Peptides and Strains
The recombinant AMP-17 protein was obtained using a prokaryotic expression system and purified by nickel ion metal chelator affinity chromatography. The minimal inhibitory concentration (MIC) of recombinant AMP-17 toward C. albicans was 20 μg/mL as detected using the micro-liquid dilution method (Ma et al. 2020).C. albicans (ATCC10231) was stored in 30% glycerol at −80 °C and subcultured twice on Sabouraud dextrose agar (SDA) plate. Before each experiment, cells were cultured in Sabouraud dextrose broth (SDB) for 18 h on a shaking incubator (200 rpm) at 37 °C.
Analysis of cell morphology
The effect of AMP-17 on the ultrastructure of C. albicans cells was analyzed using TEM. C. albicans cells at an initial density of 1.0~5.0 × 106 colony-forming units (CFU)/mL were treated with 40 μg/mL AMP-17 at 37 °C for 16 h, and then collected by centrifugation (5,000 rpm for 10 min). Untreated cells served as the control. Samples from each group were washed twice with phosphate buffer saline (PBS) and fixed in 1 mL of 2.5% glutaraldehyde at 4 ℃ overnight. The fixed samples were washed twice with PBS and dehydrated in 50%, 75%, and 100% ethanol. Finally, the cells were observed under a Hitachi H-7650 TEM (Tokyo, Japan).
Cell cycle analysis by flow cytometry using PI
C. albicans cells (1.0~5.0 × 106 CFU/mL) were treated with AMP-17 at a final concentration of 0 (control), 20, 40, or 80 μg/mL. The cells were incubated at 37 °C for 12 h, collected by centrifugation at 3,000 rpm for 5 min, and washed twice with PBS. Each group of cells was resuspended in precooled 70% ethanol and fixed at 4 °C for more than 18 h. The fixed cells were collected by centrifugation at 3,000 rpm for 5 min at 4 °C and washed twice with sterile PBS. After incubation with ribonuclease A (RNAse A) at 37 °C for 2 h, Propidium iodide (PI) staining solution was added to the cells at a final concentration of 0.1 mg/mL, followed by incubation at 4 °C for 18 h in the dark. The cell cycle was then analyzed by flow cytometry.
Measurement of ROS generation in C. albicans
The dye 2',7'-dichlorofluorescein diacetate (DCFH-DA) from Sigma Chemicals (St Louis, MO, USA) was used to detect the generation of intracellular ROS (Chang et al. 2011; Li et al. 2015). C. albicans cells at an initial density of 1.0~5.0 × 106 CFU/mL were treated with 0 (control), 20, 40, or 80 μg/mL AMP-17 at 37 °C for 12 h. Cells treated with fluconazole (FLC) under the same conditions served as the positive control. Following treatment, the cells were collected, fixed, and stained with DCFH-DA at a final concentration of 20 μg/mL. After 30 min of incubation at 30 °C, the cells were collected, washed twice, and then diluted to 1 × 106 CFU/mL with SDB. The fluorescence intensity (excitation and emission at 485 and 530 nm, respectively) of the cells was measured with a microplate reader (Berthold Biotechnologies, Bad Wildbad, Germany) and fluorescence images were obtained by confocal laser scanning microscopy (CLSM) using an FITC filter (Olympus FV1000, Olympus, Tokyo, Japan). To test the effect of antioxidants on AMP-17-induced ROS accumulation, these experiments were also conducted in the presence of 5 and 50 mM ascorbic acid (AA) and glutathione (GSH). To determine whether the ROS production induced by AMP-17 is involved in the antifungal activity of the peptide, we examined the anti-C. albicans activity of AMP-17 without and with the ROS scavengers, AA and GSH. The MIC was determined using the Clinical and Laboratory Standards Institute (CLSI) method (Ma et al. 2020). All samples were analyzed in triplicate, and the experiment was repeated three times.
Detection of the correlation between AMP-17-induced ROSaccumulation and necrosis using DCFH-DAand PI double staining
C. albicans cells (1.0~5.0 × 106 CFU/mL) were cultured in SDB containing 80 μg/mL AMP-17 at 37 °C for 12 h. Sterile water was used as a negative control. The cells were centrifuged for 10 min at 5,000 rpm, incubated with 10 μg/mL DCFH-DA (a probe for detecting intracellular ROS) and 20 μg/mL PI (an indicator dye for cell necrosis) for 30 min in dark, and then analyzed by CLSM.
Measurement of the MMP
A JC-1 kit (Beyotime, Shanghai, China) was used to analyze changes in MMP. C. albicans cells (1.0~5.0 × 106 CFU/mL) were treated with 0 (control), 20, 40, or 80 μg/mL AMP-17 at 37 °C for 12 h. The positive control sample was incubated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) under the same conditions. The cell suspensions were then stained with 5 μmol/L JC-1 at 37 °C for 30 min in the dark. JC-1 fluorescence (green and red) was monitored at Ex/Em = 490/525 nm and 490/590 nm with a microplate reader (Berthold Biotechnologies, Bad Wildbad, Germany). The results were presented as the mean values of triplicate measurements from three independent experiments.
Analysis of apoptosis and necrosis induced by AMP-17
C. albicans cells at a concentration of 1.0~5.0 × 106 CFU/mL were treated with 0 (control), 20, 40, or 80 μg/mL AMP-17 and incubated at 37 °C for 12 h. The cells were then collected by centrifugation (5,000 rpm for 10 min) and washed twice in PBS. Each sample was processed according to the instructions of the Annexin V/FITC Kit (BD pharmingen, NJ, USA). The stained cells were observed by CLSM.
All data were analyzed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Differences were analyzed by one-way ANOVA using Dunnett’s analysis. Data are expressed as mean±standard deviation (Mean±SD), and p value of <0.05 was considered statistically significant.