, Jia N, Gao L, Fang W & Golubovic L (2013) Effects of antimicrobial peptide revealed by simulations: translocation, pore formation, membrane corrugation and Euler buckling. Int J Mol Sci 14: 7932–7958. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
42
Guilhelmelli F, Vilela N, Albuquerque P, Derengowski LD, Silva-Pereira I & Kyaw CM (2013) Antibiotic development challenges: the various mechanisms of action of antimicrobial peptides and of bacterial resistance. Front Microbiol 4: 353. Habermann E (1972) Bee and wasp venoms. Science 177: 314–322. Hajri A, Metzger E, Vallat F, Coffy S, Flatter E, Evrard S, Marescaux J & Aprahamian M (1998) Role of nitric oxide in pancreatic tumour growth: in vivo and in vitro studies. Br J Cancer 78: 841–849. Kasahara Y, Iwai K, Yachie A, Ohta K, Konno A, Seki H, Miyawaki T & Taniguchi N (1997) Involvement of reactive oxygen intermediates in spontaneous and CD95 (Fas/APO-1)-mediated apoptosis of neutrophils. Blood 89: 1748–1753. Lee J, Hwang JS, Hwang B, Kim JK, Kim SR, Kim Y & Lee DG (2010) Influence of the papiliocin peptide derived from Papilio xuthus on the perturbation of fungal cell membranes. FEMS Microbiol Lett 311: 70–75. Lee J, Hwang JS, Hwang IS, Cho J, Lee E, Kim Y & Lee DG (2012) Coprisin-induced antifungal effects in Candida albicans correlate with apoptotic mechanisms. Free Radic Biol Med 52: 2302–2311. Lee MT, Sun TL, Hung WC & Huang HW (2013) Process of inducing pores in membranes by melittin. P Natl Acad Sci USA 110: 14243–14248. Lehrer RI & Ganz T (1999) Antimicrobial peptides in mammalian and insect host defence. Curr Opin Immunol 11: 23–27. Lohner K (2009) New strategies for novel antibiotics: peptides targeting bacterial cell membranes. Gen Physiol Biophys 28: 105–116. Merrifield B (1986) Solid phase synthesis. Science 232: 341–347.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
J. Lee & D.G. Lee
M€ uhl H, Sandau K, Br€ une B, Briner VA & Pfeilschifter J (1996) Nitric oxide donors induce apoptosis in glomerular mesangial cells, epithelial cells and endothelial cells. Eur J Pharmacol 317: 137–149. Park C & Lee DG (2010) Melittin induces apoptotic features in Candida albicans. Biochem Biophys Res Commun 394: 170–172. Pinton P & Rizzuto R (2006) Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum. Cell Death Differ 13: 1409–1418. Reddy KV, Yedery RD & Aranha C (2004) Antimicrobial peptides: premises and promises. Int J Antimicrob Agents 24: 536–547. Shami PJ, Sauls DL & Weinberg JB (1998) Schedule and concentration-dependent induction of apoptosis in leukemia cells by nitric oxide. Leukemia 12: 1461–1466. Sheppard R (2003) The fluorenylmethoxycarbonyl group in solid phase synthesis. J Pept Sci 9: 545–552. Shirtliff ME, Krom BP, Meijering RA, Peters BM, Zhu J, Scheper MA, Harris ML & Jabra-Rizk MA (2009) Farnesolinduced apoptosis in Candida albicans. Antimicrob Agents Chemother 53: 2392–2401. Smiley ST, Reers M, Mottola-Hartshorn C, Lin M, Chen A, Smith TW, Steele GD Jr & Chen LB (1991) Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. P Natl Acad Sci USA 88: 3671–3675. Whiteman M & Halliwell B (1997) Thiourea and dimethylthiourea inhibit peroxynitrite-dependent damage: nonspecificity as hydroxyl radical scavengers. Free Radic Biol Med 22: 1309–1312.
FEMS Microbiol Lett 355 (2014) 36–42
Melittin triggers apoptosis in Candida albicans through the reactive oxygen species-mediated mitochondria/caspasedependent pathway Juneyoung Lee* & Dong Gun Lee School of Life Sciences, KNU Creative BioResearch Group (BK21 Plus Program), College of Natural Sciences, Kyungpook National University, Daegu, Korea
Correspondence: 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 702701, Korea. Tel.: +82 53 9505373; fax: +82 53 9555522; e-mail: [email protected] *Present address: Juneyoung Lee, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Japan
MICROBIOLOGY LETTERS
Received 16 February 2014; revised 5 April 2014; accepted 23 April 2014. Final version published online 13 May 2014. DOI: 10.1111/1574-6968.12450
Abstract Melittin is one of the best-studied antimicrobial peptides, and many studies have focused on the membrane underlying its membrane-disruptive activity. We previously showed that melittin could cause some hallmarks of apoptosis in Candida albicans. Here, we first report the exact mechanism of melittin-induced fungal apoptosis. We first characterized the reactive oxygen species generated by melittin. The results showed that melittin strongly produced highly reactive hydroxyl radicals (˙OH), which contribute to cell death. Next, we showed that melittin also disrupted the mitochondrial membrane potential (DΨm) and induced the Ca2+ release from the endoplasmic reticulum and its remarkable accumulation in mitochondria. Finally, we investigated the role of caspase in the apoptotic pathway. The results showed that melittin activated metacaspase, which was mediated by cytochrome c release. To summarize, melittin is involved in the mitochondria- and caspase-dependent apoptotic pathway in C. albicans. Our findings suggest that melittin possesses a dual antimicrobial mechanism, including membrane-disruptive and apoptotic actions.
Editor: Stefan Olsson Keywords antimicrobial peptide; fungal; programmed cell death.
Introduction Antimicrobial peptides (AMPs) are a major component of the innate immune systems in numerous organisms. AMP deficiency may lead to infection by pathogenic microorganisms or even death (Lehrer & Ganz, 1999). The classic mechanism of AMP is disruption of the microbial plasma membrane (Reddy et al., 2004). Briefly, AMPs interact directly with the bacterial membrane through electrostatic forces between positively charged amino acids, specifically arginine (R) and lysine (K), and negatively charged phospholipids in the bacterial membrane, such as phosphatidylglycerol (PG) and zwitterionic phosphatidylethanolamine (Lohner, 2009). Because the extracellular side of the mammalian cell membrane bilayer contains abundant neutral phospholipids, such as phosphatidylcholine (PC) and ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
sphingomyelin, the antimicrobial activity of most AMPs is microbial cell-specific (Guilhelmelli et al., 2013). Most AMPs exert their activity by disrupting target microorganism membranes via the barrel-stave model or the toroidal model. In the toroidal model, peptides insert into the membrane by forming a bundle, resulting in pore formation (Chen et al., 2013). Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2), the principal active component in the venom of the honeybee, Apis mellifera (Habermann, 1972), is a prototype toroidal poreforming peptide (Guilhelmelli et al., 2013), and its membranolytic mechanism has been well-established in many studies. At nanomolar concentrations, melittin induces the formation of transient pores, which causes leakage of atomic ions and, at micromolar concentrations, melittin forms larger pores that allow the leakage of kilodalton-
FEMS Microbiol Lett 355 (2014) 36–42
37
Novel mechanism of melittin as an apoptosis inducer
sized molecules (Lee et al., 2013). The molecular mechanism of melittin-induced pore formation has been further characterized employing an aspirated giant unilamellar vesicle experiment, fully hydrated multi-layers of peptidelipid (PC/PG) mixtures, and grazing-angle X-ray anomalous diffraction. Specifically, the procedure of stable pore formation, above a critical P/L (peptide-to-lipid molar ratio), the changes in the mechanism that occur as a result of differences in peptide concentration, and the lipid structure of the pore have been thoroughly investigated (Lee et al., 2013). Owing to its potent activity and welldefined effect against the plasma membrane, melittin is always used as a positive control peptide in AMP studies. Previously, we focused on the distinctive effects of melittin in addition to its membrane-disruptive action, and suggested its potential as an apoptosis inducer in the human pathogenic fungus, Candida albicans. After treatment of 2.5 lM melittin, we observed some apparent morphologic changes during apoptosis, such as the appearance of phosphatidylserine on the outer leaflet of cell membranes and both DNA degradation and nuclear fragmentation (Park & Lee, 2010). However, the exact apoptotic pathway remained elusive. Therefore, in this study, we investigated the pathway of melittin-induced fungal apoptosis. In line with our previous study, we used the same concentration (2.5 lM) of melittin in this study. Finally, we demonstrated the potential of AMPs with dual mechanisms.
buffered saline (PBS) with 5 lM 2-[6-(40 -hydroxy)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (HPF). The cells were washed in PBS and analyzed using an FACSCalibur flow cytometer (Becton-Dickinson). In this study, thiourea was used in millimolar levels, as ˙OH scavenger (Whiteman & Halliwell, 1997). For that, 150 mM thiourea was treated simultaneously with 2.5 lM melittin or 10 mM H2O2. The cells were treated with 2.5 lM melittin or 10 mM H2O2 with or without thiourea and cultured in yeast extract peptone dextrose (YPD) media plates for 4 h at 28 °C. Finally, the colony was counted. Data are presented from three independent experiments using the means SD. JC-1 staining for the detection of mitochondrial membrane potential (DΨm)
JC-1 dye (Molecular Probes) was used to detect the change of mitochondrial membrane potential. Candida albicans (ATCC 90028) cells (2 9 104 cells mL1) were treated with 2.5 lM melittin or 10 mM H2O2 for 2 h at 28 °C. The treated cells were washed in PBS and then suspended in 200 lL staining solution containing 2 lg mL1 JC-1 for 20 min at 37 °C. The cells were centrifuged at 500 g for 5 min and the pellet was re-suspended in 1 mL PBS. The cells were analyzed using an FACSCalibur flow cytometer (Becton-Dickinson) (Lee et al., 2012).
Materials and methods
Fura-2, AM and Rhod-2, AM staining for the detection of cytosolic and mitochondrial Ca2+
Solid-phase peptide synthesis
Candida albicans (ATCC 90028) cells (2 9 104 cells mL1) were treated with 2.5 lM melittin or 10 mM H2O2, for 2 h at 28 °C. The cells were washed twice with Krebs buffer (pH 7.2) and treated with 1 mL Krebs buffer, including both 0.01% Pluronic F127 (Molecular Probes) and 1% bovine serum albumin. Thereafter, the cells were treated with 5 lM Fura-2, AM (Molecular Probes) or 10 lM Rhod-2, AM (Molecular Probes) and incubated for 30 min at 28 °C. The cells treated with dyes were then washed with calcium-free Krebs buffer (pH 7.2). Finally, the stained cells were observed with a fluorescence microscope (Nikon Eclipse Ti-S, Japan) and the fluorescence intensity was detected with a spectrofluorophotometer (Shimadzu, RF-5301PC; Arduino et al., 2009). Data are presented from three independent experiments using the means SD.
Melittin was chemically synthesized by Anygen Co. Ltd (Gwangju, Korea). Briefly, the assembly of peptides consisted of a 60-min cycle for each residue at ambient temperature as follows: (1) the 2-chlorotrityl (or 4-methylbenzhydrylamine amide) resin was charged in a reactor and then washed with dichloromethane and N,N-dimethylformamide (DMF), respectively, and (2) a coupling step with vigorous shaking using a 0.14 mM solution of FmocL-amino acids and Fmoc-L-amino acids pre-activated for c. 60 min with a 0.1 mM solution of 0.5 M HOBt/DIC in DMF. Finally, the peptide was cleaved from the resin using a trifluoroacetic acid cocktail solution at ambient temperature (Merrifield, 1986; Sheppard, 2003). HPF staining for the detection of hydroxyl radical generation
The intracellular hydroxyl radical (˙OH) levels were measured by incubating the C. albicans (ATCC 90028) cells (2 9 104 cells mL1) with 2.5 lM melittin (Park & Lee, 2010) or 10 mM H2O2 for 2 h at 28 °C, in phosphateFEMS Microbiol Lett 355 (2014) 36–42
Metacaspase activation and cytochrome c release detection
Activated metacaspases in C. albicans (ATCC 90028) cells were measured using the CaspACETM FITC-VAD-FMK In ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
38
Situ Marker (Promega). The cells (2 9 104 cells mL1) were treated with 2.5 lM melittin or 10 mM H2O2 for 2 h at 28 °C. The treated cells were washed in PBS, suspended in 200 lL of staining solution containing 10 lM CaspACETM FITC-VAD-FMK In Situ Marker, and incubated for 30 min at room temperature in the dark. The cells were re-suspended in PBS and metacaspase activation was analyzed with an FACSCalibur flow cytometer (Becton-Dickinson; Lee et al., 2012). To investigate cytochrome c release after the treatment of melittin, C. albicans (ATCC 90028) cells were treated with 2.5 lM melittin or 10 mM H2O2 for 2 h at 28 °C. The cells were harvested and homogenized in buffer A [50 mM Tris, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.5]. To remove the impurities, the mixture was supplemented with 2% glucose and centrifuged. The supernatant was collected, centrifuged at 30 000 g for 45 min and then used to examine cytoplasmic cytochrome c. To obtain pure mitochondria, the pel-
J. Lee & D.G. Lee
let was washed in buffer B (50 mM Tris, 2 mM EDTA, pH 5.0). Mitochondria was suspended in 2 mg mL1 of Tris-EDTA buffer. After the treatment of 500 mg mL1 ascorbic acid for 5 min, the cytochrome c levels in the cytosolic or mitochondrial fractions were determined at 550 nm with spectrophotometer (Beckman, DU530; Barbu et al., 2013). Data are presented from three independent experiments using the means SD.
Results and discussion Hydroxyl radical-mediated oxidative stress caused by melittin
AMPs are thought to be a novel candidate as a substitute for conventional antibiotics. The general mechanism of AMPs is a potent membrane-disruptive action; however, we hypothesize that AMPs can induce other additional effects after the cell entry. In this study, for the first time,
(a)
(b)
Fig. 1. Hydroxyl radical (˙OH) generation by melittin in Candida albicans. (a) Candida albicans cells were treated with 2.5 lM melittin or 10 mM H2O2 for 2 h at 28 °C, with 5 lM HPF dye, to specifically detect ˙OH. The cells stained were analyzed by FACSCalibur flow cytometer. (b) The survival rates of C. albicans after melittin treatment for 4 h at 28 °C, in the presence of 150 mM thiourea as a scavenger of ˙OH, were examined. The black bars indicate no treatment of thiourea, whereas the white bars indicate the treatment of thiourea. Data are presented from three independent experiments using the means SD. ***P < 0.001 (Student’s t-test).
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Lett 355 (2014) 36–42
39
Novel mechanism of melittin as an apoptosis inducer
Fig. 2. JC-1 staining assay for detecting the change of mitochondrial membrane potential (DΨm) after melittin treatment.
(a)
(b)
Fig. 3. Melittin significantly increased mitochondrial Ca2+ levels in Candida albicans, compared to cytosolic Ca2+. (a) Fluorescence microscopy of Fura-2AM and Rhod-2AM for detection of cytosolic Ca2+ and mitochondrial Ca2+, respectively. The gray images indicate the DIC (differential interference contrast) images. (b) Percentages of Fura-2AM-positive cells or Rhod-2AM-positive cells were assessed. Data are presented from three independent experiments using the means SD. **P < 0.01, ***P < 0.001 (Student’s t-test).
FEMS Microbiol Lett 355 (2014) 36–42
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
40
(a)
(d)
J. Lee & D.G. Lee
(b)
(c)
(e)
Fig. 4. Metacaspase activation and cytochrome c release by melittin in Candida albicans. (a–d) Candida albicans cells were treated with 2.5 lM melittin or 10 mM H2O2 for 2 h at 28 °C, with 10 lM CaspACETM FITC-VAD-FMK In Situ Marker, to detect metacaspase. The cells stained were analyzed by an FACSCalibur flow cytometer. (e) Cytochrome c release from cytosol to mitochondria was examined and the patterns showed mutual release patterns between cytosol and mitochondria. Data are presented from three independent experiments using mean SD. *P < 0.05, **P < 0.01 (Student’s t-test).
we propose a melittin-induced apoptotic mechanism in C. albicans. Reactive oxygen species (ROS) has been focused on, due to their pivotal role in the apoptosis of many different cell types. For example, hydrogen peroxide (H2O2), an ROS, induced CD95 (Fas/APO-1)-mediated neutrophil apoptosis (Kasahara et al., 1997). Nitric oxide (NO˙), another component of ROS, also induced apoptosis in various cells, including mesangial cells, epithelial cells, endothelial cells (M€ uhl et al., 1996) and, by extension, cancer cells (Hajri et al., 1998; Shami et al., 1998). Previously, we suggested that endogenous ROS generation was involved in the antifungal activity of melittin against C. albicans. In this study, among the ROS, we focused on hydroxyl radical (˙OH), as it is a highly reactive molecule and a crucial mediator of apoptosis (Lee et al., 2012). We used HPF staining to detect melittin-induced ˙OH and investigated the survival rates in the presence of thiourea, which mimics the role of an ˙OH scavenger. The ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
results showed that melittin induced higher ˙OH generation (76.12%) than induced by the negative control (4.51%). H2O2 (97.27%) showed more potent inductive activity than melittin; however, the activity of melittin was also remarkable considering the established effect of H2O2 (Fig. 1a). To further investigate the ˙OH generation, we investigated the survival rates after treatment with melittin and H2O2 in the presence of thiourea, as a scavenger of ˙OH. Although there are some discrepancies between H2O2 and melittin, the increased survival rates after treatment with both melittin and thiourea compared with those after treatment with melittin alone, demonstrated that hydroxyl radicals constitute a significant portion of melittin-induced ROS (Fig. 1b). Melittin-induced mitochondrial dysfunction
We subsequently investigated mitochondrial involvement in melittin-induced apoptosis, using JC-1 dye. MitoFEMS Microbiol Lett 355 (2014) 36–42
41
Novel mechanism of melittin as an apoptosis inducer
chondria is the most important site regarding the occurrence of apoptotic key events. In particular, the loss of mitochondrial membrane potential (DΨm) and mitochondrial permeability transition are distinctive features of early apoptosis. In normal cells, JC-1 containing delocalized positive charges can be observed as red fluorescence in the mitochondrial matrix. In contrast, in apoptotic cells, the dye remains in the cytoplasm as fluorescent green (Smiley et al., 1991). The Y mean/X mean ratio of the control was 5.1; however, the ratios in melittin-treated cells (0.6) and H2O2-treated cells (0.4) were remarkably decreased (Fig. 2). These decreases indicated that melittin greatly reduced the mitochondrial membrane potential, triggering the mitochondrial dysfunction of C. albicans. Mitochondrial Ca2+ and melittin-induced fungal apoptosis
In the regulation of apoptosis, Ca2+ homeostasis in both the endoplasmic reticulum (ER) and mitochondria is involved in the localization of regulator proteins, Bcl-2 (Pinton & Rizzuto, 2006). For this reason, investigations of Ca2+ homeostasis or communication between the ER and mitochondria through Ca2+, are critical in apoptosis studies. In this study, to investigate the change in Ca2+ as a mediator of ER-mitochondrial communication and its essential role in apoptosis, we used two dyes, Fura-2AM and Rhod-2AM, to assess the cytosol and mitochondria, respectively. The results showed that melittin caused a slight increase in intracellular Ca2+ levels (Fura-2AM levels), whereas melittin remarkably increased mitochondrial Ca2+ levels (Rhod-2AM levels), even compared with H2O2 (Fig. 3a and b). This mitochondrial Ca2+ overload would be one of the main factors to induce mitochondrial damage through perturbation or rupture by the decrease of DΨm. Namely, we confirm that the mitochondria are primarily involved in the apoptotic pathway of melittin in C. albicans. Effect of melittin on intracellular cytochrome c release and caspase activation
Caspase, an intracellular protease, is activated when apoptosis is induced; specifically, metacaspase 1 (CaMCA1), which encodes a putative caspase in C. albicans, is activated (Shirtliff et al., 2009). We examined the metacaspase dependency of melittin-induced apoptosis. The results showed that the melittin-exposed cells exhibited increased fluorescence intensity of metacaspase (34.17%) similar to that of H2O2 (39.08%) (Fig. 4a–d). This indicates that as a central regulator of apoptosis, metacaspase is associated with melittin-induced fungal FEMS Microbiol Lett 355 (2014) 36–42
cell death. We subsequently investigated the release of cytochrome c, an important stimulus for caspase activation. Cytochrome c is released from the mitochondria into the cytosol before caspase activation during apoptosis (Bossy-Wetzel et al., 1998). Therefore, we investigated the translocation of cytochrome c after peptide treatment. The results showed that after melittin exposure, the cytochrome c concentration in the mitochondria decreased. Conversely, the concentration of cytochrome c in the cytosol increased (Fig. 4e). This could demonstrate that metacaspase activation is induced by cytochrome c leakage. Moreover, the results also suggested that these activities were correlated with mitochondrial membrane potential disruption, as noted previously. In the present study we suggested that melittin-induced apoptosis in C. albicans is triggered by hydroxyl radial generation, mitochondrial membrane permeabilization, and metacaspase activation. It is well-established that melittin disrupts the fungal cell membrane of C. albicans, with potent activity (Lee et al., 2010). Here, we showed a novel mechanism of melittin through the apoptotic pathway. Although the relationship between these two mechanisms remains to be investigated, this study suggests that melittin could trigger fungal apoptosis after membrane damage utilizing a dual mechanism against pathogenic microorganisms.
Acknowledgements This work was supported by a grant from the Next-Generation BioGreen 21 Program (no. PJ008158), Rural Development Administration, Republic of Korea.
References Arduino DM, Esteves AR, Domingues AF, Pereira CM, Cardoso SM & Oliveira CR (2009) ER-mediated stress induces mitochondrial-dependent caspases activation in NT2 neuron-like cells. BMB Rep 42: 719–724. Barbu EM, Shirazi F, McGrath DM, Albert N, Sidman RL, Pasqualini R, Arap W & Kontoyiannis DP (2013) An antimicrobial peptidomimetic induces mucorales cell death through mitochondria-mediated apoptosis. PLoS ONE 8: e76981. Bossy-Wetzel E, Newmeyer DD & Green DR (1998) Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J 17: 37–49. Chen L, Jia N, Gao L, Fang W & Golubovic L (2013) Effects of antimicrobial peptide revealed by simulations: translocation, pore formation, membrane corrugation and Euler buckling. Int J Mol Sci 14: 7932–7958. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
42
Guilhelmelli F, Vilela N, Albuquerque P, Derengowski LD, Silva-Pereira I & Kyaw CM (2013) Antibiotic development challenges: the various mechanisms of action of antimicrobial peptides and of bacterial resistance. Front Microbiol 4: 353. Habermann E (1972) Bee and wasp venoms. Science 177: 314–322. Hajri A, Metzger E, Vallat F, Coffy S, Flatter E, Evrard S, Marescaux J & Aprahamian M (1998) Role of nitric oxide in pancreatic tumour growth: in vivo and in vitro studies. Br J Cancer 78: 841–849. Kasahara Y, Iwai K, Yachie A, Ohta K, Konno A, Seki H, Miyawaki T & Taniguchi N (1997) Involvement of reactive oxygen intermediates in spontaneous and CD95 (Fas/APO-1)-mediated apoptosis of neutrophils. Blood 89: 1748–1753. Lee J, Hwang JS, Hwang B, Kim JK, Kim SR, Kim Y & Lee DG (2010) Influence of the papiliocin peptide derived from Papilio xuthus on the perturbation of fungal cell membranes. FEMS Microbiol Lett 311: 70–75. Lee J, Hwang JS, Hwang IS, Cho J, Lee E, Kim Y & Lee DG (2012) Coprisin-induced antifungal effects in Candida albicans correlate with apoptotic mechanisms. Free Radic Biol Med 52: 2302–2311. Lee MT, Sun TL, Hung WC & Huang HW (2013) Process of inducing pores in membranes by melittin. P Natl Acad Sci USA 110: 14243–14248. Lehrer RI & Ganz T (1999) Antimicrobial peptides in mammalian and insect host defence. Curr Opin Immunol 11: 23–27. Lohner K (2009) New strategies for novel antibiotics: peptides targeting bacterial cell membranes. Gen Physiol Biophys 28: 105–116. Merrifield B (1986) Solid phase synthesis. Science 232: 341–347.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
J. Lee & D.G. Lee
M€ uhl H, Sandau K, Br€ une B, Briner VA & Pfeilschifter J (1996) Nitric oxide donors induce apoptosis in glomerular mesangial cells, epithelial cells and endothelial cells. Eur J Pharmacol 317: 137–149. Park C & Lee DG (2010) Melittin induces apoptotic features in Candida albicans. Biochem Biophys Res Commun 394: 170–172. Pinton P & Rizzuto R (2006) Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum. Cell Death Differ 13: 1409–1418. Reddy KV, Yedery RD & Aranha C (2004) Antimicrobial peptides: premises and promises. Int J Antimicrob Agents 24: 536–547. Shami PJ, Sauls DL & Weinberg JB (1998) Schedule and concentration-dependent induction of apoptosis in leukemia cells by nitric oxide. Leukemia 12: 1461–1466. Sheppard R (2003) The fluorenylmethoxycarbonyl group in solid phase synthesis. J Pept Sci 9: 545–552. Shirtliff ME, Krom BP, Meijering RA, Peters BM, Zhu J, Scheper MA, Harris ML & Jabra-Rizk MA (2009) Farnesolinduced apoptosis in Candida albicans. Antimicrob Agents Chemother 53: 2392–2401. Smiley ST, Reers M, Mottola-Hartshorn C, Lin M, Chen A, Smith TW, Steele GD Jr & Chen LB (1991) Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. P Natl Acad Sci USA 88: 3671–3675. Whiteman M & Halliwell B (1997) Thiourea and dimethylthiourea inhibit peroxynitrite-dependent damage: nonspecificity as hydroxyl radical scavengers. Free Radic Biol Med 22: 1309–1312.
FEMS Microbiol Lett 355 (2014) 36–42
