In Vitro and In Vivo Studies on the Antibacterial Activity and Safety of a New Antimicrobial Peptide Dermaseptin-AC

Abstract

Antimicrobial resistance has been an increasing public health threat in recent years. Antimicrobial peptides are considered as potential drugs against drug-resistant bacteria because they are mainly broad-spectrum and are unlikely to cause resistance. In this study, a novel peptide was obtained from the skin secretion of Agalychnis callidryas using the "shotgun" cloning method. The amino acid sequence, molecular weight, and secondary structure of Dermaseptin-AC were determined. The in vitro antimicrobial activity, hemolysis, and cytotoxicity of Dermaseptin-AC were evaluated. MICs and minimum bactericidal concentrations (MBCs) of Dermaseptin-AC against seven different bacterial strains ranged between 2 ∼ 4 μM and 2 ∼ 8 μM. The HC₅₀ (50% maximum hemolysis concentration) of Dermaseptin-AC against horse erythrocytes was 76.55 μM. The in vivo anti-MRSA effect was tested on immune-suppressed MRSA pneumonia in mice. Dermaseptin-AC showed anti-MRSA effects similar to the same dose of vancomycin (10 mg/kg body weight). Short-term (7 days of intraperitoneal injection, 10 mg/kg body weight) in vivo safety evaluation of Dermaseptin-AC was tested on mice. The survival rate during the 7-day injection was 80%. Dermaseptin-AC showed no obvious effect on the liver, heart, spleen, kidney, and blood, but did induce slight pulmonary congestion. The skin safety of Dermaseptin-AC was evaluated on wounds on the back skin of a rat, and no irritation was observed. IMPORTANCE In this study, we discovered a new antimicrobial peptide, Dermaseptin-AC, and studied its in vitro and in vivo antimicrobial activity. These studies provide some data for finding new antimicrobial peptides for overcoming antimicrobial resistance. Dermaseptin-AC showed strong broad-spectrum antibacterial activity and relatively low hemolysis, and was more cytotoxic to cancer cells than to normal cells. Dermaseptin-AC was active in vivo, and its anti-MRSA effect was similar to that of vancomycin when administered by intraperitoneal injection. Safety studies found that continuous injection of Dermaseptin-AC may cause mild pulmonary congestion, while there was no obvious irritation when it was applied to skin wounds. Chronic wounds are often accompanied by high bacterial burdens and, at the same time, antimicrobial resistance is more likely to occur during repeated infections and treatments. Therefore, developing Dermaseptin-AC to treat chronic wound infection may be an attractive choice.

Keywords: Dermaseptin; antimicrobial peptides; bacterial resistance.

FIG 1

Discovery of Dermaseptin-AC. (a) Nucleotide sequence of the cDNA-encoding precursor of the novel peptide Dermaseptin-AC, and the amino acid sequence translated by the open reading frame. Signal peptide sequence is double underlined. Mature peptide sequence is underlined. (b) Amino acid sequence alignment of Dermaseptin-AC precursor and the top 5 similar peptide precursors. (c) Amino acid sequence alignment of the mature peptide of Dermaseptin-AC and the top 5 similar peptides. Symbols: * (asterisk), conserved residues; : (colon), very similar residues; . (period), similar residues.

FIG 2

Synthesis and purification of Dermaseptin-AC. (a) HPLC chromatogram of synthetic peptide. The y axis indicates absorbance at λ = 214 nm. Linear gradient elution from 100% solution A (80% ACN, 19.95% water, 0.05% TFA) to 100% solution B (0.05% TFA, 99.95% water), 80 min, flow rate 1 mL/min. (b) MALDI-TOF spectrum of purified peptide. (c) HPLC chromatogram of purified peptide. The y axis indicates absorbance at λ = 214 nm. Linear gradient elution from 70% solution A (80% ACN, 19.95% water, 0.05% TFA) to 100% solution B (0.05% TFA, 99.95% water), 80 min, flow rate 1 mL/min.

FIG 3

Structure of Dermaseptin-AC. (a) Amino acid composition of Dermaseptin-AC presented by HeliQuest. (b) Circular dichroism spectra recorded for the purified synthetic Dermaseptin‐AC in 50 μM NH₄Ac, 100 μM NH₄Ac, 50 μM TFE, and 100 μM TFE water solution. (c) 3D structure models of Dermaseptin-AC as predicted by I-TASSER.

FIG 4

In vitro antimicrobial activity of Dermaseptin‐AC. (a) The in vitro antimicrobial activity of Dermaseptin‐AC. All strains were incubated with the peptide in Mueller-Hinton broth (MHB) at 37°C for 18 ∼ 20 h. (b) Effect of serum on the antimicrobial activity of Dermaseptin‐AC. Bacteria were incubated with the peptide in MHB or MHB with 5% fetal bovine serum at 37°C for 18 ∼ 20 h. (c) Membrane permeability of bacteria under Dermaseptin‐AC treatment. Bacteria were incubated with peptide or 70% isopropanol at 37°C for 2 h and then stained by SYTOX Green. Bacteria treated with 70% isopropanol were set as 100% permeabilized. Permeability was calculated from the measured fluorescence intensity. (d) Inhibition and eradication activities of Dermaseptin‐AC against MRSA biofilm. Forming biofilm was incubated with peptide in tryptic soy broth (TSB) at 37°C for 24 h. Mature biofilm was cultured in TSB at 37°C for 48 h and then incubated with peptide at 37°C for 24 h. Floating bacteria were rinsed off and the biofilm was stained with 0.1% crystal violet solution. B represents blank control (sterile medium), V represents vehicle control (1% DMSO), and G represents growth control. All data were analyzed by GraphPad Prism (Version 8.0.2), shown as mean ± SEM of 3 individual experiments with 3 replicates in each.

FIG 5

Hemolysis and cytotoxicity of Dermaseptin-AC. (a) Hemolysis of Dermaseptin-AC against horse erythrocytes. Hemolysis was calculated based on the measured OD value. (b) Cell viability under Dermaseptin-AC treatment (MTT assay). Cells were cultured with peptide at 37°C for 24 h and then incubated with MTT at 37°C for 4 h. Cell viability was calculated from measured OD values and untreated cells were set as 100% viable. (c) Cytotoxicity of Dermaseptin-AC against HMEC-1 and U251MG cells (LDH assay). Cells were incubated with peptide at 37°C for 24 h, and LDH release was calculated from the measured OD value. (d) Cell proliferation under Dermaseptin‐AC treatment. Cells were incubated with peptide at 37°C for 24 h and then enumerated. P represents positive control (1% Triton X-100) and N represents negative control (PBS). All data were analyzed by GraphPad Prism (Version 8.0.2), shown as mean ± SEM of 3 individual experiments with 3 replicates in each.

FIG 6

In vivo anti-MRSA activity of Dermaseptin-AC. Mice were nasally infected with MRSA (1 × 10⁹ CFU/mL, 20 μl) and treated by intraperitoneal injection after 24 h. Mice were euthanized after 24 h treatment. Viable MRSA in the BALF (a) and lung homogenate (b) of mice were counted. The left upper lobes of mice were weighed immediately and then dried at 60°C for 72 h. (c) Lung wet/dry ratio (ratio of fresh lung weight to dried lung weight) was calculated to estimate the degree of pulmonary edema. (d) Spleen index (fresh spleen weight/body weight) was calculated to estimate the immune status. All data were analyzed by GraphPad Prism (Version 8.0.2), shown as mean ± SEM with 8 replicates. Significant differences are indicated with asterisks (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001). “ns” indicates no significance (P > 0.05).

FIG 7

Intraperitoneal injection safety evaluation of Dermaseptin-AC. Mice were kept in SPF conditions for 3 days and then intraperitoneally injected with 10 mg/kg peptides or normal saline with equal volume for 7 days. Start and end days of the injection are indicated by arrows (↑). Mice were euthanized 3 days after final administration. Body weight (a) and % survival (b) were recorded every day. (c) The main organs were weighed immediately and organ index (ratio of fresh organ weight to body weight) was calculated (normal saline n = 5, peptide n = 4). (d) Blood cells, including white blood cells (WBC), red blood cells (RBC), and platelets (PLT) in mouse whole blood were counted (n = 4). (e) Photos (200×) of main organs tissues stained with hematoxylin and eosin were taken. Lung congestion is indicated by a yellow arrow. N represents normal saline and P represents peptide (10 mg/kg body weight). All data were analyzed by GraphPad Prism (Version 8.0.2), shown as mean ± SEM. Significant differences are indicated with asterisks (**, P < 0.01). “ns” indicates no significance (P > 0.05).

FIG 8

Skin wound safety evaluation of Dermaseptin-AC. The rat was kept in an SPF environment for 3 days and then de-haired with pet depilatory cream. Twenty-four hours after hair removal, the back skin of the rat was carefully scratched and six “#”-shaped wounds were made. After 24 h, a volume of 0.5 mL peptide solution of different concentrations and PBS was applied evenly. The treatment was then carried out every 24 h for a total of four times. The rat was euthanized on the fifth day after wound establishment and skin tissues were collected. Photos of newly established wounds (a), treated wounds (b), and tissues stained with hematoxylin and eosin (200×) (c).