Dermaseptin-PH: A Novel Peptide with Antimicrobial and Anticancer Activities from the Skin Secretion of the South American Orange-Legged Leaf Frog, Pithecopus (Phyllomedusa) hypochondrialis

Abstract

The dermaseptin peptides, mainly derived from the skin secretions of Hylidae frogs, belong to a superfamily of antimicrobial peptides and exhibit diverse antimicrobial and anticancer activities with low cytotoxicity. Here, we reported a novel dermaseptin peptide, from the South American orange-legged leaf frogs, Pithecopus (Phyllomedusa) hypochondrialis, processing the shortest peptide length, namely Dermaseptin-PH. The complementary DNA (cDNA) encoding biosynthetic precursor of Dermaseptin-PH was initially identified by the rapid amplification of cDNA ends PCR (RACE-PCR) technique from the skin secretion. The predicted primary structure was confirmed by a combination of reverse-phase high performance liquid chromatography (RP-HPLC) and MS/MS fragmentation from the skin secretion. Chemically-synthetic Dermaseptin-PH was investigated using a range of bioactivity assessment assays to evaluate the biological activities and cytotoxicity of Dermaseptin-PH. Dermaseptin-PH inhibited the growth of Gram-negative bacteria, Gram-positive bacteria, and pathogenic yeast Candidaalbicans. In addition, Dermaseptin-PH showed a broad-spectrum of anticancer activities against several cancer cell lines including MCF-7, H157, U251MG, MDA-MB-435S, and PC-3. The potent antimicrobial and anticancer activities of Dermaseptin-PH make it a promising candidate in the discovery of new drugs for clinical applications, and the relatively short sequence of Dermaseptin-PH can provide new insight for the research and structural modification of new peptide drugs.

Keywords: amphibian skin secretion; anticancer; antimicrobial; dermaseptin; molecular cloning.

Figure 1

Nucleotide and translated open-reading frame amino acid sequences of Dermaseptin-PH from the skin secretion of Pithecopus hypochondrialis. The putative signal peptide is double-underlined, the mature peptide is single-underlined, and the stop codon is marked by an asterisk.

Figure 2

Alignments of the amino acid sequences of the prepropeptides of Dermaseptin-1, Dermaseptin-H1, Dermaseptin-H2, and Dermaseptin-PH comprise five domains, 1: putative N-terminal signal peptide region; 2: acidic amino acid rich spacer peptide region; 3: endoproteolytic cleavage point; 4: mature peptide progenitor region; 5: extension of biosynthetic precursor. Amino acid indicated with bold type serves as an amide donor for C-terminal amidation. The identical amino acid residues are indicated by the asterisks.

Figure 3

Alignments of amino acid sequences of Dermaseptin-1 (Accession No. P84596), Dermaseptin-2 (Accession No. P84597), Dermaseptin-3 (Accession No. P84598), Dermaseptin-4 (Accession No. P84599), Dermaseptin-5 (Accession No. P84600), Dermaseptin-6 (Accession No. P84601), Dermaseptin-7 (Accession No. P84880), Dermaseptin-H1 (Accession No. Q0VZ36), Dermaseptin-H2 (Accession No. Q0VZ37) and Dermaseptin-PH derived from the frog Pithecopus hypochondrialis are displayed. The identical amino acids are shaded in black; the amino acid with slight changes are indicated in bold type; the gaps are used to maximise alignments.

Figure 4

Reverse-phase high performance liquid chromatography (RP-HPLC) chromatogram of lyophilised skin secretion of Pithecopus hypochondrialis. The arrow indicates the retention time/elution position of the fraction containing a peptide with a mass coincident with that of Dermaseptin-PH. The absorbance was set at λ = 214 nm. The X-axis indicates the retention time in minutes and the Y-axis indicates the relevant absorbance in arbitrary units.

Figure 5

MS/MS fragmentation datasets derived from ions coincident with the mass of Dermaseptin-PH. Predicted singly and doubly charged b-ions and y-ions arising from MS/MS fragmentation of Dermaseptin-PH. Ions detected by MS/MS fragmentation are indicated in red and blue.

Figure 6

Circular dichroism (CD) spectra of Dermaseptin-PH (50 μM) in 10 mM ammonium acetate/water solution (blue line) and in 50% 2,2,2-trifluoroethanol (TFE)/10 mM ammonium acetate/water solution (red line).

Figure 7

The antimicrobial activities of Dermaseptin-PH against the growth of (a) E. coli; (b) P. aeruginosa; (c) S. aureus; (d) MRSA; (e) E. faecalis; and (f) C. albicans in a range of concentrations from 1 μM to 512 μM. Data represent means ± stand error of the mean (SEM) of 5 replicates.

Figure 7

The antimicrobial activities of Dermaseptin-PH against the growth of (a) E. coli; (b) P. aeruginosa; (c) S. aureus; (d) MRSA; (e) E. faecalis; and (f) C. albicans in a range of concentrations from 1 μM to 512 μM. Data represent means ± stand error of the mean (SEM) of 5 replicates.

Figure 8

The haemolytic activity of Dermaseptin-PH was tested on horse red blood cells. The positive control group was treated with 1% Triton X-100 lysis buffer.

Figure 9

The biofilm formation inhibitory activity of Dermaseptin-PH against (a) E. coli and (b) S. aureus, and the biofilm eradication activity of Dermaseptin-PH against (c) E. coli and (d) S. aureus. Negative control was obtained following incubation with relevant culture medium. Data represent means ± SEM of 5 replicates.

Figure 10

Cell membrane permeability effects of Dermaseptin-PH on (a) E. coli; (b) P. aeruginosa; (c) S. aureus; (d) MRSA; (e) E. faecalis; and (f) C. albicans, by using SYTOX Green (Life technologies, Carlsbad, CA, USA) assay at peptide concentrations corresponding to MIC results. Positive control was obtained following incubation with 70% isopropyl alcohol. Negative control (NC) was obtained following incubation with 5% tryptic soy broth (TSB) in 0.85% NaCl solution. Data represent means ± SEM of 5 replicates. The levels of significance are: * p < 0.05, *** p < 0.001.

Figure 11

The effects of Dermaseptin-PH on proliferation of human cancer cell lines (a) MCF-7; (b) H157; (c) U251MG; (d) MDA-MB-435S; and (e) PC-3 and (f) human dermal microvascular endothelium cell HMEC-1; after treatment with Dermaseptin-PH for 24 h at a range of concentrations from 10⁻⁹ to 10⁻⁴ M. The levels of significance are: * p < 0.05, *** p < 0.001. The negative control group was treated with relevant serum-free culture medium.

Figure 12

Cell membrane permeability effects of Dermaseptin-PH on MCF-7 cell by using SYTOX Green (1 μM) (Life technologies) at peptide concentrations from 1 to 100 μM. Positive control was obtained following incubation with 0.5% Triton X-100. Negative control was obtained following incubation with relevant serum-free culture medium. Data represent means ± SEM of 5 replicates.