Abstract

CGA-N9 is a peptide derived from the N-terminus of human chromogranin A comprising amino acids 47–55. Minimum inhibitory concentration (MIC) assays showed that CGA-N9 had antimicrobial activity and exhibited time-dependent inhibition activity against Candida tropicalis, with high safety in human red blood cells (HRBCs) and mouse brain microvascular endothelial cells (bEnd.3). According to the results of transmission electron microscopy (TEM), flow cytometry and confocal microscopy, CGA-N9 accumulated in cells without destroying the integrity of the cell membrane; the peptide was initially localized to the cell membrane and subsequently internalized into the cytosol. An investigation of the cellular internalization mechanism revealed that most CGA-N9 molecules entered the yeast cells, even at 4°C and in the presence of sodium azide (NaN3), both of which block all energy-dependent transport mechanisms. In addition, peptide internalization was affected by the endocytic inhibitors 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), cytochalasin D (CyD) and heparin; chlorpromazine (CPZ) also had some effect on CGA-N9 internalization. Similar results were obtained in the MIC assays, whereby the anticandidal activity of CGA-N9 was blocked to different degrees in the presence of EIPA, CyD, heparin or CPZ. Therefore, most CGA-N9 passes through the C. tropicalis cell membrane via direct cell penetration, whereas the remainder enters through macropinocytosis and sulfate proteoglycan-mediated endocytosis, with a slight contribution from clathrin-mediated endocytosis.

Introduction

Candida is regarded as the third most common pathogen responsible for clinical diseases ranging from superficial infections of the vaginal and oral mucosa to life-threatening systemic diseases in immunocompromised patients [13]. Candida tropicalis has become one of the most common clinical species of this genus, after Candida albicans, causing ∼3–66% of clinical candidiasis, especially in America and Asia [46]. With the continuous emergence of conventional antibiotic-resistant strains, the morbidity and mortality rates due to candidiasis are gradually increasing [1,7]. Thus, there is an urgent need to discover novel and more effective antifungal agents to combat potentially fatal pathogenic fungi [810].

Based on their advantages over the limited number of clinical compounds used to treat fungal infections and the side effects of conventional antifungal drugs, antimicrobial peptides (AMPs) have received much attention in recent years and show a wide range of activity, low toxicity and low resistance development [11]. Various AMPs with diverse structures and antimicrobial properties have been discovered from different sources, such as plants, animals and microorganisms [1215].

Chromogranin A (CGA), a member of the chromogranin family, is ubiquitously distributed in the chromaffin granules of neuroendocrine cells. It is highly conserved and widely presents in many mammals, such as humans, bovine and mice, especially in the N-terminal and C-terminal regions [16]. CGA exhibits antibacterial and antifungal activities, particularly in the N-terminal domain [17]. CGA-derived peptides are involved in innate immunity [18]. In this study, we identified the AMP CGA-N9, which is an N-terminal derivative of human CGA consisting of amino acids 47–55.

The first target of AMPs is the cell membrane [1921]. The electrostatic interaction between the peptide and cell membrane, followed by entry via the membrane and effects on intracellular organelles, appears to be the basic mechanism by which most AMPs perform their antimicrobial function [19,22,23]. Although the mechanism of action of most AMPs is reported to include membrane lysis and intracellular activity [2429], the exact membrane process responsible for non-pore-dependent mechanisms remains poorly understood due to the complicated physical and chemical properties of these peptides.

Here, we investigate the routes by which CGA-N9 enters C. tropicalis.

Materials and methods

Microorganisms, cell line and reagents

Candida glabrata (ATCC90525), Candida parapsilosis (ATCC20224), Candida krusei (ATCC6258), C. tropicalis (ATCC20138), Candida albicans (ATCC2048), Cryptococcus neoformans (ATCC14116), Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923), Bacillus subtilis (ATCC5230), Listeria monocytogenes (ATCC13932) and Pseudomonas aeruginosa (ATCC35554) were supplied by the China Academy of Chinese Medical Sciences (Beijing, China). Fungi were sub-cultured onto Sabouraud dextrose (SD) agar at 30°C for 48 h. Bacteria were cultured on Luria-Bertani (LB) agar at 37°C for 16 h. The bacteria and fungi were maintained at 4°C for short-term storage.

The mouse brain microvascular endothelial cell line (bEnd.3) was provided by the Shaanxi Key Laboratory of Natural Products Chemistry and Biology, College of Chemistry & Pharmacy, Northwest A&F University.

CGA-N9 (NH2-RILSILRHQ-COOH) was synthesized using a solid-phase method. One milligram of peptide was dissolved in 15 µl of dimethyl sulfoxide, and 985 µl of phosphate-buffered saline (PBS) (20 mmol/l, pH 6.0) was added to a total volume of 1 ml; an appropriately diluted sample was used for subsequent analysis.

Antimicrobial assay

The antimicrobial activity of peptide CGA-N9 was evaluated by employing the broth micro-dilution method [30], with minor modifications. In brief, fungi were cultured in SD liquid medium at 28°C for logarithmic growth, and bacteria were cultured in LB liquid medium at 37°C for logarithmic growth. Cells were suspended in medium, and the concentration was adjusted to 1 × 106 cfu/ml for fungal inocula and 1 × 105 cfu/ml for bacterial inocula. A 100-µl volume of CGA-N9 solution (1 mg/ml) was added to the wells of a 96-well plate and serially diluted twofold with PBS. The final concentrations of the peptide mixture ranged from 1000 to 1.95 µg/ml. Each well was inoculated with equal volumes of microbial cells. After incubation for 16 h for bacteria and 20 h for fungi, 10 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) solution (5 mg/ml in PBS) was added to each well to detect live cells. Absorbance at 570 nm (A570) was measured. The MIC100 was defined as the lowest concentration resulting in no visible growth compared with control cells [7]. The cytotoxicity kinetics of CGA-N9 against C. tropicalis was defined as the cell viability kinetics measured at 4-h intervals. Experiments were conducted in triplicate.

Fungicidal assay

The minimum fungicidal concentration (MFC) was determined following the incubation of CGA-N9 with C. tropicalis in the MIC assay by removing 150 µl of sample from each well, plating the samples onto SD agar plates and culturing for 20–36 h at 28°C. The resulting colonies were counted. MFC was defined as the lowest concentration of CGA-N9 that killed 99.9% of the initial inoculum [25].

Hemolytic assay

The hemolytic activity of CGA-N9 was tested by a previously reported method [31]. Briefly, fresh HRBCs (human red blood cells) from healthy volunteers were washed thrice with normal saline, and HRBS suspensions were prepared at a final concentration of 2% for this assay. One hundred microliters of double-diluted CGA-N9 (0–500 µg/ml) was added to each well of a 96-well plate, followed by 100 µl of 2% HRBC suspension in each well. After incubation for 30 min at 37°C, 150 µl of supernatant was transferred to a new 96-well plate, and the amount of hemoglobin released at 540 nm was measured. One-percent Triton X-100 was used as a positive control, and normal saline was used as a negative control. The percentage of hemolysis was calculated by the following equation: 
formula

Cell counting kit-8 method

An in vitro mammalian cell cytotoxicity test of CGA-N9 was performed with a mouse brain microvascular endothelial cell line (bEnd.3) using the CCK8 method (Cell Counting Kit-8) [32,33]. 4 × 103 bEnd.3 cells were seeded in each well of a 96-well plate. After the cells were incubated at 37°C in 5% CO2 for 10 h, different concentrations of CGA-N9 (0–80 times the MIC100) were added in the wells and further incubated for 48 h. The toxicity of CGA-N9 towards bEnd.3 cells was determined using CCK8 (MedChem Express, Shanghai, China). Absorbance was measured by an ELISA plate reader at 450 nm. Cells that were not incubated with CGA-N9 were used as a negative control, and DMEM containing 5% FBS was used as a blank control. Cell viability was calculated with the following equation: 
formula

Transmission electron microscopy

C. tropicalis cells were observed by transmission electron microscopy (TEM) after CGA-N9 treatment [28]. Briefly, 1 × 106 cfu/ml mid-log phase C. tropicalis cells were incubated with CGA-N9 at a concentration of 3.9 µg/ml (MIC100) at 28°C. C. tropicalis cells in 1 ml of culture were collected after each 4-h interval and fixed overnight in 500 µl of 5% glutaraldehyde in PBS at 4°C. The cells were then further fixed in 1 ml of osmium acid for 1.5 h at room temperature. The samples were dehydrated and embedded in resin. Ultra-thin sections were stained with uranyl acetate followed by lead citrate. The specimens were observed by TEM (Hitachi H-7650; Hitachi, Ltd, Tokyo, Japan). C. tropicalis cells that had not been exposed to CGA-N9 were used as controls.

Flow cytometry

Propidium iodide (PI) can bind nucleic acids after penetrating the compromised cell membrane of dead, membrane-damaged and apoptotic cells. The effect of CGA-N9 on the membrane permeability of C. tropicalis was determined by flow cytometry using PI and the method outlined by Li et al. [28]. Briefly, 1 × 106 cfu/ml logarithmic C. tropicalis cells in SD broth were incubated with CGA-N9 (3.9 µg/ml) at 28°C. After incubation for 4, 8, 12 and 16 h, the cells were incubated with 50 µg/ml PI in the dark for more than 10 min at room temperature. The samples were analyzed using a FACSCalibur flow cytometer (BD, NJ, U.S.A.). C. tropicalis cells that had not undergone treatment with CGA-N9 or had been treated with 0.3% Triton X-100 were used as negative or positive controls, respectively. The results were analyzed using the FACSDiva version 6.1.3 software package.

Calcein leakage assay

The effect of CGA-N9 on the membrane integrity of C. tropicalis was characterized according to the leakage rate of calcein from liposomes. Calcein-loaded liposomes were prepared according to a previously reported method [34] with minor modifications. Dioleoyl phosphatidylcholine and dioleoyl phosphoethanolamine (Sigma–Aldrich, Shanghai, China) (1 : 1.27, w/w) were employed to mimic the logarithmic-phase Candida lipid bilayer [35]. A suspension of calcein-loaded liposomes was incubated with 3.9 µg/ml CGA-N9 at 28°C to evaluate pore formation and membrane integrity by measuring dye leakage every 1 h using a fluorescence spectrophotometer (Cary Eclipse, CA, U.S.A.) at λex/λem = 492/517 nm. In this assay, 20 mmol/l PBS (pH 7.0) was selected as a negative control, and 10 mmol/l H2O2 was used as a positive control. All samples were examined in triplicate. After 10 h in the presence of 0.1% Triton X-100 in Tris buffer, 100% of the dye was released from liposomes.

Peptide labeling and cellular internalization

To assess cellular uptake of CGA-N9, the peptide was labeled by covalent modification of its N-terminus with fluorescein 5-isothiocynate (FITC) using the method described by Ruissen et al. [36].

Logarithmic-phase C. tropicalis cells (1 × 106 cfu/ml) were incubated with the FITC-CGA-N9 conjugate at a final concentration of 2.9 µg CGA-N9/ml (MIC50) at 28°C for 0, 4, 8, 12 and 16 h. After incubation, the cells were washed with pH 8.5 PBS and further incubated with 20 mmol/l Dil for 25 min to stain the cell membrane. The cells were washed and examined using a confocal laser microscope (Olympus, Tokyo, Japan) at λex/λem = 549 nm/565 nm for Dil and λex/λem = 495 nm/519 nm for FITC to observe CGA-N9 internalization.

Confocal microscopy to assay the effects of energy on cellular peptide uptake

The effects of energy on cellular peptide uptake were determined using a previously reported method [37]. A total of 1 × 106 cfu/ml logarithmic-phase C. tropicalis cells were pre-incubated at 4 and 30°C for 12 h with the FITC-CGA-N9 conjugate at a concentration of 2.9 µg CGA-N9/ml (MIC50). The cells were examined via confocal microscopy at λex/λem = 495/519 nm. C. tropicalis cells that had not undergone treatment with FITC-CGA-N9 or with 0.65 mg/ml NaN3 in the presence of FITC-CGA-N9 at 30°C for 12 h were used as negative and positive controls, respectively.

Confocal microscopy assay to identify the cellular peptide uptake pathway

Pathways involved in CGA-N9 uptake by C. tropicalis cells were investigated using various endocytic inhibitors [38]. First, 1 × 106 cfu/ml logarithmic-phase C. tropicalis cells were incubated for 1 h with one of the following endocytic inhibitors: methyl-β-cyclodextrin (Mβ-CD, 3.75 mg/ml), cytochalasin D (CyD, 5.08 µg/ml), chlorpromazine (CPZ, 7.11 µg/ml), chloroquine (CQ, 1.72 µg/ml), NaN3 (0.65 mg/ml), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) (10 µg/ml) and heparin (100 µg/ml). The FITC-CGA-N9 conjugate at a concentration of 3.9 µg CGA-N9/ml was then added after the edocytic inhibitors were washed off, and the samples were further incubated for 12 h. Uptake of the FITC-CGA-N9 conjugate was determined using a confocal laser microscope (Olympus, Tokyo, Japan). The fluorescence value was recorded after analysis using the FV31S-SW software. Uptake of FITC-CGA-N9 by C. tropicalis cells in the absence of endocytic inhibitors was used as a control.

Assessment of the effects of endocytic inhibitors on peptide anticandidal activity

The effect of endocytic inhibitors on the anticandidal activity of CGA-N9 was detected by the MIC assay [38], with minor modifications. A total of 1 × 106 cfu/ml logarithmic-phase C. tropicalis cells were treated with Mβ-CD (3.75 mg/ml), CyD (5.08 µg/ml), CPZ (7.11 µg/ml), CQ (1.72 µg/ml), NaN3 (0.65 mg/ml), EIPA (10 µg/ml) or heparin (100 µg/ml) for 1 h at 28°C [39]. Subsequently, CGA-N9 was added at concentrations of 1.95 µg/ml (MIC5), 2.9 µg/ml (MIC50) and 3.9 µg/ml (MIC100) for 16 h at 28°C after the edocytic inhibitors were washed off, and the samples were then subjected to the MIC assay. All experiments were performed in triplicate. In this assay, 1.95 µg/ml CGA-N9 was used to identify the endocytosis inhibitors that exerted a primary effect against CGA-N9 endocytosis. The antifungal activity of CGA-N9 against C. tropicalis cells in the absence of endocytic inhibitors was used as a control.

Statistical analysis

SPSS version 21.0 was used for the statistical analysis (ANOVA and Tukey's test). All data are presented as the mean ± standard deviation. A P-value <0.05 indicated a difference, a P-value <0.01 indicated a significant difference and a P-value <0.001 indicated an extremely significant difference.

Results

Antimicrobial activity of CGA-N9

The cytotoxicity of CGA-N9 against microbes was determined via the MIC assay and SD agar plate culture. The MIC assay results indicated that CGA-N9 rapidly reduced the viability of C. tropicalis cells in a dose-dependent manner, with MIC50 and MIC100 values of 2.9 and 3.9 µg/ml, respectively (Table 1).

Table 1
Antimicrobial spectrum of the AMP CGA-N9

MIC values were determined by MIC assay. MIC100 was defined as the lowest concentration resulting in no visible growth compared with control cells.

Strains MIC100 (µg/ml) 
C. glabrata 500.00 
C. parapsilosis 250.00 
C. krusei 15.63 
C. tropicalis 3.90 
C. neoformans 3.90 
E. coli — 
S. aureus — 
B. subtilis 15.63 
L. monocytogenes 31.25 
P. aeruginosa — 
Strains MIC100 (µg/ml) 
C. glabrata 500.00 
C. parapsilosis 250.00 
C. krusei 15.63 
C. tropicalis 3.90 
C. neoformans 3.90 
E. coli — 
S. aureus — 
B. subtilis 15.63 
L. monocytogenes 31.25 
P. aeruginosa — 

‘—’ indicates no detectable antimicrobial effect at CGA-N9 concentrations >1000 µg/ml.

As shown in Figure 1, cell viability dramatically increased at CGA-N9 concentrations <3.9 µg/ml. To evaluate C. tropicalis viability, cells were cultured on SD agar plates after treatment with different concentrations of CGA-N9. No colonies grew in the presence of concentrations >3.9 µg/ml, suggesting that exposure to CGA-N9 killed the Candida cells.

Cytotoxicity of CGA-N9 against C. tropicalis at different concentrations.

Figure 1.
Cytotoxicity of CGA-N9 against C. tropicalis at different concentrations.

Approximately 1 × 106 logarithmic-phase C. tropicalis cells were incubated with CGA-N9 for 18 h at 28°C. MFC values were determined by the formation of colonies after culturing on SDA plates. MFC was defined as the lowest concentration of antifungal agent that caused at least 99.9% killing of the initial inoculum. Data represent the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test).

Figure 1.
Cytotoxicity of CGA-N9 against C. tropicalis at different concentrations.

Approximately 1 × 106 logarithmic-phase C. tropicalis cells were incubated with CGA-N9 for 18 h at 28°C. MFC values were determined by the formation of colonies after culturing on SDA plates. MFC was defined as the lowest concentration of antifungal agent that caused at least 99.9% killing of the initial inoculum. Data represent the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test).

Figure 2 presents the cytotoxicity kinetics of CGA-N9. The results revealed that CGA-N9 cytotoxicity was time-dependent, with C. tropicalis cell killing beginning after 8 h of CGA-N9 treatment.

Cytotoxicity kinetics of CGA-N9 against C. tropicalis.

Figure 2.
Cytotoxicity kinetics of CGA-N9 against C. tropicalis.

Approximately 1 × 106 logarithmic-phase C. tropicalis cells were incubated with 3.9 µg/ml CGA-N9 at 28°C. The cytotoxicity kinetics of CGA-N9 was based on the cell survival rate at different times after CGA-N9 treatment. Data represent the mean ± standard deviation of three independent experiments. **P < 0.01, ***P < 0.001 (Student's t-test).

Figure 2.
Cytotoxicity kinetics of CGA-N9 against C. tropicalis.

Approximately 1 × 106 logarithmic-phase C. tropicalis cells were incubated with 3.9 µg/ml CGA-N9 at 28°C. The cytotoxicity kinetics of CGA-N9 was based on the cell survival rate at different times after CGA-N9 treatment. Data represent the mean ± standard deviation of three independent experiments. **P < 0.01, ***P < 0.001 (Student's t-test).

Hemolytic activity

The hemolytic activity of CGA-N9 (0–500 µg/ml) is revealed in Figure 3. A 5% hemolysis rate for CGA-N9 occurred at the concentration of 105.62 µg/ml (27 times the MIC100). At a concentration >16 times the MIC100 (62.5 µg/ml), the hemolysis rate was only 0.26%. The results showed that CGA-N9 exhibits biosafety in HRBCs.

The hemolytic activity of CGA-N9.

Figure 3.
The hemolytic activity of CGA-N9.

The hemolysis of CGA-N9 was measured by the release of hemoglobin. HRBCs were incubated with different concentrations of CGA-N9 (0–500 µg/ml) for 30 min at 37°C. One-percent Triton X-100 was used as a positive control, and normal saline was used as a negative control. Five-percent hemolysis occurs in red blood cells post-treatment with 105.62 µg/ml CGA-N9 (27 times the MIC100). Data represent the mean ± standard deviation of three independent experiments. ***P < 0.001 (Student's t-test).

Figure 3.
The hemolytic activity of CGA-N9.

The hemolysis of CGA-N9 was measured by the release of hemoglobin. HRBCs were incubated with different concentrations of CGA-N9 (0–500 µg/ml) for 30 min at 37°C. One-percent Triton X-100 was used as a positive control, and normal saline was used as a negative control. Five-percent hemolysis occurs in red blood cells post-treatment with 105.62 µg/ml CGA-N9 (27 times the MIC100). Data represent the mean ± standard deviation of three independent experiments. ***P < 0.001 (Student's t-test).

Cytotoxicity to mammalian cells

Using the CCK8 method, CGA-N9 showed non-toxicity to bEnd.3 cells after treatment for 48 h at concentrations of 10–50 times the MIC100. CGA-N9 did not display any bEnd.3 cell growth inhibition until reaching concentrations of 60–80 times the MIC100, at which the cell viability rate was still more than 70%. Interestingly, the viabilities of bEnd.3 cells at concentrations that were 10–40 times the MIC100 of CGA-N9 were higher than that of the control group, which may be due to the nutritional benefits of CGA-N9 as a peptide bEnd.3 cell growth (Figure 4). These data are sufficient to illustrate a high safety of CGA-N9 towards mammalian cells.

Cytotoxicity of CGA-N9.

Figure 4.
Cytotoxicity of CGA-N9.

Cytotoxicity of CGA-N9 to mammalian cells in vitro was tested by using the CCK8 method with mouse brain microvascular endothelial cells (bEnd.3). After 4 × 103 bEnd.3, cells were incubated in each well of a 96-well plate at 37°C in 5% CO2 for 10 h, different concentrations of CGA-N9 (0–80 times the MIC100) were added and further incubated for 48 h. Absorbance was measured by an ELISA plate reader at 450 nm. Cells that were not incubated with CGA-N9 were used as a negative control, and DMEM containing 5% FBS was used as a blank control. The cell viability of bEnd.3 was calculated. Data represent the mean ± standard deviation of three independent experiments. **P <0.01, ***P <0.001 (Student's t-test).

Figure 4.
Cytotoxicity of CGA-N9.

Cytotoxicity of CGA-N9 to mammalian cells in vitro was tested by using the CCK8 method with mouse brain microvascular endothelial cells (bEnd.3). After 4 × 103 bEnd.3, cells were incubated in each well of a 96-well plate at 37°C in 5% CO2 for 10 h, different concentrations of CGA-N9 (0–80 times the MIC100) were added and further incubated for 48 h. Absorbance was measured by an ELISA plate reader at 450 nm. Cells that were not incubated with CGA-N9 were used as a negative control, and DMEM containing 5% FBS was used as a blank control. The cell viability of bEnd.3 was calculated. Data represent the mean ± standard deviation of three independent experiments. **P <0.01, ***P <0.001 (Student's t-test).

Ultrastructure of C. tropicalis cells post-treatment with CGA-N9

The impact of 3.9 µg/ml CGA-N9 on the ultrastructure of C. tropicalis cells was observed via TEM (Figure 5). Compared with the control, CGA-N9 exhibited time-dependent effects on C. tropicalis cells, which was in accordance with the observed cytotoxicity kinetics. The cells displayed cytoplasmic vacuolization, mitochondrial structural damage and loss of nuclear envelope integrity after CGA-N9 treatment. Moreover, the cell wall and outer membrane of cells treated with CGA-N9 remained intact, without visible disruption, even after 16 h of treatment. These results indicate that CGA-N9 may penetrate the cell membrane without disrupting the integrity of the membrane bilayer and subsequently interact with cellular organelles.

Effect of CGA-N9 on the ultrastructure of C. tropicalis cells.

Figure 5.
Effect of CGA-N9 on the ultrastructure of C. tropicalis cells.

A total of 1 × 106 logarithmic-phase C. tropicalis cells were incubated with 3.9 µg/ml CGA-N9 at 28°C for 4, 8, 12 and 16 h. Cell ultrastructure was observed via transmission electron microscopy. C. tropicalis cells that had not undergone CGA-N9 treatment were used as controls. Scale bar = 1 µm.

Figure 5.
Effect of CGA-N9 on the ultrastructure of C. tropicalis cells.

A total of 1 × 106 logarithmic-phase C. tropicalis cells were incubated with 3.9 µg/ml CGA-N9 at 28°C for 4, 8, 12 and 16 h. Cell ultrastructure was observed via transmission electron microscopy. C. tropicalis cells that had not undergone CGA-N9 treatment were used as controls. Scale bar = 1 µm.

Effects of CGA-N9 on the outer membrane permeability of C. tropicalis cells

PI penetrates only damaged membranes and embeds in double-stranded nucleic acids, emitting red fluorescence (λex/λem = 535 nm/615 nm); therefore, PI is widely used to detect disruption of the C. tropicalis outer membrane. According to our results (Figure 6), the PI fluorescent probe was unable to cross the outer membrane of C. tropicalis cells, even following treatment with CGA-N9 at 3.9 µg/ml for 16 h. In contrast, PI fluorescence was detected in C. tropicalis after treatment with the membrane-disrupting agent Triton X-100. The results suggest that CGA-N9 did not disrupt the permeability of the outer membrane of C. tropicalis.

Effect of CGA-N9 on the outer membrane permeability of C. tropicalis cells.

Figure 6.
Effect of CGA-N9 on the outer membrane permeability of C. tropicalis cells.

The nucleic acid dye PI was incubated with C. tropicalis cells after treatment with 3.9 µg/ml CGA-N9 or 0.3% Triton X-100 at 28°C. The fluorescence of PI that had entered the cell and bound to nucleic acids was recorded using flow cytometry. An increase in fluorescence was defined as an increase in membrane permeability. Full entry was observed upon the addition of the membrane-disrupting agent Triton X-100. C. tropicalis cells that had not been exposed to CGA-N9 were used as controls. Data represent the mean ± standard deviation of three independent experiments. ***P <0.001 (Student's t-test).

Figure 6.
Effect of CGA-N9 on the outer membrane permeability of C. tropicalis cells.

The nucleic acid dye PI was incubated with C. tropicalis cells after treatment with 3.9 µg/ml CGA-N9 or 0.3% Triton X-100 at 28°C. The fluorescence of PI that had entered the cell and bound to nucleic acids was recorded using flow cytometry. An increase in fluorescence was defined as an increase in membrane permeability. Full entry was observed upon the addition of the membrane-disrupting agent Triton X-100. C. tropicalis cells that had not been exposed to CGA-N9 were used as controls. Data represent the mean ± standard deviation of three independent experiments. ***P <0.001 (Student's t-test).

Effect of CGA-N9 on membrane integrity

Pore formation in the membrane was investigated by examining calcein release from liposomes treated with CGA-N9. The results showed that exposure to 3.9 µg/ml CGA-N9 did not cause leakage of calcein encased in liposomes (Figure 7), indicating that no pores were formed in the neutral liposome bilayers after treatment with CGA-N9. In contrast, complete leakage from liposomes was observed at 10 h after the addition of the membrane-disrupting agent Triton X-100.

Effect of CGA-N9 on membrane integrity.

Figure 7.
Effect of CGA-N9 on membrane integrity.

Calcein-loaded liposomes were incubated with 3.9 µg/ml CGA-N9 or 0.3% H2O2 at 28°C. The fluorescence of calcein released from the liposomes was recorded using a fluorescence spectrophotometer. An increase in fluorescence indicated an increase in membrane permeability. Complete leakage from liposomes was observed upon the addition of the membrane-disrupting agent Triton X-100 (0.1%) at 10 h. Calcein-loaded liposomes that had undergone PBS treatment were used as negative controls. Data represent the mean ± standard deviation of three independent experiments.

Figure 7.
Effect of CGA-N9 on membrane integrity.

Calcein-loaded liposomes were incubated with 3.9 µg/ml CGA-N9 or 0.3% H2O2 at 28°C. The fluorescence of calcein released from the liposomes was recorded using a fluorescence spectrophotometer. An increase in fluorescence indicated an increase in membrane permeability. Complete leakage from liposomes was observed upon the addition of the membrane-disrupting agent Triton X-100 (0.1%) at 10 h. Calcein-loaded liposomes that had undergone PBS treatment were used as negative controls. Data represent the mean ± standard deviation of three independent experiments.

CGA-N9 internalization

Internalization of FITC-labeled peptides into C. tropicalis was further examined by laser confocal microscopy, as shown in Figure 8. Confocal microscopy analysis showed that FITC-CGA-N9 was initially localized at the periphery of C. tropicalis cells, with further incubation resulting in internalization and intracellular accumulation in a time-dependent manner. CGA-N9 was initially internalized into cells in a nondisruptive manner and subsequently induced cytotoxicity.

CGA-N9 internalization in C. tropicalis cells.

Figure 8.
CGA-N9 internalization in C. tropicalis cells.

A total of 1 × 106 logarithmic-phase C. tropicalis cells were incubated with MIC50 of CGA-N9 (2.9 µg/ml) at 28°C for 4, 8, 12 and 16 h. Entry of the FITC-CGA-N9 conjugate into C. tropicalis cells was observed using confocal microscopy in cells co-stained with the membrane dye Dil. CGA-N9 entered C. tropicalis cells in a time-dependent manner. Initially, the FITC-CGA-N9 conjugate accumulated around the cell membrane. Upon further incubation, FITC-CGA-N9 was translocated into the cytoplasm, as indicated by increased intracellular green fluorescence intensity. Scale bar = 10 µm.

Figure 8.
CGA-N9 internalization in C. tropicalis cells.

A total of 1 × 106 logarithmic-phase C. tropicalis cells were incubated with MIC50 of CGA-N9 (2.9 µg/ml) at 28°C for 4, 8, 12 and 16 h. Entry of the FITC-CGA-N9 conjugate into C. tropicalis cells was observed using confocal microscopy in cells co-stained with the membrane dye Dil. CGA-N9 entered C. tropicalis cells in a time-dependent manner. Initially, the FITC-CGA-N9 conjugate accumulated around the cell membrane. Upon further incubation, FITC-CGA-N9 was translocated into the cytoplasm, as indicated by increased intracellular green fluorescence intensity. Scale bar = 10 µm.

Effect of energy on CGA-N9 internalization

An environmental temperature of 4°C blocks all energy-dependent pathways [37], and NaN3, an inhibitor of cytochrome oxidase in the mitochondrial respiratory chain, can inhibit mitochondrial ATP (adenosine triphosphate) production. In contrast with their behavior at 30°C, the FITC-CGA-N9 conjugate was incompletely internalized into C. tropicalis cells after being cultured at 4°C for 12 h. NaN3 had the same effect as 4°C on the cellular uptake of CGA-N9. As shown by confocal microscopy, although CGA-N9 entered most of the cells, it remained localized at the periphery in some cells, binding only to the membrane (Figure 9). These results confirmed that CGA-N9 internalization mostly involves energy-independent direct cell penetration, with a slight contribution of energy-dependent endocytosis.

Effect of energy on CGA-N9 internalization.

Figure 9.
Effect of energy on CGA-N9 internalization.

A total of 1 × 106 logarithmic-phase C. tropicalis cells were incubated with the MIC50 of CGA-N9 (2.9 µg/ml) for 12 h. Cells in the control group that had not undergone CGA-N9 treatment and in the NaN3-treated group were cultured at 28°C. Uptake of the FITC-CGA-N9 conjugate was assessed by confocal microscopy. Compared with that at 30°C, cellular uptake of CGA-N9 by C. tropicalis cells was partially blocked by 4°C and NaN3. Scale bar = 10 µm.

Figure 9.
Effect of energy on CGA-N9 internalization.

A total of 1 × 106 logarithmic-phase C. tropicalis cells were incubated with the MIC50 of CGA-N9 (2.9 µg/ml) for 12 h. Cells in the control group that had not undergone CGA-N9 treatment and in the NaN3-treated group were cultured at 28°C. Uptake of the FITC-CGA-N9 conjugate was assessed by confocal microscopy. Compared with that at 30°C, cellular uptake of CGA-N9 by C. tropicalis cells was partially blocked by 4°C and NaN3. Scale bar = 10 µm.

Effects of endocytic inhibitors on CGA-N9 internalization

Endocytosis pathways are mediated by many factors, such as cholesterol, Na+/H+ ion pumps, clathrin and actin [38,4042]. Endocytosis inhibitors, such as CPZ, CQ, Mβ-CD, NaN3, CyD, EIPA and heparin, inhibit these factors to suppress endocytic pathways. CPZ inhibits clathrin-mediated endocytosis, and CQ prevents endocytosis by blocking internal acidification of the endosome. Mβ-CD suppresses pinocytosis through caveolin, NaN3 disrupts endocytosis by inhibiting mitochondrial ATP production and CyD and EIPA are inhibitors of macropinocytosis. CyD interacts with the cytoskeleton, inhibiting actin polymerization, and EIPA blocks Na+/H+ exchange. Heparin is a competitive inhibitor of sulfate proteoglycans on the cell surface that competes with endocytic agents for binding the sulfated-protein receptor, thereby preventing endocytic agents from aggregating on the membrane and reducing endocytosis [38,39,43].

We further explored the relationship between endocytic pathways in C. tropicalis cells and CGA-N9. Logarithmic-phase C. tropicalis cells were pretreated with CPZ (7.11 µg/ml), CQ (1.72 µg/ml), Mβ-CD (3.75 mg/ml), NaN3 (0.65 mg/ml), CyD (5.08 µg/ml), EIPA (10 µg/ml) or heparin (100 µg/ml), followed by incubation with 3.9 µg/ml (MIC100) FITC-CGA-N9 conjugate without the removal of the inhibitor. As observed via laser confocal microscopy (Figure 10A) and the recorded FITC fluorescence values (Figure 10B), CQ and Mβ-CD had no effect on cellular uptake of CGA-N9, whereas CPZ, NaN3, CyD, EIPA and heparin all inhibited uptake. These results suggest that the endocytic pathways involved in CGA-N9 internalization are ATP-dependent macropinocytosis, clathrin-mediated endocytosis and sulfate proteoglycan receptor binding.

Effects of endocytic inhibitors on CGA-N9 internalization.

Figure 10.
Effects of endocytic inhibitors on CGA-N9 internalization.

A total of 1 × 106 logarithmic-phase C. tropicalis cells were incubated with the MIC100 of CGA-N9 (3.9 µg/ml) at 28°C for 12 h. Uptake of the FITC-CGA-N9 conjugate by C. tropicalis cells in the presence of endocytic inhibitors was assessed (A), and the fluorescence intensity of FITC was recorded (B) using confocal microscopy, Scale bar = 10 µm. Uptake of the FITC-CGA-N9 conjugate by C. tropicalis cells in the absence of endocytic inhibitors was used as the control. Compared with the control, cellular uptake of CGA-N9 by C. tropicalis was partially blocked by endocytic inhibitors. Pre-incubation with CyD, an inhibitor of actin polymerization, dramatically blocked the internalization of 3.9 µg/ml CGA-N9. A similar inhibitory effect was observed in cells pretreated with ethylisopropylamiloride (EIPA), a Na+/H+ exchange inhibitor, and heparin, a competitive inhibitor of sulfate proteoglycan binding. Additionally, NaN3 reduced CGA-N9 endocytosis by inhibiting ATP production, and CPZ, a clathrin-dependent endocytosis inhibitor, showed a slight inhibitory effect on CGA-N9 cellular uptake. In contrast, chloroquine (CQ), an endosomal internal acidification inhibitor, and methyl-β-cyclodextrin (Mβ-CD), a caveolae-mediated endocytosis inhibitor, had no effect on cell penetration by CGA-N9. Data represent the mean ± standard deviation of three independent experiments. ***P < 0.001 (Student's t-test).

Figure 10.
Effects of endocytic inhibitors on CGA-N9 internalization.

A total of 1 × 106 logarithmic-phase C. tropicalis cells were incubated with the MIC100 of CGA-N9 (3.9 µg/ml) at 28°C for 12 h. Uptake of the FITC-CGA-N9 conjugate by C. tropicalis cells in the presence of endocytic inhibitors was assessed (A), and the fluorescence intensity of FITC was recorded (B) using confocal microscopy, Scale bar = 10 µm. Uptake of the FITC-CGA-N9 conjugate by C. tropicalis cells in the absence of endocytic inhibitors was used as the control. Compared with the control, cellular uptake of CGA-N9 by C. tropicalis was partially blocked by endocytic inhibitors. Pre-incubation with CyD, an inhibitor of actin polymerization, dramatically blocked the internalization of 3.9 µg/ml CGA-N9. A similar inhibitory effect was observed in cells pretreated with ethylisopropylamiloride (EIPA), a Na+/H+ exchange inhibitor, and heparin, a competitive inhibitor of sulfate proteoglycan binding. Additionally, NaN3 reduced CGA-N9 endocytosis by inhibiting ATP production, and CPZ, a clathrin-dependent endocytosis inhibitor, showed a slight inhibitory effect on CGA-N9 cellular uptake. In contrast, chloroquine (CQ), an endosomal internal acidification inhibitor, and methyl-β-cyclodextrin (Mβ-CD), a caveolae-mediated endocytosis inhibitor, had no effect on cell penetration by CGA-N9. Data represent the mean ± standard deviation of three independent experiments. ***P < 0.001 (Student's t-test).

Effect of endocytic inhibitors on CGA-N9 cytotoxicity

To further explore the effects of endocytosis inhibitors on CGA-N9-induced cytotoxicity and the primary endocytic pathways involved, logarithmic-phase C. tropicalis cells were treated with 1.95, 2.9 and 3.9 µg/ml CGA-N9 in the presence of CPZ (7.11 µg/ml), CQ (1.72 µg/ml), Mβ-CD (3.75 mg/ml), NaN3 (0.65 mg/ml), CyD (5.08 µg/ml), EIPA (10 µg/ml) or heparin (100 µg/ml), and cell viability was then determined using the MIC assay (Figure 11). Compared with the survival rate of control cells, survival rates were significantly enhanced by treatment with EIPA and heparin, followed by CyD, and then NaN3 and CPZ. In the CQ- and Mβ-CD-treatment groups, the survival rate of C. tropicalis cells was nearly the same as that of the control, suggesting that CQ and Mβ-CD had no effect on CGA-N9 internalization. Moreover, at low CGA-N9 concentrations, the primary endocytic pathway was mediated by heparin, followed by EIPA and then CyD. At killing concentrations of CGA-N9, CPZ and NaN3 also exhibited some effect. The results indicate that CGA-N9 entered the cells partly via the endocytosis pathway in an ATP-dependent manner; in contrast, endocytic inhibitors, with the exception of CQ and Mβ-CD, partially ameliorated the cytotoxicity of CGA-N9 in C. tropicalis.

Effects of endocytic inhibitors on the antifungal activity of CGA-N9.

Figure 11.
Effects of endocytic inhibitors on the antifungal activity of CGA-N9.

A total of 1 × 106 logarithmic-phase C. tropicalis cells were incubated with CGA-N9 at 28°C for 16 h at concentrations of 3.9 µg/ml (MIC100) and 2.9 µg/ml (MIC50) (A) and 1.95 µg/ml (MIC5) (B). The antifungal activity of CGA-N9 against C. tropicalis cells in the presence of endocytic inhibitors was assessed using the MIC assay. The antifungal activity of CGA-N9 against C. tropicalis cells in the absent of endocytic inhibitors was used as a control. Compared with the control, pre-incubation with heparin, a competitive binding inhibitor of sulfate proteoglycans, dramatically blocked CGA-N9 activity. Inhibitory effects were also detected in cells pretreated with ethylisopropylamiloride (EIPA), a Na+/H+ exchange inhibitor, and CyD, an inhibitor of actin polymerization. Additionally, CPZ, a clathrin-dependent endocytosis inhibitor, and NaN3, an ATP production inhibitor, partly reduced CGA-N9 endocytosis. In contrast, CQ, an endosomal internal acidification inhibitor, and methyl-β-cyclodextrin (Mβ-CD), a caveolae-mediated endocytosis inhibitor, had no effect on the penetration of CGA-N9. It is interesting that EIPA and heparin showed primary inhibitory activity at low CGA-N9 concentrations. Data represent the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test).

Figure 11.
Effects of endocytic inhibitors on the antifungal activity of CGA-N9.

A total of 1 × 106 logarithmic-phase C. tropicalis cells were incubated with CGA-N9 at 28°C for 16 h at concentrations of 3.9 µg/ml (MIC100) and 2.9 µg/ml (MIC50) (A) and 1.95 µg/ml (MIC5) (B). The antifungal activity of CGA-N9 against C. tropicalis cells in the presence of endocytic inhibitors was assessed using the MIC assay. The antifungal activity of CGA-N9 against C. tropicalis cells in the absent of endocytic inhibitors was used as a control. Compared with the control, pre-incubation with heparin, a competitive binding inhibitor of sulfate proteoglycans, dramatically blocked CGA-N9 activity. Inhibitory effects were also detected in cells pretreated with ethylisopropylamiloride (EIPA), a Na+/H+ exchange inhibitor, and CyD, an inhibitor of actin polymerization. Additionally, CPZ, a clathrin-dependent endocytosis inhibitor, and NaN3, an ATP production inhibitor, partly reduced CGA-N9 endocytosis. In contrast, CQ, an endosomal internal acidification inhibitor, and methyl-β-cyclodextrin (Mβ-CD), a caveolae-mediated endocytosis inhibitor, had no effect on the penetration of CGA-N9. It is interesting that EIPA and heparin showed primary inhibitory activity at low CGA-N9 concentrations. Data represent the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test).

Discussion

CGA-N9 displays antimicrobial activity, particularly C. tropicalis, with high safety in HRBCs and mouse brain microvascular endothelial cells. Here, the cytotoxicity of this peptide against C. tropicalis is investigated based on the routes by which CGA-N9 crosses cell membranes.

The membrane activity of AMPs can be roughly divided into two categories: membrane destruction and non-membrane destruction. Most AMPs kill bacteria by destroying the cell membrane, though a few kill microbes via non-membrane destruction [44,45]. In this study, CGA-N9, a hydrophobic peptide with no typical structural characteristics, killed C. tropicalis in a time-dependent manner without destroying the integrity of the cell membrane, including an absence of pore formation.

Numerous studies have investigated the membrane processes associated with cell-penetrating peptides (CPPs) [46,47], the entry pathways of which are classified into two groups: energy-independent direct penetration and energy-dependent endocytosis [4850].

The direct penetration process has unique features; for example, it is not only energy-independent but also occurs at low temperatures and in the presence of endocytic inhibitors [50]. Direct penetration across the cell membrane primarily occurs in cases involving high concentrations of highly basic and arginine-rich peptides [40,51,52]. In this study, cellular internalization of CGA-N9 by C. tropicalis cells was assessed using an FITC-CGA-N9 conjugate at 4°C and in the presence of NaN3 at a concentration of 2.9 µg of CGA-N9/ml (MIC50). Our results showed incomplete internalization of the peptide in C. tropicalis cells under these conditions.

Endocytosis is a natural and energy-dependent process that occurs in all cells and may occur via several different pathways: macropinocytosis, clathrin- or caveolin-mediated endocytosis or clathrin-/caveolin-independent endocytosis [47,51,5355]. The endocytic inhibitors CyD, EIPA, heparin and CPZ inhibit endocytosis pathways by different mechanisms. In our investigation, CyD, EIPA, heparin and CPZ also influenced CGA-N9 internalization, indicating that CGA-N9 internalization is associated with the macropinocytosis pathway, sulfate proteoglycans on the cell surface and the clathrin-dependent endocytosis pathway. At concentrations below the MIC50, the macropinocytosis pathway is the primary endocytic pathway for CGA-N9, a conclusion also supported by its antimicrobial activity. In addition, CPZ and NaN3reduced the antimicrobial activity of CGA-N9, though their effect was reduced compared with that of EIPA, heparin or CyD.

Overall, a single CPP can exploit different routes to enter cells and these routes may occasionally operate concomitantly, depending on the differing physicochemical properties, sizes and concentrations of diverse CPPs [5658]. Based on the results of our CGA-N9 internalization experiments, except for a few peptides that require ATP for internalization, CGA-N9 molecules pass through the cell membrane via direct penetration. That is, energy-independent direct penetration is the primary internalization pathway, accompanied by energy-dependent macropinocytosis, sulfate proteoglycan-mediated endocytosis and weak clathrin-mediated endocytosis.

Studies have revealed that arginine residues contribute more to cellular uptake than do lysines [44,59,60]. The 9-amino-acid sequence of CGA-N9 contains two arginine residues, and these residues facilitate peptide binding to the membrane through electrostatic interactions.

In conclusion, CGA-N9 appears to first bind to the cell membrane through electrostatic forces, after which it passes through the membrane via direct penetration, accompanied by macropinocytosis, sulfate proteoglycan-mediated endocytosis and weak clathrin-mediated endocytosis with ATP consumption.

Abbreviations

     
  • AMPs

    antimicrobial peptides

  •  
  • ATCC

    American Type Culture Collection

  •  
  • ATP

    adenosine triphosphate

  •  
  • bEnd.3

    mouse brain microvascular endothelial cell line

  •  
  • CCK8

    cell counting kit-8

  •  
  • Cfu

    colony forming unit

  •  
  • CGA

    chromogranin A

  •  
  • CGA-N9

    the sequence from the 47th to the 55th amino acid of the N-terminus of chromogranin A

  •  
  • CPP

    cell-penetrating peptide

  •  
  • CPZ

    chlorpromazine

  •  
  • CQ

    chloroquine

  •  
  • CyD

    cytochalasin D

  •  
  • EIPA

    5-(N-ethyl-N-isopropyl)-amiloride

  •  
  • FITC

    fluorescein isothiocyanate

  •  
  • HRBCs

    human red blood cells

  •  
  • LB

    Luria-Bertani

  •  
  • MFC

    minimum fungicidal concentration

  •  
  • MIC

    minimum inhibitory concentration

  •  
  • MTT

    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

  •  
  • Mβ-CD

    methyl-β-cyclodextrin

  •  
  • NaN3

    sodium azide

  •  
  • PBS

    phosphate-buffered saline

  •  
  • PI

    propidium iodide

  •  
  • SD

    Sabouraud dextrose

  •  
  • SPSS

    statistical product and service solutions

  •  
  • TEM

    transmission electron microscopy

Author Contribution

Ruifang Li drafted the manuscript and participated in the design and coordination of the experiments. Chen Chen carried out the experiments and drafted the manuscript. Sha Zhu provided guidance for the experiments. Xueqin Wang and Yanhui Yang participated in the final editing of the manuscript. Weini Shi, Sijia Chen, Congcong Wang, Lixing Yan and Jiaofan Shi participated in the experiments. All authors have read and approved the final manuscript.

Funding

The National Natural Science Foundation of China (31071922 and 31572264), the Innovative Research Team (in Science and Technology) at the University of Henan Province (19IRTSTHN008), the Fundamental Research Funds for Henan Provincial Colleges and Universities at the Henan University of Technology (2015RCJH03), the Henan Provincial Science and Technology Research Project (162102310404) and the National Engineering Laboratory for Wheat and Corn Further Processing at the Henan University of Technology (NL2016010) all provided financial support for the authors.

Competing Interests

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

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