Communications between various organelle–organelles play an essential role in cell survival. The cross-talk between mitochondria and vacuoles comes up with the vital roles of the intercompartmental process. In this study, we found a couple of cell death features, membrane damage, and apoptosis using antimicrobial peptide from American Cockroach. Periplanetasin-4 (LRHKVYGYCVLGP-NH2) is a 13-mer peptide derived from Periplaneta americana and exhibits phosphatidylserine exposure and caspase activation without DNA fragmentation. Apoptotic features without DNA damage provide evidence that this peptide did not interact with DNA directly and exhibited dysfunction of mitochondria and vacuoles. Superoxide radicals were generated from mitochondria and converted to hydrogen peroxide. Despite the enhancement of catalase and total glutathione contents, oxidative damage disrupted intracellular contents. Periplanetasin-4 induced cell death associated with the production of superoxide radicals, calcium uptake in mitochondria and disorder of vacuoles, such as increased permeability and alkalization. While calcium movement from vacuoles to the mitochondria occurred, the cross-talk with these organelles proceeded and the inherent functionality was impaired. To sum up, periplanetasin-4 stimulates superoxide signal along with undermining the mitochondrial functions and interfering in communication with vacuoles.
Intracellular pH is a tightly regulated physiological parameter in all living processes. In yeast, intracellular pH dynamics affect cellular function [1,2]. In mammalian cells, the endoplasmic reticulum (ER) is shown to be near neutral, identical with that of the cytosol. Vesicular compartments of the pathway acidify through the Golgi [1,2] and nuclear pH is similar to cytoplasmic pH . Mitochondrial matrix pH is reported to be around 8 . For the difference between yeast and higher eukaryotes, the pH in the organelle is slightly different. Measurements in yeast have always been significantly lower around 7.5 . Interaction among vacuoles with mitochondria , vacuoles and Golgi , ER with Golgi , ER with mitochondria , and is processed. However, further studies on the intercellular organelle function are needed .
Insects use both cellular and humoral mechanisms in their defense against the pathogens, as well as innate immunity, which is dominant in the final category. They are also increasingly recognized as sources of microbial contamination and remarkably resistant to microbes; following exposure, a complex genetic cascade is activated, leading to the production of a series of the antimicrobial compound that is released into the hemolymph [7,8]. Cockroaches frequently live in close contact with humans and are one of the oldest winged insects. Owing to their lifestyle, they are frequently exposed to potential parasites and pathogenic microorganisms; however, few encounters result in infection suspected vectors of pathogenic microbes [7–9]. They exist in environments with high amounts of toxic substances, including microbial toxins, pollutants, xenobiotics and insecticides . The most common domestic species of cockroach, the American cockroach Periplaneta americana, is a notorious and obnoxious insect with a strong ability to adapt to complex environments [9,10]. Previous studies of P. americana have mainly focused on the reproduction, digestive characteristics, effects of adipokinetic hormones, sexually dimorphic glomeruli, and related interneurons . In contrast, studies that are vital for the biological control of the species has not been well reported .
Antimicrobial peptides (AMPs) from insects play an essential role in the defense against invading pathogens, especially those that lack adaptive immunity . Normally, in response to microbial infection or body injury, AMPs are synthesized in epithelia, fat bodies or by certain cells in the hemolymph, and then rapidly secreted into the hemolymph to kill microorganisms [7,11]. AMPs are small, amphipathic, cationic molecules  that are integral components of the innate host defense system. AMPs are commonly 12–50 amino acids in length, contain excessive positively charged amino acids and fold into a diverse array of amphipathic structures upon contact with microbial membranes [7,12]. Recently, an antimicrobial protein isolated from the hemolymph of P. americana was reported . A transcriptome analysis of uninfected and Escherichia coli-infected P. americana was performed, and several AMP candidates were identified  and designated it periplanetasin-4 (LRHKVYGYCVLGP-NH2). Herein, we focused on the antimicrobial properties and mode of actions of a novel AMP, periplanetasin-4.
Materials and methods
Whole transcriptome sequencing of E. coli-infected P. americana, and the identification of potential AMPs were performed by Kim et al. . Among the identified AMPs, periplanetasin-4 was selected for further analysis, and its antimicrobial properties were compared with those of the toxin melittin from bee venom, which was used as a positive control. The peptide was synthesized by Anygen Co. (Gwangju, Korea). Periplanetasin-4 and melittin were treated by 5 µM. Ascorbic acid which was used as a ROS scavenger was treated by 5 mM.
Microbial strains and antimicrobial susceptibility testing
Candida albicans (ATCC 90028) and Candida parapsilosis (ATCC 22019) were obtained from American Type Culture Collection (ATCC; Manassas, VA). Malassezia furfur (KCTC 7744), Trichosporon beigelii (KCTC 7707), Aspergillus fumigatus (KCTC 6145) and Aspergillus flavus (KCTC 6905) were obtained from the Korean Collection for Type Cultures (KCTC). Fungal strains were cultured in YPD broth (BD) at 28°C with aeration, and M. furfur was cultured in YM broth (BD) containing 1% olive oil at 32°C. Bacterial strains were cultured in LB broth (BD) at 37°C with aeration.
Cells (2 × 105 cells/ml) in the exponential phase of growth were inoculated into 0.1 ml of medium and then dispensed into microtiter plates. The minimum inhibitory concentration (MIC) of peptides was determined with two-fold serial dilutions, based on the method of the Clinical and Laboratory Standards Institute (CLSI) . After 24 h of incubation, growth was measured using a microtiter BioTek ELx800 Absorbance Reader (BioTek Instruments, Winooski, VT, U.S.A.) by monitoring the absorption at 600 nm. The MIC values were defined as the lowest concentration of the peptide at which 90% of the isolates were inhibited.
Propidium iodide (PI) uptake and membrane depolarization assay
C. albicans cells (2 × 106 cells/ml) were collected and suspended in phosphate-buffered saline (PBS) with periplanetasin-4 and melittin. After incubation for 2 and 4 h, cells were harvested by centrifugation and resuspended in PBS. To assess membrane permeability, the cells were stained with 9 µM PI and incubated for 5 min at room temperature. Then, the cells were analyzed using a FACSVerse flow cytometer (Becton Dickinson, Franklin Lakes, NJ, U.S.A.) . To establish membrane depolarization, the incubated cells were stained with 50 µg/ml bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] (Molecular Probes, Eugene, OR). Flow cytometric analysis was performed using a FACSVerse flow cytometer .
Determination of intracellular reactive oxygen species (ROS) level
Intracellular ROS accumulation was assessed using the 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes). Cells (2 × 106cells/ml) were incubated in the presence of periplanetasin-4 for 2 and 4 h. Then, the cells were suspended in PBS, and 10 µM H2DCFDA was added to the suspension. After incubation for 1 h, the cells were washed with PBS, and the fluorescent cells were detected with a Shimadzu RF-5301PC spectrofluorophotometer . H2DCFDA fluorescence was measured by excitation at 485 nm and emission at 535 nm.
According to the manufacturer's instructions, fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit (BD Pharmingen, San Diego, CA) was used to detect the externalization of phosphatidylserine (PS) in the membrane of apoptotic cells. C. albicans protoplast cells (2 × 106 cells/ml) were prepared and were harvested after 2 h of periplanetasin-4 treatment as reported previously . After incubation with periplanetasin-4, the cells were analyzed using a FACSVerse flow cytometer DNA fragmentation was analyzed using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay via an in situ cell death detection kit (Roche) using the FACSVerse flow cytometer. Caspases were measured by FITC-VAD-FMK staining. After incubation with periplanetasin-4, the cell samples were washed twice and incubated with CaspACE FITC-VAD-FMK for 20 min. Fluorescence intensity was determined using the FACSVerse flow cytometer .
Cell cycle analysis
DNA content of the cells stained with the DNA-binding fluorescent dye SYTOX green was quantified by flow cytometry. After treatment with 5 µM periplanetasin-4 for 4 h, the cells (2 × 106 cells/ml) were harvested and fixed with 70% ethanol overnight at 4°C. The cells were washed twice with 50 mM sodium citrate buffer (pH 7.4) and treated with 200 mg/ml of RNase A. The mixture was incubated for 2 h at 37°C and washed twice with sodium citrate buffer. For DNA staining, the cells were incubated with 1 µM SYTOX green in the dark for 20 min. DNA content of each cell was determined using a FACSVerse flow cytometer .
Detection of mitochondrial functions
We used JC-1 to assess the effect of periplanetasin-4 on mitochondrial membrane potential. After the cells (2 × 106 cells/ml) had been incubated with the peptide for 2 h, they were washed with warm PBS and incubated with JC-1 (Molecular Probes, CA). Fluorescence intensity at FL-1 and FL-2 were recorded using a flow cytometer, and the results were described as a ratio of the mean values of FL-1 and FL-2 .
MitoTracker Green was utilized to measure mitochondrial mass . The compound passively diffuses across the plasma membrane and accumulates in active mitochondria, defining their number and mass. Log-phased C. albicans cells (2 × 106 cells/ml) were cultured in YPD medium at 28°C and harvested in PBS. The cell suspensions were then treated with periplanetasin-4 for 2 h at 28°C and then was washed with PBS. The cells were incubated with 0.1 µM MitoTracker Green for 30 min at 28°C. The stained cells were analyzed using the FACSVerse flow cytometer, fluorescence microscopy (Nikon Eclipse Ti-5; Nikon, Minato, Japan).
Vacuolar membrane permeability detection
To determine vacuolar response to periplantetasin-4, the fluorescent dye BCECF is utilized . Yeast cells (2 × 106cells/ml) were treated with periplanetasin-4 at 28°C for 2 h. After incubation, the cells were harvested at 13,000 g and suspended in 1 mg/ml 5 µM BCECF at 28°C for 30 min. Stained cells were then washed three times with PBS, and the fluorescent cells were analyzed using a FACSVerse flow cytometer.
Vacuolar membrane permeability was assessed the cell-permeant tracer 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate (C-DCFDA, Invitrogen). After 2 h incubation with periplanetasin-4 (5 µM), the cell suspensions (2 × 106 cells/ml) were stained with 5 µl of C-DCFDA (1 mg/ml) at 30°C for 30 min [4,22] and then cells were washed with PBS. The cells were analyzed using a fluorescence microscope and flow cytometry .
Detection of superoxide radicals, hydrogen peroxide and hydroxyl radicals
Superoxide radicals and hydroxyl radical levels were measured using the MitoSOX Red Superoxide Indicator (Molecular Probes, Eugene, OR, U.S.A.) and 3′-(p-hydroxyphenyl) fluorescein (HPF, Invitrogen) according to the manufacturer's instructions, respectively. Yeast cells (2 × 106 cells/ml) were treated with periplanetasin-4 at 28°C for 2 h. After incubation, the cells were harvested at 13,000 g and suspended in 5 µM MitoSOX Red and 5 µM HPF at 28°C for 30 min, respectively. Stained cells were then washed three times with PBS, and the fluorescent cells were analyzed with a FACSVerse flow cytometer .
Hydrogen peroxide level was measured using the Amplex Red Kit (Invitrogen) according to the manufacturer's instructions. C. albicans cells (2 × 106 cells/ml) were treated with periplanetasin-4 described above and incubated for 2 h at 28°C. The cell suspension was centrifuged at 13,000 g for 5 min and then the cell pellet was extracted using lysis buffer (10 mM Tris–HCl, 1 mM EDTA [pH 8.0], 100 mM NaCl, and 0.1% Triton X-100). The absorbance of Amplex red as measured using an ELISA microplate Reader at 570 nm.
Measurement of lipid peroxidation
Lipid peroxidation was quantified based on malondialdehyde (MDA) levels. After treatment with periplanetasin-4, the suspensions (2 × 106 cells/ml) were then cultured for 2 h and sampled. The pellet was broken with lysis buffer (10 mM Tris–HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl, 2% Triton X-100, 1% SDS). The cell suspension was incubated with 5% trichloroacetic acid and centrifuged. The supernatant was mixed with an equal volume of 0.5% (w/v) thiobarbituric acid solution. The mixture was heated at 95°C for 30 min and then cooled on ice. The absorbance of the reaction mixture was measured at 532 and 600 nm [16,22].
For superoxide dismutase (SOD) and catalase activity assay, C. albicans cells (2 × 106cells/ml) were treated with periplanetasin-4 described above and incubated for 2 h at 28°C. The cell suspension was centrifuged at 13,000 g for 5 min and then the cell pellet was extracted using lysis buffer (10 mM Tris–HCl, 1 mM EDTA [pH 8.0], 100 mM NaCl, and 0.1% Triton X-100). After centrifugation, the supernatants were used for SOD and CAT assays using the SOD Assay Kit (Sigma–Aldrich) and the Amplex Red Catalase Assay Kit (Molecular Probes), respectively.
Total glutathione quantification
After C. albicans cells (2 × 106 cells/ml) were treated with periplanetasin-4 for 2 h, each sample was sonicated. Deproteinated supernatants, using 5% trichloroacetic acid, were analyzed using a glutathione assay kit (Sigma–Aldrich). The absorbance of the reaction mixture was monitored at 415 nm with a microtiter ELISA reader (Molecular Devices Emax, Sunnyvale, CA) .
Assessment of calcium homeostasis
Cytosolic and mitochondrial of calcium was measured as described previously. To measure cytosolic and mitochondrial calcium levels, Fura-2AM (Molecular Probes) and Rhod-2AM were used, respectively. After incubating C. albicans cells (2 × 106 cells/ml) with periplaneatsin-4 for 2 h, they were washed in Krebs buffer (pH 7.4) and treated with 1% bovine serum albumin and 0.01% pluronic acid F-127 (Molecular Probes). The cell suspensions were then stained with 5 µM Fura-2AM or 10 µM Rhod-2AM and incubated at 37°C for 40 min, allowing the cells to completely hydrolyze the acetoxymethyl ester. The cells were washed in calcium-free Krebs buffer and maintained in calcium- and dye-free medium. The fluorescence intensities of Fura-2AM (excitation = 335 nm, emission = 505 nm) and Rhod-2AM (excitation = 550 nm, emission = 580 nm) were analyzed using a spectrofluorophotometer (Shimadzu RF-5301PC, Shimadzu, Japan) .
Membrane fluidity determination
Cell membrane fluidity was determined by measuring fluorescence intensity with 1,6-diphenyl-1,3,5-hexatriene (DPH; Sigma, St. Louis, MO). The suspension of C. albicans cells (2 × 106 cells/ml) was incubated as described above. The cells were fixed with 0.37% formaldehyde and washed with cold PBS, then subjected to two freeze–thaw cycles in liquid nitrogen and warm PBS. The cell suspensions were incubated with 0.6 mM DPH for 45 min at 28°C, then washed three times with PBS. Fluorescence intensity was immediately measured with an RF-5301PC spectrofluorophotometer (Shimadzu, Kyoto, Japan). The excitation and emission wavelengths were 350 and 425 nm, respectively .
Estimation of pore size in artificial liposomes and live cells
Artificial liposomes were prepared as reported previously . A suspension of liposomes treated with the peptides (1 ml, final volume) was stirred for 10 min in the dark and then centrifuged at 13,000 g for 10 min. Dye leakage from the LUVs was monitored by measuring the fluorescence intensity at an excitation and emission wavelengths of 494 nm and 520 nm, respectively, using a spectrofluorophotometer (Shimadzu RF-5301PC). The percentage of liposome integrity after treatment was determined by comparison to the amount of dye release by liposomes treated with 1% Triton X-100 (set to 100%) . The percentage of dye leakage caused by the peptides was calculated as follows: , where F is the fluorescence intensity after incubation with the peptides, and F0 and Ft are the fluorescence intensities incubated without any compound and with Triton X-100, respectively .
Cells (2 × 106cells/ml) were suspended in PBS, periplanetasin-4 and melittin were added, and the mixtures were incubated for 4 h. After incubation, the cells were harvested by centrifugation and resuspended in PBS. Then, soluble fluorescent molecules, including FD4 and FD10, were added to the cells at final concentrations of 0.1 mg/ml . The influx of fluorescent molecules was observed using a fluorescent microscope (Nikon Eclipse Ti-S, Japan).
Measurement of hemolysis and lactate dehydrogenase (LDH) release
The hemolytic activity of the peptides was evaluated by determining the release of hemoglobin from 8% human erythrocytes as the change in turbidity at 414 nm, measured with an ELISA plate reader. A fresh human blood sample diluted with PBS (35 mM phosphate buffer and 150 mM NaCl [pH 7.4]) was centrifuged at 400 g for 10 min and washed three times. The erythrocyte suspension was transferred to sterilize, 96-well plates and two-fold serial dilutions of the test compound were added to the wells. The plates were incubated at 37°C for 1 h, and then centrifuged at 400 g for 10 min. An aliquot of the supernatant was taken, and the hemolytic activity of the peptides was evaluated by measuring the release of hemoglobin from human erythrocytes with an ELISA reader. The levels of hemolysis in PBS alone and with 0.1% Triton X-100 were set at 0 and 100%, respectively. The percentage of hemolysis was calculated with the following equation: hemolysis (%) = [(Abs414 nm in peptide solution – Abs414 nm in PBS)/(Abs414 nm in 0.1% Triton X-100 – Abs414 nm in PBS)] × 100 .
The 8% erythrocyte suspension was loaded into 96-well plates and treated with peptides at 1.25–80.0 µM. After incubation at 37°C for 1 h, the plate was centrifuged at 400 g for 10 min. The supernatant was transferred to a fresh 96-well plate, and LDH release was measured by the CytoTox96 non-radioactive cytotoxicity assay (Promega, Madison, WI). The percentage of LDH release was calculated as follows: , where At is the absorbance value of the sample, A100 is the absorbance of lysis buffer and A0 is the absorbance of untreated cells .
All experiments were performed in triplicate and the data were represented as the means ± SD (standard deviation). Statistical significance was determined via Student's t-test. P < 0.05 was considered to indicate statistical significance.
Results and discussion
Characterization of periplanetasin-4 and its antimicrobial activity
Cockroaches are a potential health problem to humans as well as wild and domestic animals. Owing to their unsanitary lifestyle, cockroaches can pick up, carry, and transfer a plethora of bacteria and fungi . Therefore, they have adapted to produce potent AMPs as a means of protecting their own health. In our previous study, AMP candidates corresponding to the transcriptome of the cockroach P. americana were reported. Among them, periplanetasin-4, LRHKVYGYCVLGP-NH2, was synthesized, and its purity and molecular mass were 95.8% and 1503.8 Da, respectively. Its net charge at physiological pH was +3 (Table 1). Hydrophobicity was calculated by Eisenberg and Weiss method and calculated hydrophobicity was −0.83. Periplanetasin-4 has the lowest molecular mass, which makes its synthesis cost-efficient [7,20]. To investigate its antimicrobial effects, a susceptibility test was performed using several pathogenic strains causing superficial fungal infection. The antimicrobial properties were compared with those of the toxin melittin from bee venom, which was used as a positive control. As shown in Table 2, MIC of periplanetasin-4 is in the range of 2.5–10 µM against pathogenic fungi. Periplanetasin-4 possessed antifungal activity against a source of fungal superficial infection. Candida spp. are highly evolved for interaction with, and survival in the human host. They are the main cause of disseminated fungal infections and rank fourth among the most common nosocomial pathogens. Both adaptive and innate immunity contribute to host resistance to candidiasis . Hence, C. albicans was selected as a model organism to investigate the mode of antifungal action.
|Peptide||Amino acid sequence||Purity||Molecular mass (Da)||Net charge (physiological pH)||Hydrophobicity|
|Peptide||Amino acid sequence||Purity||Molecular mass (Da)||Net charge (physiological pH)||Hydrophobicity|
|Microbial strains||MIC (µM)|
|Candida albicans ATCC 90028||5||2.5|
|Candida parapsilosis ATCC 22019||5||1.25|
|Trichosporon beigelii KCTC 7707||2.5||1.25|
|Malassezia furfur KCTC 7744||5||2.5|
|Aspergillus flavus KCTC 6905||10||2.5|
|Aspergillus fumigatus KCTC 6145||5||1.25|
|Microbial strains||MIC (µM)|
|Candida albicans ATCC 90028||5||2.5|
|Candida parapsilosis ATCC 22019||5||1.25|
|Trichosporon beigelii KCTC 7707||2.5||1.25|
|Malassezia furfur KCTC 7744||5||2.5|
|Aspergillus flavus KCTC 6905||10||2.5|
|Aspergillus fumigatus KCTC 6145||5||1.25|
Periplanetasin-4 reveals features of programmed cell death (PCD) and necrosis
Since necrosis and apoptosis may occur independently, sequentially and in parallel, the clear distinction between them is important. In many cases, the type or dose of the stress signal including heat, radiation, hypoxia and certain medications determine the type of death . Necrosis is firstly described by the lack of morphological features characteristic of apoptosis and autophagy. Nowadays, necrotic cells are morphologically characterized by organelles and cell swelling, lysosomal permeabilization and breaking the continuity of the cell membrane destruction that results in the outflow of the cell contents to the outside [29,30]. In addition, necrotic cells are characterized by a rapid decrease in energy level, random degradation of DNA and causing inflammation in the surrounding tissue by inducing the infiltration of macrophages, neutrophils and dendritic cells, and post-inflammatory cytokine secretion . The effect of periplanetasin-4 on the integrity of cell membranes was examined using PI, which can only enter cells that have damaged membranes. Therefore, we determined the impact on the plasma membrane. Compared with untreated cells, melittin-treated cells showed an increase in fluorescence intensity in a time-dependent manner (Figure 1A). Periplanetasin-4 showed little PI influx at 2 h, but at 4 h, the PI was significantly elevated. The ability of periplanetasin-4 to disrupt cytoplasmic membrane potentials was investigated using DiBAC4(3), which binds to the cell membrane and allows estimation of the membrane potential [16,32,33]. Periplanetasin-4- and melittin-treated cells also showed an accumulation of DiBAC4(3), and the accumulation of cell treated with periplanetasin-4 was lower than that of melittin. The result indicated that periplanetasin-4 caused depolarization of the membrane (Figure 1B). When periplanetasin-4 exposed in C. albicans cells, changes in membrane potential occurred earlier than membrane permeabilization. Although melittin had more potent membrane disruption activity than periplanetasin-4, PI influx changes membrane depolarization suggested that other cause may influence on cell death according to time.
Impact of periplanetasin-4 on membrane function and ROS level of C. albicans.
The overproduction of ROS leads to mitochondrial transmembrane potentials and enhances the susceptibility to apoptotic stimuli . To identify whether perplanetasin-4 increases intracellular ROS levels during perplanetasin-4-induced cell death, we used H2DCFDA, a fluorescent oxidant-specific probe that undergoes oxidation and cleavage of its acetate groups, reducing it to fluorescent 2′,7′-dichlorofluorescein [17,24]. The results showed that periplanetasin-4 elevated intracellular ROS levels compared with the levels in untreated cells (Figure 1C). The major intracellular events associated with yeast apoptosis are exposure of PS residues on the outside, caspase activation and DNA fragmentation [29,35]. In apoptotic cells, DNA is degraded at internucleosomal linker sites, yielding DNA fragments in multiples of 180 bp . Periplanetasin-2 from P. americana activates mitochondria-mediated apoptotic signals including DNA fragmentation . On the other hand, periplanetasin-4 exhibits PS exposure and caspase activation, whereas DNA-strand break (DSB) does not exhibit (Figure 2). The formation of breaks in the DNA strand occurs after treatment with periplanetasin-2 . Cyclin-dependent kinase (CDK) activity was required for efficient DSB repair in fission yeast. Cell cycle is driven by CDKs that form a negative feedback loop oscillator . Cell cycle checkpoints activated by DSBs are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells have to detect the break and either transiently block cell cycle progression, to allow time for the repair, or exit the cell cycle . To compare cell cycle progression of untreated cells and cells upon exposure to periplanetasin-4, flow cytometric analysis was used and the result indicated that cell cycle did not alter (Figure 2D). Cell cycle arrest prevents duplication and segregation of damaged nucleic acids at a critical stage of DNA replication or cell division . Taken together, periplanetasin-4 has simultaneously features of apoptosis and necrosis without DNA damage.
Apoptotic features of periplanetasin-4.
Periplanetasin-4 interfere function of cellular compartments, mitochondria and vacuoles
The difference of morphological and biochemical features between necrosis and PCD is rupture of membrane, total disintegration of cellular organelles [29–31]. PCD is important for intracellular organelles communication. This communication is grafted into the connection of the organelles. Compounds that have a strong effect on certain molecular targets involved in mitochondrial function will cause a much more modest change in membrane potential. A major challenge in monitoring the mitochondrial membrane potential in primary screening is the relatively small change expected when highly oxidized phosphorylation is regulated . The destruction of the mitochondrial transmembrane potential has been suggested as a point of no return from apoptotic signaling . The disruption of the mitochondrial membrane potential depends on the transport of electrons from NADH to the molecular oxygen and the proton transfer mediated by the F0F1–ATPase complex. The energy stored in the electrochemical gradient is used by F0F1–ATPase to convert ADP to ATP during oxidative phosphorylation . JC-1 is a membrane-permeant dye that can selectively enter mitochondria and reversibly changes a fluorescence emission shift from green (monomers) to red (J-aggregates) [4,34,43]. The ratio of red to green fluorescence represents the status of mitochondrial membrane potential in the cell population . The periplanetasin-4-treated cells exhibited collapse mitochondrial membrane potential compared with the control. The mitochondrial membrane potential was significantly increased in cells relative to periplanetasin-4 (Figure 3A). Mitochondrial hyperpolarization by periplanetasin-4 indicates influencing on mitochondrial function in contrast with mechanisms of periplanetaisin-2 . Mitochondrial hyperpolarization is associated with an increase in mitochondrial ROS production . Furthermore, the elevation of mitochondrial membrane potential, i.e. mitochondrial hyperpolarization, hyperpolarization precedes PS externalization and occurs in the early phase of Fas-induced apoptosis of Jurkat human leukemia T cells and normal human PBL (peripheral blood lymphocytes) , but mitochondrial hyperpolarization is a rare phenomenon in yeast.
Membrane dysfunctions by periplanetasin-4.
Mitochondria are highly dynamic cellular organisms that continue to undergo two opposing processes of cleavage and fusion. Mitochondrial dynamics affects not only the mitochondrial morphology but also mitochondrial biogenesis, bioenergetics, intracellular mitochondrial distribution within the cell, and cell death [4,34]. Consistent with such an interpretation, the extent of inter-mitochondrial interactions can be led to mitochondrial fusion, reflects an augmentation in the functional activity of these mitochondria. Mitochondrial biomass and morphological changes were observed using the MitoTracker Green. The fluorescence intensity of periplanetasin-4-treated cells increased in comparison with that of untreated cells. As determined by fluorescence microscopy, periplanetasin-4 leads to a decrease in tubular mitochondria with an increase in mitochondrial biomass and an increase in fragmented mitochondria. Indeed, periplanetasin-4 increases fission activity with the mitochondrial biomass (Figure 3B,C). Increase in mitochondrial membrane potential is correlated with an increase in mitochondrial size .
In fungal cells, vacuoles and the mitochondria are essential for physiological processes and involved in oxidative stress response. The fungal vacuoles are an acidic organelle containing abundant hydrolytic enzymes and high levels of ions, functioning in nutrient storage, maintenance of ion homeostasis, autophagy, adaptation to environmental stresses, and cell differentiation [4,46]. To verify vacuolar membrane permeability, we analyzed cells labeled with BCECF by flow cytometry. BCECF fluorescence intensity increases vacuoles alkalization as the pH increases from 5.0 to 8.0 . Periplanetasin-4 indicated increase in fluorescence intensity and it suggested the evidence that the vacuoles membrane is permeabilized and is alkalinized. It has the potential for inhibition of the vacuolar-type H+-ATPase (V-ATPase), which is ubiquitous proton pump coupling ATP hydrolysis to proton translocation across the membranes of intracellular compartments . To determine the permeability of the vacuolar membrane changes and localization more detail, we employed the fluorogenic compound C-DCFDA, which becomes fluorescent and membrane-impermeant upon hydrolysis of the acetate groups by esterases in the lumen of the vacuole . The membrane-permeable compound, C-DCFDA, can easily diffuse into the vacuoles and hydrolyze by acidic esterases in this compartment, generating the membrane-impermeant fluorescent agent. However, during the process of promoting the permeability of the vacuolar membrane, this agent can leak out of the vacuoles, resulting in diffusion of fluorescence into the cytosol. Therefore, it is an ideal indicator of vacuolar membrane permeabilization . The cells exposed to periplanetasin-4 indicated an increase in fluorescence intensity and the vacuoles fragmentation (Figure 4). Taken together, the intracellular response by periplanetasin-4 makes dysfunction in mitochondria and vacuoles.
Vacuolar response by periplanetasin-4.
ROS-induced periplanetasin-4 play a key role in communication between intracellular organelles
ROS, such as the superoxide radicals, hydrogen peroxide, and the hydroxyl radical, typically arise because of electron leakage from the electron transport chain to oxygen . There are three main reactions leading to the formation of intracellular ROS, the mitochondrial electron transport, the vacuolar Fenton reaction and the formation of disulfide bonds for oxidative protein folding in the ER [22,49]. Mitochondria have long been established as a major source of the superoxide radicals, which is generated from dioxygen by electron leakage originating in the mitochondrial transport chain during respiration. In yeast, specific sources of superoxide in the mitochondrial chain . Mitochondrial superoxide production analyzed with the fluorescent dye MitoSox, which selectively targets mitochondria, where it is rapidly oxidized by superoxide but not by other nitrogen species or reactive oxygen . We observed an increase superoxide radicals and hydrogen peroxide in periplanetasin-4 treated cells, measured by MitoSox and Amplex red fluorescence (Figure 5A,B), whereas the level of hydroxyl radical remained unchanged, measured by HPF (Figure 5C). The accumulation of cellular ROS, particularly hydroxyl radicals, inevitably propagate the synthesis of further hydroperoxides and other reactive derivatives, all of which can inflict extensive oxidative damage to cell biomolecules such as proteins, DNA, and lipids . Periplanetasin-4 did not cause DNA damage. Despite the ROS accumulation, it was confirmed that intracellular oxidative damage in cells was occurred by measuring lipid peroxidation. MDA is a biomarker of lipid peroxidation and a decomposition product of polyunsaturated fatty acid hydroperoxidase [50,51]. As shown in Figure 5D, the MDA levels in the periplanetasin-4-treated cells were increased compared with that in control cells. The generated superoxide radicals lead to intracellular oxidative damage and peroxidation of phospholipid . Superoxide radicals from the mitochondria accounted for the majority of ROS and they affect phospholipid.
Origin of periplanetasin-4 induced ROS and oxidative damage.
Excessive accumulation of intracellular ROS can damage the cell structure and directly or indirectly cause cell death . Periplanetasin-4 induced cell death did not accompany by a significant increase in hydroxyl radical. Biomolecules oxidized by hydroxyl radicals can become radicals themselves, which propagate even further non-specific cell oxidative damage. In fact, most oxidative damage in cells is mediated by hydroxyl radicals, which is far more toxic than its precursors superoxide radicals and hydrogen peroxide . However, the mitochondria and vacuoles show their dysfunctions that seem to respiration and control the proton gradient. Hence, we checked the antioxidant defense systems, both enzymatic and non-enzymatic antioxidant action. ROS-scavenging enzymes like SOD, CAT have been shown to supply some protection against oxidative stress. SOD is the first defense enzyme against oxidative stress that produces hydrogen peroxide from superoxide radicals. The SOD-derived hydrogen peroxide can, in turn, be degraded to water and oxygen by the redox-sensitive heme groups of catalase . CAT1, the single catalase gene in C. albicans, plays a decisive role in scavenging hydrogen peroxide [4,53]. When cells were treated with periplanetasin-4, SOD activity did not exhibit a significant change compared with untreated cells (Figure 5E), whereas the catalase activity was increased (Figure 5F). While mitochondrial superoxide radicals were increased, SOD activity is maintained and then catalase activity detoxified the toxicity of hydrogen peroxide, catalyzing water and oxygen.
Calcium levels can affect ROS and glutathione through multiple mechanisms, while ROS generated in different compartments by different means can in turn strongly affect calcium levels through different mechanisms. Thus, there appear to be a well-regulated but complex interplay of these different molecules that is still not entirely understood . To determine non-enzymatic antioxidant defense systems, glutathione levels were determined in cell extracts. The result indicated to increase the total cellular glutathione level (Figure 6A). Both of catalase and glutathione action can interrupt the hydroxyl radical toxicity. To determine whether calcium signaling was involved in cell death, we used intracellular calcium levels with Fura-2AM and Rhod-2AM, the membrane-permeable derivative of the ratiometric calcium indicators. The cytosolic-free calcium concentration in C. albicans in the periplanetasin-4-treated group was not shown a significant difference with the control group (Figure 6B). Whereas the fluorescence intensity of Rhod-2AM, a mitochondrially targeted free calcium, increased in cells treated with periplanetasin-4 (Figure 6C). Free calcium levels in the cytoplasm were maintained and mitochondrial calcium level was increased in cells treated with periplanetasin-4 (Figure 6B,C). These results demonstrate that the increase in mitochondrial membrane potential leads to enhance superoxide production in mitochondria and reflect in an elevation of calcium uptake .
Antioxidant systems and intracellular signaling by periplanetasin-4 of C. albicans.
Cross-talk between intracellular organelles can involve signaling pathways or direct physical contact. Furthermore, contact sites between the mitochondria and vacuoles are identified in yeast . We hypothesized that the origin of mitochondrial calcium ion is cytosol from vacuoles and cytosolic calcium ion can maintain due to leak other calcium storage, vacuoles. Vacuoles are the primary intracellular store of calcium in yeast and have putative vacuolar Ca2+/H+ exchanger and V-ATPase, as a vacuolar proton pump for maintenance of cytosolic calcium homeostasis . In previous experiments, the vacuolar membrane is permeabilized and alkalized. Calcium release from vacuoles to cytosol is the response to an alkaline pulse . It implied the disruption of vacuoles ion store capacity or ion homeostasis. Another rationale for this is Fe2+ stress . Since the production of hydroxyl radical is dependent upon the presence of iron, it would require the organism to store iron . If Fenton reaction could not regulate, toxic ferrous ion and high level of superoxide is continuously accumulated . Thus, the accumulation of superoxide was restricted through antioxidant using ascorbic acid and the change in the vacuoles was observed (Figure 7A). Ascorbic acid-cotreated cells showed a lower dysfunction compared with alone periplanetasin-4 treated cells (Figure 7B,C). The dysfunction of vacuoles remains through the scavenging of superoxide radicals. Additionally, loss of V-ATPase activity results in a striking induction of iron import . The relationship between V-ATPase and iron homeostasis remains to be elucidated . Thus, the intracellular process inducing periplanetasin-4 interferes with communication between mitochondria and vacuoles.
Oxidative damage by periplanetasin-4 reduced by ROS scavenger.
Periplanetasin-4 contributes to interact with plasma membrane
Membrane integrity collapses after a series of events. The dynamics of the membrane were observed through DPH to determine the cell status after the intracellular process. The fluorescence intensity of DPH decreased in periplanetasin-4-treated cells compared with that in untreated cells, and the cell membrane became more unstable. Melittin showed decrease in DPH fluorescence intensity on C. albicans cells (Figure 8A). This decrease in DPH intensity revealed perturbation of the cell membrane following periplanetasin-4 treatment. The mechanistic actions of AMP on membrane instability have been established mainly for peptides with helical properties . Hydrophobic properties of periplanetasin-4 rigidify membrane fluidity, leading to increase membrane permeability and leak intracellular content. The alteration of membrane fluidity could be revealed due to the presence of hydrophobic compounds that penetrate in the lipid bilayer of the cytoplasmic membrane . To determine whether periplanetasin-4 causes membrane alteration through a direct physical interaction with the phospholipid bilayer, artificial membranes with lipid compositions were prepared. Artificial membranes containing using molecules of different weights and radius were measured . Model membranes exposed to periplanetasin-4 allowed leakage of intracellular probes. As shown in Figure 8B, periplanetasin-4 caused a substantial release of calcein and intermediate release at FD4. Treatment with periplanetasin-4 did not release FD10 from the liposomes, mimics C. albicans membranes. These results indicated that the degree of disturbance was between 1.4 and 2.3 nm on artificial membranes. Charge, secondary structure, hydrophobicity, amphipathicity and hydrophobic moment are all essential to function. Altering one property will often result in significant changes to one or more of the others because they are so interdependent . When the C. albicans cells were treated with periplanetasin-4, FD4 permeated into the cytoplasm, but larger molecules with a radius of 2.3 nm were unable to pass on surface of C. albicans. Melittin caused leakage of all fluorescent dyes, indicating that the size is larger than 2.3 nm (Figure 8C). This phenomenon correlated with the results of the artificial membrane experiment, which similarly showed that membrane disturbance was induced. Periplanetasin-4 does not allow molecules with radius of up to 2.3 nm to cross the bilayer and it may allow movement of intracellular ions. These observations indicated that this disruption of membrane integrity might directly interact with the phospholipid bilayer, which destabilizes the cytoplasmic membrane, resulting in cell death.
Impact of periplanetasin-4 on membrane action of C. albicans.
Periplanetasin-4 has lower cytotoxicity against human eukaryotes
One of the prerequisites for developing antibiotics is no fatal action by the drug on human cells or that is at least kept within a minimum range free of treatment-free side effects. So far, AMPs are thought to be free of cytotoxicity due to such a non-specific membrane-lytic mechanism. However, not all peptides are in this class. Detrimental cytotoxicity of AMPs has been reported. The cytotoxicity of melittin on different mammalian cells including erythrocytes is well known . Melittin was used as a positive control to compare the cytotoxicity. However, it is still not confirmed whether periplanetasin-4 acts as a signal of danger. To assess hemolysis and LDH release, the peptides were treated with various concentrations (1.3–80.0 µM). Periplanetasin-4 showed no hemolytic activity towards human erythrocytes at any tested concentrations and induced almost no LDH release (Table 3), whereas melittin exhibited potent hemolytic effect and LDH release. Taken together, periplanetasin-4 exerted broad-spectrum antimicrobial activities without hemoglobin or LDH release from human erythrocytes.
|μM||% Hemolysis||% LDH release|
|μM||% Hemolysis||% LDH release|
American Type Culture Collection
Clinical and Laboratory Standards Institute
bis-1,3-dibutylbarbituric acid trimethine oxonol
Korean Collection for Type Cultures
large unilamellar vesicle
minimum inhibitory concentration
programmed cell death
reactive oxygen species
transferase dUTP nick end labeling
Study conception and design: H.L., J.S.H., D.G.L.; acquisition of data: H.L., D.G.L.; analysis and interpretation of data: H.L., J.S.H., D.G.L.; drafting of manuscript: H.L., D.G.L.; critical revision: H.L., J.S.H., D.G.L.
This work was supported by a grant from the Next-Generation BioGreen 21 Program (Project No. PJ01325603), Rural Development Administration, Republic of Korea.
The Authors declare that there are no competing interests associated with the manuscript.
These authors contributed equally to this work and should be considered co-first authors.