The antimicrobial peptide CGA-N12 (NH2-ALQGAKERAHQQ-COOH) is an active peptide derived from chromogranin A (CGA) and consists of the 65th to 76th amino acids of the N-terminus. The results of our previous studies showed that CGA-N12 exerts anti-Candida activity by inducing apoptosis without destroying the integrity of cell membranes. In this study, the effect of CGA-N12 on the cell membrane structure of Candida tropicalis was investigated. CGA-N12 resulted in the dissipation of the membrane potential, the increase in membrane fluidity, and the outflow of potassium ions in C. tropicalis without significantly changing the ergosterol level. Fluorescence quenching was applied to evaluate the membrane channel characteristics induced by CGA-N12 through detection of the following: membrane permeability of hydrated Cl (ϕ ≈ 0.66 nm) using the membrane-impermeable halogen anion-selective fluorescent dye lucigenin, passage of the membrane-impermeable dye carboxyfluorescein (CF) (ϕ ≈ 1 nm) through the membrane, and membrane permeation of H3O+ based on the membrane non-permeable pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS). In conclusion, CGA-N12 can induce the formation of non-selective ion channels <1 nm in diameter in the membranes of C. tropicalis, resulting in the leakage of potassium ions, chloride ions, and protons, among others, leading to dissipation of the membrane potential. As a result, the fluidity of membranes is increased without destroying the synthesis of ergosterol is not affected.

## Introduction

The incidence of fungal infections has risen sharply over the past two decades, causing high morbidity and mortality [1]. In particular, a very large number of urinary tract infections (UTIs) are caused by Candida, especially in patients in intensive care units. Candida tropicalis is one of the most common strains causing UTIs [2], and UTIs caused by C. tropicalis may develop into severe candidemia [3]. Furthermore, routine clinical antifungal drugs such as triazoles and liposome polyene have been found to be ineffective in the treatment of UTIs [3]. The widespread use of broad-spectrum antibiotics is believed to be the main reason for the rise in microbial resistance [4,5]. In addition to the over- and misuse of antifungals [6], increases in the range and diversity of infective pathogenic fungi have led to the growing number of patients with mycosis [7]. Indeed, the resistance of fungal pathogens to traditional antifungal compounds has recently increased to a disturbing extent [8,9].

At present, the main types of antifungal drugs include the following: azoles, which restrict ergosterol synthesis; polyenes, which target ergosterol in fungal membranes; and acanthomycin, which targets cell wall synthesis [10]. Therefore, the development of new antifungal drugs with novel modes of action is important for maintaining human health. As potential antibiotics, antimicrobial peptides (AMPs) possess many excellent physical and chemical properties, such as multiple action sites, multiple action mechanisms, a low tendency for developing new drug resistance mechanisms and low toxicity [11,12]. Given the mechanism of some common antifungal agents, the development of new, non-toxic antifungal drugs is more challenging than is the development of other types of antimicrobial agents because of the similarity between fungal structures and the corresponding human protein or cellular structures [13].

CGA-N12 is an antimicrobial peptide derived from chromogranin A and consists of the 65th to 76th amino acids of the N-terminus of the protein [14]. Previous studies have shown that CGA-N12 can disturb the synthesis of cell wall of C. tropicalis by inhibiting the β-1,6-glucanase activity of KRE9 [15], enter Candida cells and inhibit their growth by inducing apoptosis [16] without destroying the integrity of the cell membrane. However, it was not clear how CGA-N12 affects the cell membrane after entering the cells. Thus, the effects on membrane fluidity, ergosterol level, membrane potential, and membrane channel characteristics induced by CGA-N12 were examined in the present study.

## Materials and methods

### Microorganism and materials

C. tropicalis (ATCC20138) was supplied by the China Academy of Chinese Medical Sciences (Beijing, China). The fungi were cultured on Sabouraud dextrose (SD) agar at 28°C for 48 h and maintained at 4°C for short-term storage.

CGA-N12 was synthesized via solid-phase peptide synthesis with free N- and C-termini. Peptide purification was performed by high-performance liquid chromatography, and the mass of the peptide was confirmed by mass spectrometry. The anti-Candida activity of CGA-N12 was assessed by the broth micro-dilution method to define the minimum inhibitory concentration (MIC), which was 75 µM [17].

### Protoplasts preparations

Cell wall-free protoplasts of C. tropicalis were prepared using Zymolyases-20T following the manufacturer's instructions. Mid-exponential phase cells were collected by centrifugation (5000 rpm for 5 min) and washed twice with PBS (0.02 M, pH 7.2), and the wet weight of the cells was recorded. The cell pellet was resuspended in TE buffer (100 mM Tris, pH 8.0, 100 mM EDTA; 1.4 ml/g), and the final concentration was adjusted to 3.5 ml/g by adding ddH2O. To remove the mannan layer from the outer layer of the cells, 0.5% (v/v) β-mercaptoethanol was added, and the cells were incubated at 30°C for 45 min with gentle shaking. The cell pellet was collected by centrifugation at 5000 rpm for 5 min, washed twice, and then resuspended in buffer S (1.0 M sorbitol, 10 mM PIPES, pH 6.5; 4.0 ml/g). Zymolyase-20T (50 U/g wet weight cells) was added, and the samples were incubated at 30°C for 60 min with gentle shaking. The samples were then washed twice with buffer S to obtain of C. tropicalis protoplasts.

### Membrane fluidity assessment

Membrane fluidity was evaluated by examining anisotropic (γ) changes of TMA-DPH (APExBIO, U.S.A.), a fluorescent membrane probe [18]. Protoplasts (106 CFU) were treated with CGA-N12 (1×MIC) for 0, 2, 4, 6, 8 and 10 h, after which 4 ml of the fluorescent probe (TMA-DPH, final concentration 2 µM) was added. The samples were then incubated for 30 min at 30°C in a water bath. The fluorescence anisotropy γ (λex = 355 nm, λem = 430 nm) of each sample was measured using an LS-55 fluorescence spectrometer (PerkinElmer, U.S.A.). Each experiment was performed in triplicate.

### Ergosterol level determination

The ergosterol level in the C. tropicalis plasma membrane was determined as reported by Kocsis et al. [19]. A total of 106 CFU of C. tropicalis cells in SD medium were incubated with 1 × MIC of CGA-N12 for 0, 5, 10, 15, 20 and 25 h at 28°C. After incubation, the samples were centrifuged to collect the cells, which were then suspended in alcoholic KOH (25% w/v, 3 ml) for 1 h in an 85°C water bath. The samples were cooled to ambient temperature, and sterols were isolated by adding 1 ml of distilled water and 3 ml of n-heptane. After 3 min of vortex, the heptane layer was recovered, and ethanol was used to dilute the samples prior to spectrophotometric analysis (Shimadzu UV 2450, Japan) between 240 nm and 300 nm. PBS (0.02 M, pH 7.2) and Fluconazole (1 µg/ml) were set as negative and positive control, respectively. Each experiment was performed in triplicate.

### Membrane depolarization assay

The release of DiSC3(5) (3,3-dipropylthiadicarbocyanine iodide) (Sigma–Aldrich, Shanghai, China), a fluorescent probe, from the C. tropicalis cell membrane as a result of peptide (CGA-N12) treatment was monitored to study the effect of the peptide on the cell membrane potential. C. tropicalis (106 CFU) was resuspended in 5 mM HEPES buffer (pH 7.2) to OD600 = 0.05, and the samples were assessed using a fluorescence spectrophotometer (λexem = 600 nm/675 nm) (Cary Eclipse, Hitachi Japan). After a stable fluorescence signal was obtained, a solution of DiSC3(5) in DMSO was added to a final concentration of 1.6 µM. Following continuous detection to a steady fluorescence intensity, an appropriate amount of CGA-N12 (1 × MIC) was added to each sample. A sample without CGA-N12 was used as the blank control, and a sample with 1 µM NaN3 was used as the positive control. Each experiment was repeated three times.

### Intracellular potassium efflux measurement

C. tropicalis cells cultured in SD to mid-exponential phase were collected, washed twice with PBS (0.02 M, pH 7.2) and resuspended. CGA-N12 was added to a final concentration of 1 × MIC and incubated at 28°C for 0, 5, 10, 15, 20 and 25 h. The supernatant was collected by centrifugation and passed through a 0.22 µm filter. After filtration, the K+ level was determined using an inductively coupled plasma optical emission spectrometer (Agilent 5110, U.S.A.). A fungal suspension not containing any antibacterial agent was used as the blank control; for the positive control, an equal amount of melittin was added to the suspension. Each experiment was performed in triplicate.

### Fluorescent dye-loaded liposomes preparation and purification

Lucigenin powder (Sigma–Aldrich, Shanghai, China) was dissolved in 50 mM NaNO3 (pH 7.0) to a final concentration of 1 mM. CF powder (Sigma–Aldrich, Shanghai, China) was dissolved in Millipore water to a final concentration of 40 mM. HPTS powder (Sigma–Aldrich, Shanghai, China) was dissolved in 50 mM NaNO3 (pH 7.0) to a final concentration of 0.1 mM. All dyes were filter sterilized.

Using the mass ratio DOPC : DOPE = 1 : 1.27 for the yeast cell membrane [20], DOPC and DOPE were accurately weighed to a total mass of 8 mg and dissolved in a round-bottom flask containing 4 ml of chloroform. The fluorescent dye-loaded liposomes were assembled on a rotary evaporator. After the vacuum of the instrument was slowly increased to its maximum (0.1 MPa), the solution was continuously evaporated for 60 min (100 rpm, 45°C) until a film formed on the bottle wall. The film was dissolved in 4 ml of ether, and 100 µl of the filter-sterilized fluorescent dye stock solution was injected under the liquid surface. The sample was ultrasonicated for 6 min (intermittent time 4 s, working time 4 s, 200 W) in an ice-water bath, and a uniform, milky white solution was obtained after a second rotary evaporation step (same method as above, 60 min). Three millilitres of filter-sterilized 50 mM NaNO3 (pH 7.0) was added to the flask, and the mixture was rotary evaporated at 45°C for 30 min under normal pressure. Finally, the solution was passed through a 0.2 µm Nucleopore filter to obtain a liposome solution loaded with the fluorescent dye, and the solution was stored at 4°C in the dark.

The fluorescent dye-loaded liposomes were purified by gel filtration (Sephadex G-25), and a 50 mM NaNO3 (pH 7.0) solution was used as an extra-vesicle aqueous solution to separate and remove the free fluorescent dye outside the liposomes. The purified liposomes were dispersed in a 50 mM NaNO3 (pH 7.0) solution to a final lipid concentration of 0.3 mM. The HPTS liposomes were dispersed in 50 mM NaNO3 (pH 6.0) to a final lipid concentration of 0.3 mM (final concentration of DOPE).

### Fluorimeter-based lucigenin-quenching assays

CGA-N12 was dissolved in 150 mM NaCl to prepare a stock solution. A total of 1980 µl of lucigenin liposomes was placed in a quartz colorimetric dish with four transparent sides and incubated with 20 µl CGA-N12 (1×MIC) stock solution as an experimental group at CGA-N12 : liposome = 1 : 3 (molar ratio); 20 µl NaCl (150 mM) was used as a blank control. When the fluorescence intensity was stable, 20 µl of 20% Triton-X100 was added to establish 100% leakage. A fluorescence spectrophotometer (λexem = 368 nm/505 nm) was used to monitor the samples for 30 min. The percentage of quenched lucigenin was determined as follows:
$quenchingrate(%)=[(It−I0)/(I∞−I0)]×100%$
where It is the fluorescence intensity at a given time of the addition of the peptide, I0 is the intensity before peptide addition, and I is the intensity after the addition of 20% Triton X-100. Each experiment was performed in triplicate.

### Fluorimeter-based HPTS assays

CGA-N12 stock solutions were prepared in Millipore water and diluted with 50 mM NaNO3 (pH 7.0) to 1 × MIC. First, we acquired the HPTS spectra under neutral and acidic conditions. A 1980 µl aliquot of HPTS-preloaded liposomes was placed in a quartz colorimetric dish with four transparent sides. For the blank control, 20 µl of 50 mM NaNO3 (pH 7.0) was added; for the experimental groups, 20 µl of CGA-N12 stock solution (1 × MIC) was added at CGA-N12 : liposomes = 1 : 3 (molar ratio). The fluorescence signal was measured at λex = 455 nm and λex = 404 nm over the full wavelength range (λem = 514 nm); fluorescence was also observed with a fluorescence confocal microscope (Olympus, Tokyo, Japan). Each experiment was performed in triplicate.

### Fluorimeter-based CF leakage assays

CGA-N12 stock solutions were prepared in Millipore water and diluted with 50 mM NaNO3 (pH 7.0) to 1 × MIC. A total of 1980 µl aliquot of CF liposomes was placed in a quartz colorimetric dish with four transparent sides. For the blank control, 20 µl of 50 mM NaNO3 was added; for the experimental groups, 20 µl of the CGA-N12 (1 × MIC) stock solution was added at CGA-N12 : liposomes = 1 : 5 (molar ratio); for the positive control, 20 µl of melittin (1 × MIC) was added. After 10, 20 µl of 20% Triton-X100 was added to establish 100% leakage. A fluorescence spectrophotometer (λexem = 489 nm/515 nm) was used to monitor the fluorescence intensity every 5 h for 15 h. The percentage of CF leakage was determined as follows:
$leakage(%)=[(It−I0)/(I∞−I0)]×100%$
where It is the fluorescence intensity at a given time of the addition of the peptide, I0 is the intensity before the addition of the peptide, and I is the intensity after the addition of 20% Triton X-100. Each experiment was carried out three times.

### Statistical analysis

SPSS version 21.0 was used for the statistical analysis (ANOVA and Tukey's test). Data are means ± standard deviation (SD), and differences of P < 0.05 were considered to be significant.

## Results

### CGA-N12 affected cell membrane fluidity

TMA-DPH is a fluorescent membrane probe, membrane fluidity can be characterized by the rotational motion of TMA-DPH, whereby faster rotation of the excited-state fluorophore indicates smaller steady-state anisotropy, i.e. a decrease in the anisotropy corresponds to an increase in the membrane fluidity [21]. To detect the effects of CGA-N12 on cell membrane fluidity, changes in the steady-state anisotropy of cell membrane-bound TMA-DPH were analysed by spectroscopic methods. The results are shown in Figure 1. Within 10 h of CGA-N12 treatment, the fluorescence anisotropy (γ) of the protoplasts in the experimental group decreased significantly compared with that of the control group (Figure 1). The results showed that the membrane fluidity of C. tropicalis was enhanced by the action of CGA-N12.

#### CGA-N12 reduced the steady state anisotropy of TMA-DPH inserted into the plasma membrane.

Figure 1.
CGA-N12 reduced the steady state anisotropy of TMA-DPH inserted into the plasma membrane.

Protoplasts (1 × 106 CFU) were treated with CGA-N12 (1 × MIC) for 0, 2, 4, 6, 8 and 10 h. An amount of 4 ml of the fluorescent probe (TMA-DPH, final concentration 2 µM) was added, and the samples were incubated for 30 min at 30°C in a water bath. The fluorescence anisotropy γ (λex = 355 nm, λem = 430 nm) of the samples was measured using an LS-55 fluorescence spectrometer. Protoplasts treated with PBS (0.02 M, pH 7.0) were used as a control group. Data represent the mean ± standard deviation of three independent experiments. 0.01 < P < 0.05 (Student's t-test).

Figure 1.
CGA-N12 reduced the steady state anisotropy of TMA-DPH inserted into the plasma membrane.

Protoplasts (1 × 106 CFU) were treated with CGA-N12 (1 × MIC) for 0, 2, 4, 6, 8 and 10 h. An amount of 4 ml of the fluorescent probe (TMA-DPH, final concentration 2 µM) was added, and the samples were incubated for 30 min at 30°C in a water bath. The fluorescence anisotropy γ (λex = 355 nm, λem = 430 nm) of the samples was measured using an LS-55 fluorescence spectrometer. Protoplasts treated with PBS (0.02 M, pH 7.0) were used as a control group. Data represent the mean ± standard deviation of three independent experiments. 0.01 < P < 0.05 (Student's t-test).

### CGA-N12 had no effect on the plasma membrane ergosterol level

Ergosterol, a sterol unique to fungi, is an important component of fungal cell membranes for maintaining cell integrity and membrane fluidity [22]. The ergosterol level in the C. tropicalis cell membrane was measured by absorbance at 281.5 nm. 24(28)-Dehydroergosterol, another component of the fungal cell membrane, also has a maximum absorption at 281.5 nm in addition to 230 nm. The ergosterol amount was calculated by subtracting the absorbance of 24(28)-dehydroergosterol at 230 nm [22].

As depicted in Figure 2, within 25 h of the test, the ergosterol level increased over time. Compared with the PBS control group, the ergosterol amount in the experimental group increased slightly, but the difference between the two was not significant. While the ergosterol amount in the positive control group decreased after treatment with Fluconazole. It is speculated that CGA-N12 has no significant effect on the ergosterol level in the plasma membrane of C. tropicalis; that is, ergosterol is not a direct target of CGA-N12.

#### CGA-N12 had no effect on the ergosterol level in the plasma membrane.

Figure 2.
CGA-N12 had no effect on the ergosterol level in the plasma membrane.

A total of 106C. tropicalis cells in SD medium were incubated at 28°C with 1 × MIC CGA-N12 for 0, 5, 10, 15, 20 and 25 h. Ergosterol in the cell membrane was isolated by extraction. Spectrophotometric analysis was performed at 281.5 nm. PBS (0.02 M, pH 7.0) and Fluconazole (1 µg/ml) were as negative and positive control, respectively. Data represent the mean ± standard deviation of three independent experiments. P = 0.056 > 0.05 (Student's t-test).

Figure 2.
CGA-N12 had no effect on the ergosterol level in the plasma membrane.

A total of 106C. tropicalis cells in SD medium were incubated at 28°C with 1 × MIC CGA-N12 for 0, 5, 10, 15, 20 and 25 h. Ergosterol in the cell membrane was isolated by extraction. Spectrophotometric analysis was performed at 281.5 nm. PBS (0.02 M, pH 7.0) and Fluconazole (1 µg/ml) were as negative and positive control, respectively. Data represent the mean ± standard deviation of three independent experiments. P = 0.056 > 0.05 (Student's t-test).

### Effect of CGA-N12 on cell membrane potential

The changing process of the experimental result was shown as Figure 3, when the cell membrane depolarization probe DiSC3(5) was added, the fluorescence intensity increased dramatically. However, when DiSC3(5) was combined with a polar membrane, the fluorescence intensity decreased gradually and was monitored continuously for 11 min at which point it stabilized (Figure 3). After treatment with CGA-N12, the fluorescence intensity of the experimental group increased gradually, indicating that DiSC3(5) was dissociated from the cell membrane and that the cell membrane polarity of C. tropicalis dissipated.

#### CGA-N12 caused C. tropicalis cell membrane depolarization.

Figure 3.
CGA-N12 caused C. tropicalis cell membrane depolarization.

C. tropicalis (1 × 106 CFU) was resuspended in 5 mM HEPES buffer (pH 7.2) to OD600 = 0.05 and monitored using a fluorescence spectrophotometer (λexem = 600 nm/675 nm). Once a stable fluorescence signal was reached, DiSC3(5) was dissolved in DMSO and added to a final concentration of 1.6 µM. PBS, CGA-N12 and NaN3 were added to the blank control groups, test groups and positive controls, respectively, to determine their impacts on fluorescence (λexem = 600 nm/675 nm). Data represent the mean ± standard deviation of three independent experiments. P < 0.05 (Student's t-test).

Figure 3.
CGA-N12 caused C. tropicalis cell membrane depolarization.

C. tropicalis (1 × 106 CFU) was resuspended in 5 mM HEPES buffer (pH 7.2) to OD600 = 0.05 and monitored using a fluorescence spectrophotometer (λexem = 600 nm/675 nm). Once a stable fluorescence signal was reached, DiSC3(5) was dissolved in DMSO and added to a final concentration of 1.6 µM. PBS, CGA-N12 and NaN3 were added to the blank control groups, test groups and positive controls, respectively, to determine their impacts on fluorescence (λexem = 600 nm/675 nm). Data represent the mean ± standard deviation of three independent experiments. P < 0.05 (Student's t-test).

### CGA-N12 induced K+ leakage from C. tropicalis cells

To further evaluate the permeability of C. tropicalis membranes treated with CGA-N12, K+ release was determined. As illustrated in Figure 4, the K+ concentration in the supernatant of the blank control group was relatively low and stable; in contrast, the K+ concentration in the supernatant of the experimental group increased, and that in the supernatant of the positive group increased rapidly. These results demonstrated that when C. tropicalis was exposed to CGA-N12, membrane permeability increased, and K+ efflux occurred. It is speculated that CGA-N12 may induce potassium channel opening in C. tropicalis, causing the release of K+ without affecting the integrity of the cell membrane.

#### CGA-N12 induced K+ leakage from C. tropicalis.

Figure 4.
CGA-N12 induced K+ leakage from C. tropicalis.

The antimicrobial peptide CGA-N12 was added to a final concentration of 1 × MIC and incubated with Candida at 28°C for 0, 5, 10, 15, 20 and 25 h. The supernatants were collected by centrifugation and passed through a 0.22 µm filter, after which the K+ level was determined using ICP-OES. For the blank control, the fungal suspension did not contain any antibacterial agent; for the positive control, an equal amount of melittin was added to the suspension. Data represent the mean ± standard deviation of three independent experiments. P < 0.05 (Student's t-test).

Figure 4.
CGA-N12 induced K+ leakage from C. tropicalis.

The antimicrobial peptide CGA-N12 was added to a final concentration of 1 × MIC and incubated with Candida at 28°C for 0, 5, 10, 15, 20 and 25 h. The supernatants were collected by centrifugation and passed through a 0.22 µm filter, after which the K+ level was determined using ICP-OES. For the blank control, the fungal suspension did not contain any antibacterial agent; for the positive control, an equal amount of melittin was added to the suspension. Data represent the mean ± standard deviation of three independent experiments. P < 0.05 (Student's t-test).

### CGA-N12 induced chloride ion leakage from DOPC:DOPE liposomes

The fluorescent dye N, N’-dimethyl-9,9’-biacridinium dinitrate (lucigenin) (λexem = 368 nm/505 nm) is a membrane-impermeant halide-anion-selective dye. The combination of lucigenin with a halogen anion such as Cl results in fluorescence quenching, which can be used to detect the chloride ion permeability of liposomes after CGA-N12 treatment to determine if it induces chloride channel formation in the model membrane.

Lucigenin-treated liposomes were placed in a NaCl solution. Fluorescence quenching occurred upon treatment with CGA-N12 (Figure 5). These results showed that Cl in the external environment infiltrated the liposomes, causing the fluorescence of lucigenin to quench. CGA-N12 may form channels in the liposome membrane, allowing chloride ions to pass through.

#### CGA-N12 treatment initiated the kinetics of quenching in the fluorescence of lucigenin preloaded within DOPC : DOPE = 1 : 1.27 liposomes.

Figure 5.
CGA-N12 treatment initiated the kinetics of quenching in the fluorescence of lucigenin preloaded within DOPC : DOPE = 1 : 1.27 liposomes.

A total of 1980 µl aliquot of lucigenin liposomes was placed in a quartz colorimetric dish with four transparent sides and incubated with 20 µl CGA-N12 (1 × MIC) as an experimental group (CGA-N12:liposome = 1 : 3 (molar ratio)); 20 µl of NaCl (150 mM) was used as the blank control. When the fluorescence intensity was stable, 20 µl of 20% Triton-X100 was added to establish 100% leakage levels. A fluorescence spectrophotometer (λexem = 368 nm/505 nm) was used to monitor the samples for 30 min. Data represent the mean ± standard deviation of three independent experiments.

Figure 5.
CGA-N12 treatment initiated the kinetics of quenching in the fluorescence of lucigenin preloaded within DOPC : DOPE = 1 : 1.27 liposomes.

A total of 1980 µl aliquot of lucigenin liposomes was placed in a quartz colorimetric dish with four transparent sides and incubated with 20 µl CGA-N12 (1 × MIC) as an experimental group (CGA-N12:liposome = 1 : 3 (molar ratio)); 20 µl of NaCl (150 mM) was used as the blank control. When the fluorescence intensity was stable, 20 µl of 20% Triton-X100 was added to establish 100% leakage levels. A fluorescence spectrophotometer (λexem = 368 nm/505 nm) was used to monitor the samples for 30 min. Data represent the mean ± standard deviation of three independent experiments.

### CGA-N12 resulted in proton penetration through DOPC : DOPE liposomes

8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) (λem = 514 nm, λex = 404 nm/455 nm) is a membrane-impermeable pH-sensitive fluorescent dye; an increase in H+ reduces the fluorescence intensity of HPTS at λex = 455 nm, whereas the intensity at λex = 404 nm would be maintained or only slightly decreased. This phenomenon was used to detect the formation of H3O+channels after CGA-N12 treatment.

H3O+ has an effective hydration diameter of 0.56 nm, making it slightly smaller than Cl (0.66 nm) [23]. To examine whether the transmembrane ‘holes' induced by CGA-N12 treatment allow the passage of H3O+, we carried out fluorescence-based assays using HPTS, a membrane-impermeant pH-sensitive dye, to elucidate proton permeability across the lipid membrane.

CGA-N12 is an amphiphilic antimicrobial peptide that induces the production of H3O+, which lowers the pH of a solution. As indicated in Figure 6A, HPTS (10 µM, pH 7.0) dissolved in neutral NaNO3 (50 mM, pH 7.0) showed corresponding absorption peaks at λex = 404 nm and λex = 455 nm, but HPTS dissolved in acidic NaNO3 (pH 6.0, 50 mM) showed no fluorescence at λex = 455 nm, and the absorption at λex = 404 nm decreased only slightly. After treatment with CGA-N12 (1 × MIC), the absorption peaks of the HPTS-loaded liposome solution at λex = 455 nm disappeared rapidly, indicating that H3O+ was produced in the solution upon CGA-N12 treatment and that H3O+ entered the liposome, resulting in a decrease in the internal pH. These results demonstrated that H3O+ ions enter liposomes by passing through channels in the membranes.

#### CGA-N12 treatment initiated the kinetics of quenching in the fluorescence of HPTS preloaded within DOPC : DOPE = 1 : 1.27 liposomes.

Figure 6.
CGA-N12 treatment initiated the kinetics of quenching in the fluorescence of HPTS preloaded within DOPC : DOPE = 1 : 1.27 liposomes.

(A) HPTS scans under neutral and acidic conditions. The liposome extravesicular buffer solution was 50 mM NaNO3. HPTS pH 7.0 was used for the neutral environment. HPTS pH 6.0 was used for the acidic environment. A total of 1980 µl of HPTS-preloaded liposomes was placed in a quartz colorimetric dish with four transparent sides. A total of 20 µl of 50 mM NaNO3 (pH 7.0) was added as the blank control; 20 µl of 1 × MIC CGA-N12 stock solution was added as the experimental group. The fluorescence intensity was measured at λex = 455 nm and λex = 404 nm over the full wavelength range (λem = 514 nm). (B) Fluorescence intensities of the HPTS-preloaded liposomes under neutral and acidic conditions, and upon treatment with CGA-N12 (1 × MIC). HPTS-loaded liposomes incubated with Dil were used to demonstrate the integrity of the liposomes. Scale bar = 10 µm. Data represent the mean ± standard deviation of three independent experiments.

Figure 6.
CGA-N12 treatment initiated the kinetics of quenching in the fluorescence of HPTS preloaded within DOPC : DOPE = 1 : 1.27 liposomes.

(A) HPTS scans under neutral and acidic conditions. The liposome extravesicular buffer solution was 50 mM NaNO3. HPTS pH 7.0 was used for the neutral environment. HPTS pH 6.0 was used for the acidic environment. A total of 1980 µl of HPTS-preloaded liposomes was placed in a quartz colorimetric dish with four transparent sides. A total of 20 µl of 50 mM NaNO3 (pH 7.0) was added as the blank control; 20 µl of 1 × MIC CGA-N12 stock solution was added as the experimental group. The fluorescence intensity was measured at λex = 455 nm and λex = 404 nm over the full wavelength range (λem = 514 nm). (B) Fluorescence intensities of the HPTS-preloaded liposomes under neutral and acidic conditions, and upon treatment with CGA-N12 (1 × MIC). HPTS-loaded liposomes incubated with Dil were used to demonstrate the integrity of the liposomes. Scale bar = 10 µm. Data represent the mean ± standard deviation of three independent experiments.

Laser confocal microscopy was employed for further verification. As presented in Figure 6B, HPTS-preloaded liposomes displayed the fluorescence emission of HPTS at λex = 404 nm and λex = 455 nm in NaNO3 (50 mM) at pH 7.0; the lipid membrane stained by Dil was observed at λex=565 nm. After CGA-N12 treatment, the fluorescence intensity of the HPTS-loaded liposomes at λex = 405 nm remained unchanged, whereas that at λex = 455 nm decreased significantly. These results indicated that the internal pH of the liposomes decreased, suggesting that CGA-N12 induced the formation of proton channels in HPTS-loaded liposomes.

### Channel inner diameter of DOPC : DOPE liposomes induced by CGA-N12

5(6)-Carboxyfluorescein (CF) (λexem = 489 nm/515 nm) is a membrane-impermeable anionic fluorescent dye with a hydrodynamic diameter of ∼1 nm [24,25], which is greater than the diameter of Cl (0.66 nm) [23]. At concentrations higher than 40 mM, CF has strong fluorescence self-quenching properties.

CF-encapsulated liposomes were prepared and incubated with CGA-N12 to determine the size of the ion channels in polypeptide-treated Candida membranes based on CF leakage. From the results, the fluorescence intensity of CF did not change within 10 h (Figure 7), indicating that CGA-N12 did not result in CF leakage from the liposomes. Compared with melittin, a membrane-ruptured peptide and CF leakage rate of up to 90%,CGA-N12 induced channels with diameters no more than 1 nm in the fungal membrane model.

#### CGA-N12 treatment initiated the kinetics of leakage in the fluorescence of CF preloaded within DOPC : DOPE = 1 : 1.27 liposomes.

Figure 7.
CGA-N12 treatment initiated the kinetics of leakage in the fluorescence of CF preloaded within DOPC : DOPE = 1 : 1.27 liposomes.

A total of 1980 µl aliquot of CF liposomes was placed in a quartz colorimetric dish with four transparent sides; for the blank control, 20 µl of 50 mM NaNO3 was added; for the experimental groups, 20 µl of CGA-N12 (1 × MIC) stock solution was added; for the positive control, 20 µl of melittin (1 × MIC) was added. After 10 h, 20 µl of 20% Triton-X100 was added to establish 100% leakage levels by complete solubilization of the vesicle membranes. A fluorescence spectrophotometer (λexem = 489 nm/515 nm) was used to monitor the fluorescence intensity every 5 h for 15 h. Data represent the mean ± standard deviation of three independent experiments.

Figure 7.
CGA-N12 treatment initiated the kinetics of leakage in the fluorescence of CF preloaded within DOPC : DOPE = 1 : 1.27 liposomes.

A total of 1980 µl aliquot of CF liposomes was placed in a quartz colorimetric dish with four transparent sides; for the blank control, 20 µl of 50 mM NaNO3 was added; for the experimental groups, 20 µl of CGA-N12 (1 × MIC) stock solution was added; for the positive control, 20 µl of melittin (1 × MIC) was added. After 10 h, 20 µl of 20% Triton-X100 was added to establish 100% leakage levels by complete solubilization of the vesicle membranes. A fluorescence spectrophotometer (λexem = 489 nm/515 nm) was used to monitor the fluorescence intensity every 5 h for 15 h. Data represent the mean ± standard deviation of three independent experiments.

## Discussion

It makes it more intriguing now that CGA-N12 could form channels, with diameters less than 1 nm, on the cell membrane in addition to interacting with KRE9, a β-1,6-gluconase, to inhibit cell wall synthesis of C. tropicalis [15] and induce mitochondrial dysfunction, resulting in apoptosis without destroying the integrity of the cell membrane [16]. Therefore, CGA-N12 exerts multiple effects on its antagonistic activity on C. tropicalis.

Antimicrobial peptides have emerged as a new class of antibiotics to overcome the emergence of widespread antibiotic resistance because of their low susceptibility to the development of microbial resistance [26–28]. Antimicrobial peptides produced by various animals are known to play crucial roles in non-specific host defence and innate immunity. One of the killing mechanisms used by antimicrobial peptides is disruption of the outer and cytoplasmic membranes of bacteria via the formation of transmembrane pores or a ‘carpet mechanism', leading to of the bacterial cells lysis [27,29]. Another killing mechanism used by antimicrobial peptides is the formation of pores across the bacterial cytoplasmic membrane without causing extensive damage to host cell membranes [30]. The classical models are the ‘barrel-stave pore' model and the ‘toroidal pore' model. Transmembrane pores lead to enhanced membrane permeability and the loss of cellular inclusions (such as ATP), resulting in cell death [31]. AMPs adopt different strategies for penetrating hydrophobic cell membranes and can enter cells through pore-dependent or pore-independent mechanisms [32]. The electrostatic interaction between peptides and microbial cell membranes followed by partitioning of the biological membrane appears to be the basic antimicrobial mechanism of most AMPs [33,34]. Although most AMPs interact with and influence the integrity of microbial membranes, it is not known whether membrane permeabilization is always a lethal event or if the membrane is the only site of action. Several peptides have mechanisms of action other than membrane permeabilization. For example, buforin II kills bacteria without inducing membrane lysis and has strong affinity for DNA and RNA [35].

Membrane fluidity is a basic characteristic of biofilm structures, and it mainly refers to the movement of membrane fatty acid chains and membrane proteins. Lipids in the membrane mainly diffuse laterally, rotate, sway left and right, stretch and oscillate, and flip and undergo dissimilation at temperatures above the phase transition temperature. Membrane lipid fluidity is an indicator of the structure and function of the cell membrane. Suitable fluidity is necessary for normal cell membrane performance. In this study, we investigated changes in the membrane fluidity of C. tropicalis protoplasts upon treatment with CGA-N12. The fact that the cells exhibited significantly lower anisotropy than the control cells when treated at the experimental concentration suggested that CGA-N12 increased the mobility of the fluorophore (fluidizing effect) anchored to the head groups of the phospholipids, manifesting as a more fluid membrane structure; i.e. membrane fluidity increased. The study of amphotericin B by Younsi, M. et al. [36] proposed two reasons for the increase in membrane fluidity: local rearrangement of the membrane components and the interaction between amphotericin B and membrane components, which results in the formation of several types of channels within the membrane. At lower concentrations of amphotericin B, channels with smaller diameters are formed, causing high permeability of water and loss of K+ and Mg2+ [36] and resulting in increased membrane fluidity. This phenomenon is consistent with our experimental results. Upon interaction with the cell membranes, CGA-N12 induced the production of small ion channels (<1 nm in diameter), causing a transient increase in the permeability of water and the loss of K+ and Cl, among others, and resulting in increased membrane fluidity. In addition, to adapt to changes in their external environment, microorganisms can invoke certain mechanism [37], including (i) increasing the contents of long-chain fatty acids and unsaturated fatty acids, which leads to greater membrane fluidity and integrity [38,39], and (ii) augmenting the sterol content, which influences membrane fluidity, increasing membrane thickness and sturdiness [40]. Therefore, we speculate that the increased membrane fluidity upon CGA-N12 treatment may be a response of fungal cells to maintain cell stability.

Ergosterol, a 5,7-diene oxysterol, is the most abundant sterol in fungal cell membranes and regulates permeability and fluidity [41]. Because of its crucial functions, unique structural properties, and particular biosynthesis, ergosterol is the target of the majority of clinically available antifungal agents [42]. For example, Liu et al. [22] found that C16-fengycin disrupted ergosterol biosynthesis and reduced the ergosterol content in Fusarium graminearum in a concentration-dependent manner. The plasma membrane is considered to be the main target of C16-fengycin A. Previous works have reported that other compounds can also reduce ergosterol levels by destroying this sterol [43,44]. The mechanism of action of azoles as antifungal agents is based on the disruption of sterol biosynthesis, attenuating in ergosterol production [45]. These compounds thus prevent ergosterol from functioning in fungal membranes and disturb both the structure and function of the membrane. Because ergosterol also plays a hormone-like (‘sparking’) role, stimulating growth in fungal cells, the net effect of azoles is the inhibition of fungal growth [46]. Although ergosterol synthesis appears to be the main target of most AMPs, there are some exceptions. For instance, Kocsis et al. found that the antifungal activity of Mannich ketones has no relation to the inhibition of ergosterol synthesis [19]. In our study, the ergosterol amount increased during the first 15 h following CGA-N12 treatment and then remained stable. This is most likely because fungal ergosterol synthesis is not one of the targets of CGA-N12, and because its antifungal activity has no effect on the inhibition of ergosterol synthesis. Another possibility is that when cells are stimulated, their ergosterol amount is increased, enhancing the thickness and firmness of the membrane [40].

Antimicrobial peptides are a critical component of the innate immune system of many species, including humans [47,48]. Despite extraordinarily diverse structures, AMPs are all rich in cationic and hydrophobic amino acids, making them well suited for interacting with microbial cytoplasmic membranes, which typically have an anionic surface. When microbes are exposed to AMPs, the electrochemical potential of their cytoplasmic membranes can rapidly dissipate, allowing the permeation of large molecules, including dye markers, metabolites and cytosolic proteins, in a short time [49,50]. Exposure to most peptides causes the membrane potential of the bacterial inner membranes to dissipate within seconds, presumably due to proton and small ion (e.g. Na+ and K+) leakage [49]. Depolarization is indicative of ion movement across the cytoplasmic membrane [51]. For example, one of the mechanisms of daptomycin is dissipation of the bacterial membrane potential, resulting in disruption of multiple aspects of cellular function [52,53]. It has been suggested that membrane depolarization is not the primary target of some antimicrobial peptides, but that depolarization is required to facilitate antibiotic entry into a bacterium where these agents can then exert their activity [54]. We detected membrane potential dissipation after CGA-N12 treatment, and we speculated that cell membrane depolarization promotes the entry of CGA-N12 into cells.

Jared A. reported that the mechanism of membrane depolarization upon daptomycin exposure involves K+ efflux from a bacteria cell [51]. Cells maintain their membrane potential by establishing multiple ion gradients across the cytoplasmic membrane because proper maintenance of the K+ gradient is important for cell viability. Jared A. analysed the K+ gradient during daptomycin exposure and suggested that either K+ efflux may be responsible for membrane depolarization or that K+ may be one of several ions released during membrane depolarization caused by daptomycin [51]. In our study, the concentration of K+ in the extracellular fluid after CGA-N12 treatment was measured, and leakage of K+ was observed. This leakage may be a reason for cell membrane depolarization. In addition, the formation of small channels can lead to a transient increase in water permeability and the loss of K+ and Mg2+, among others. [36], which may increase membrane fluidity. Therefore, K+ leakage can increase membrane fluidity.

Next, we studied the leakage of other ions and measured the inner diameters of the small channels formed due to CGA-N12 treatment. Based on the proportions of membrane components of Candida cells in the exponential phase [20], we constructed a Candida-like membrane as a biofilm simulation system for studying the mechanism of action of CGA-N12 in the membrane. Recently, Hu K et al. explored interactions between ORB-1, a 15-residue disulfide-bridged AMP, and bacterial membranes using model membranes from G bacteria [23]. They found that ORB-1 allowed small anions (hydrodynamic diameter <1 nm), such as Cl and NO3, to pass through the model membrane of the G bacteria that cations of a similar size, such as H3O+ and Na+, could not pass [23]. Our experiments show that CGA-N12 induces the formation of non-selective ion channels with effective hydrodynamic diameters less than 1 nm. The CGA-N12-induced ion channels had small pore sizes, allowing chloride ions to pass through but with no selectivity for ions smaller than 1 nm, which may be related to the composition of the membrane and the structure of the peptide.

In conclusion, CGA-N12 induces the formation of non-selective ion channels less than 1 nm in diameter in C. tropicalis membranes, resulting in the leakage of e.g. K+, Cl, H+ and leading to dissipation of the membrane potential. As a result, the membranes became more fluid without losing their integrity. Additionally, this AMP does not affect the synthesis of ergosterol.

## Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

## Funding

This study was supported by the National Natural Science Foundation of China [31572264 and 31071922], the Innovative Research Team (in Science and Technology) at the University of Henan Province (19IRTSTHN008), and the National Engineering Laboratory for Wheat & Corn Further Processing, Henan University of Technology (NL2016010).

## Author Contribution

R.L. and W.S. drafted the manuscript and participated in the design of the experiments. W.S. carried out the experiments of membrane fluidity assessment, ergosterol level determination, membrane depolarization assay and intracellular potassium efflux measurement. R.Z. carried out the experiments of fluorimeter-based lucigenin-quenching assays, fluorimeter-based HPTS assays and fluorimeter-based CF leakage assays. L.H. provided guidance for experiments. A.L. participated in the membrane fluidity assessment. Y.Y. and H.J. participated in the final editing of the manuscript. M.T. assisted with the preparation and purification of the fluorescent dye-loaded liposomes. M.Z. and N.P. participated in the peptide synthesis. All authors have read and approved the final manuscript.

## Abbreviations

• AMPs

antimicrobial peptides

•
• ATCC

American Type Culture Collection

•
• ATP

•
• CF

carboxyfluorescein

•
• CFU

colony-forming unit

•
• CGA

chromagranin A

•
• CGA-N12

the amino acid sequence from the 65th to the 76th residue of the N-terminus of chromagranin A

•
• DiSC3(5)

•
• EDTA

ethylenediaminetetraacetic acid

•
• HPTS

8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt

•
• MIC

minimum inhibitory concentration

•
• ORB-1

a 15-residue disulfide-bridged AMP (LKGCWTKSIPPKPCF)

•
• PBS

phosphate-buffered saline

•
• SD

Sabouraud dextrose

•
• TMA-DPH

a fluorescent membrane probe

•
• UTIs

urinary tract infections

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