molecules Article
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 Linyuan Huang 1,† , Dong Chen 1,† , Lei Wang 1 , Chen Lin 1,2 , Chengbang Ma 1, *, Xinping Xi 1, *, Tianbao Chen 1 ID , Chris Shaw 1 and Mei Zhou 1 ID 1
2
* †
Natural Drug Discovery Group, School of Pharmacy, Queen’s University, Belfast BT9 7BL, Northern Ireland, UK;
[email protected] (L.H.);
[email protected] (D.C.);
[email protected] (L.W.);
[email protected] (C.L.);
[email protected] (T.C.);
[email protected] (C.S.);
[email protected] (M.Z.) College of Basic Medical Science, Zhejiang Chinese Medial University, Hangzhou 310053, China Correspondence:
[email protected] (C.M.);
[email protected] (X.X.); Tel.: +44-28-9097-1673 (C.M. & X.X.); Fax: +44-28-9094-7794 (C.M. & X.X.) These authors contributed equally to this work.
Received: 7 September 2017; Accepted: 22 October 2017; Published: 24 October 2017
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 Candida albicans. 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: dermaseptin; amphibian skin secretion; molecular cloning; antimicrobial; anticancer
1. Introduction Antimicrobial peptides (AMPs), as multifunctional host-defence peptides, have clear antibacterial functions against a series of bacteria, fungi, viruses, and parasites, and have gained widespread interest in recent years [1]. AMPs not only exist in the secretions of frogs and toads, but are also present in microorganisms, plants, animals, and even human bodies. In mammals, these short cationic peptides which are also called host-defence peptides (HDPs) act as natural antibiotics and participate in the immunoregulation or killing of pathogenic microorganisms after invasion [2,3]. Nevertheless, scientists found that HDPs are more inclined to induce the immune response mechanisms
Molecules 2017, 22, 1805; doi:10.3390/molecules22101805
www.mdpi.com/journal/molecules
Molecules 2017, 22, 1805
2 of 18
under physiological conditions to protect hosts from infection of pathogens, than to be directly involved in killing bacteria [4,5]. With the ever-increasing problems of antibiotic resistance and the emergence of superbacteria, the current clinical antibiotics cannot meet the demand of patients [6,7]. The various mechanisms of multidrug resistance limit the development of antibiotics. Some pathogens, including Enterococcus faecium, MRSA, and Pseudomonas aeruginosa, are drug resistant or multidrug resistant bacteria that threaten the life of immunocompromised sufferers. The ESKAPE pathogens which are comprised of six multi-drug resistant microbes are generally recommended for screening of antimicrobial drugs [8]. However, unlike the general mechanisms of most antibiotics targeting the particular enzymes in the process of pathogenic metabolism, AMPs display totally different antimicrobial mechanisms which make them one of the most promising anti-infective drug candidates that meet the customers’ need [9]. Besides, antimicrobial peptides with anticancer activities have been widely reported in some literatures, and apparently, as a result, are attracting scientists to investigate and discover new anticancer candidates. Although the development of anticancer peptides in clinical therapy is beset by difficulties, there are still a few AMPs undergoing clinical trials. LL-37 is a human source cathelicidin peptide which exists in human respiratory and reproductive system and skin [10]. A collaboration programme between the National Cancer Institute and the M.D. Anderson Cancer Centre, indicated that LL-37 was an efficient antitumour agent against melanoma, and was undergoing Phase II clinical trials at present [11]. Another candidate anticancer drug, LTX-315, an engineered lytic peptide, is currently in Phase I clinical trial for treatment against transdermally accessible tumours. It is worth noting that anticancer peptides can enhance therapeutic effects through combination with other therapeutic approaches, including surgery, chemotherapy, radiotherapy, and even immunotherapy. The combination of LTX-315 with immunotherapy for treatment of patients with transdermally accessible tumours is in the process of trials, which provide possibilities from more therapeutic aspects [12–14]. The overall objectives of this study were to isolate and identify a novel antimicrobial peptide, named Dermaseptin-PH, from the skin secretion of the South American orange-legged leaf frog, Pithecopus (Phyllomedusa) hypochondrialis [15], to measure the bioactivities of it and to assess its therapeutic potential. To this end, the complementary DNA (cDNA) encoding Dermaseptin-PH biosynthetic precursor was cloned by “shotgun” molecular cloning and subsequently, the predicted mature peptide was identified by reverse-phase high performance liquid chromatography (RP-HPLC) from the crude skin secretion and the primary structure was confirmed by MS/MS sequencing technique. After chemical synthesis of Dermaseptin-PH, antimicrobial activity was investigated using several Gram-negative and Gram-positive bacteria, and a pathogenic yeast included. In the meanwhile, anticancer activity was assessed on a wide range of cancer cell lines in vitro. The cytotoxicity of Dermaseptin-PH against eukaryotic cells was evaluated on horse red blood cells and human dermal microvascular endothelium cells. 2. Results 2.1. “Shotgun” Cloning of Dermaseptin-PH Precursor-Encoding cDNA from a Skin Secretion-Derived cDNA Library of Pithecopus hypochondrialis and Bioinformatic Analysis A full-length cDNA encoding one novel dermaseptin peptide named Dermaseptin-PH was identified from the cDNA library constructed by isolating mRNA from the skin secretion of Pithecopus hypochondrialis. The biosynthetic precursor of Dermaseptin-PH consisted of a hydrophobic signal peptide region followed by an acidic spacer and a single copy of the predicted mature peptide (Figure 1). The translated open-reading frame of this novel dermaseptin biosynthetic precursor consisted of 312 base pairs encode 70 amino acids. The prepropeptide comprises a 22-amino acid signal peptide region followed by three Glu residues, and a typical processing site Lys–Arg amino acid pairing. Following the remaining acidic spacer domain, another processing site Lys–Arg is showed before the mature Dermaseptin-PH, that contains 23 amino acid residues followed by an
Molecules 2017, 2017, 22, 22, 1805 1805 Molecules
of 18 18 33 of
Molecules 2017, 22, 1805
3 of 18
–Gly–Gly–Gln [16]. The nucleotide sequence of Dermaseptin-PH has been deposited in the GenBank amidation consensus motif –Gly–Gly–Gln [16]. The nucleotide sequence of Dermaseptin-PH has been –Gly–Gly–Gln [16]. The nucleotide sequence of Dermaseptin-PH has beencode deposited in the GenBank Database (https://www.ncbi.nlm.nih.gov/genbank/) under the accession MF805718. deposited in the GenBank Database (https://www.ncbi.nlm.nih.gov/genbank/) under the accession Database (https://www.ncbi.nlm.nih.gov/genbank/) under the accession code MF805718. code MF805718.
Figure 1. Nucleotide and translated open-reading frame amino acid sequences of Dermaseptin-PH Figure 1. Nucleotide and translated open-reading frame amino acid sequences of Dermaseptin-PH from the secretionand of Pithecopus The putative peptide isof double-underlined, Figure 1.skin Nucleotide translatedhypochondrialis. open-reading frame amino signal acid sequences 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. 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. the mature peptide is single-underlined, and the stop codon is marked by an asterisk.
Additionally, the alignments of the full length prepropeptides of Dermaseptin-PH with Additionally, the alignments of the full length prepropeptides of Dermaseptin-PH with Additionally, the alignments and of the full length prepropeptides of below Dermaseptin-PH Dermaseptin-1, Dermaseptin-H1, Dermaseptin-H2 were displayed (Figure 2). with The Dermaseptin-1, Dermaseptin-H1, and Dermaseptin-H2 were displayed below (Figure 2). The sequences Dermaseptin-1, Dermaseptin-H1, and signal Dermaseptin-H2 wereconsisting displayedof below (Figure 2). The sequences showed a highly conserved peptide domain 22 amino acid residues showed a highly conserved signal peptide domain consisting of 22 amino acid residues and the acidic sequences showed a highly conserved signal peptide domain consisting 22 amino acidor residues and the acidic spacer region comprising 20–21 residues. Besides, theyofhave a –GGQ –GEQ spacer region comprising 20–21 residues. Besides, they have a –GGQ or –GEQ extension at C-terminal. and the acidic spacer region comprising 20–21 residues. Besides, derived they have a the –GGQ or species –GEQ extension at C-terminal. The mature peptide amino acid sequences from same The mature peptide amino acid sequences derived from the same species were aligned in Figure 3. extension at C-terminal. mature peptide amino acid sequences from the species were aligned in Figure 3. The An identical Trp residue in the position threederived at N-terminal andsame a consensus An identical Trp residue in the position three at N-terminal and a consensus motif of –AA/GKAA– were in Figure amphipathic 3. An identical Trp residue inmiddle the position three N-terminal and a consensus motifaligned of –AA/GKAA– structure in the region wasatobserved. amphipathic structure in the middle region was observed. motif of –AA/GKAA– amphipathic structure in the middle region was observed.
Figure 2. Alignments of the amino acid sequences of the prepropeptides of Dermaseptin-1, Figure 2. Alignments of the aminoand acidDermaseptin-PH sequences of the prepropeptides of Dermaseptin-1, Dermaseptin-H1, Dermaseptin-H2, comprise five domains, 1: putative Dermaseptin-H1, Dermaseptin-H2, and comprise five domains, 1: putative Figure 2. Alignments of region; the amino acidDermaseptin-PH sequences of spacer the prepropeptides of3: Dermaseptin-1, N-terminal signal peptide 2: acidic amino acid rich peptide region; endoproteolytic N-terminal signal peptide region; 2: acidic amino acid rich spacer peptide region; 3: endoproteolytic Dermaseptin-H1, Dermaseptin-H2, and Dermaseptin-PH comprise five domains, 1: putative cleavage point; 4: mature peptide progenitor region; 5: extension of biosynthetic precursor. Amino acid cleavage point; 4: mature peptide progenitor region; 5: extension of biosynthetic precursor. Amino N-terminal signal peptide region; 2: acidic amino acid rich spacer peptide region; 3: endoproteolytic indicated with bold type serves as an amide donor for C-terminal amidation. The identical amino acid acid indicated with bold serves as an amide donor5:for C-terminal amidation. The identicalAmino amino cleavage point; 4: mature peptide progenitor region; extension of biosynthetic precursor. residues are indicated bytype the asterisks. residues are indicated byserves the asterisks. acid indicated with bold type as an amide donor for C-terminal amidation. The identical amino acid residues are indicated by the asterisks.
Molecules 2017, 22, 1805
Molecules 2017, 22, 1805 Molecules 2017, 22, 1805
4 of 18
4 of 18 4 of 18
Figure 3. Alignments of amino acid sequences of Dermaseptin-1 (Accession No. P84596), Figure 3. Alignments of amino acid sequences of Dermaseptin-1 (Accession No. P84596), Figure 3. Alignments amino acid Dermaseptin-3 sequences of Dermaseptin-1 (Accession P84596), Dermaseptin-2 (Accession ofNo. P84597), (Accession No. P84598),No. Dermaseptin-4 Dermaseptin-2 (Accession No. P84597), Dermaseptin-3 (AccessionNo. No.P84598), P84598), Dermaseptin-4 Dermaseptin-2 (Accession No. P84597), Dermaseptin-3 (Accession Dermaseptin-4 (Accession No. P84599), Dermaseptin-5 (Accession No. P84600), Dermaseptin-6 (Accession No. (Accession No. P84599), Dermaseptin-5 (Accession No. P84600), Dermaseptin-6 (Accession No.No. P84601), (Accession No. P84599), Dermaseptin-5 P84600), Dermaseptin-6 (Accession P84601), Dermaseptin-7 (Accession No. (Accession P84880), No. Dermaseptin-H1 (Accession No. Q0VZ36), Dermaseptin-7 (Accession No. P84880), No. Dermaseptin-H1 (Accession No. Q0VZ36), Dermaseptin-H2 P84601), Dermaseptin-7 (Accession P84880), Dermaseptin-H1 (Accession No. Q0VZ36), Dermaseptin-H2 (Accession No. Q0VZ37) and Dermaseptin-PH derived from the frog Pithecopus (Accession No. Q0VZ37) and Dermaseptin-PH from thederived frog Pithecopus hypochondrialis Dermaseptin-H2 (Accession No. Q0VZ37) and derived Dermaseptin-PH from the frog Pithecopus are hypochondrialis are displayed. The identical amino acids are shaded in black; the amino acid with slight hypochondrialis are displayed. identical amino acids are shaded in black;acid the amino with slight are displayed. The identical aminoThe acids are shaded in black; the amino with acid slight changes changes are indicated in bold type; the gaps are used to maximise alignments. changes are indicated ingaps bold type; the gaps are used to maximise alignments. indicated in bold type; the are used to maximise alignments.
2.2. Identification andand Structural Skin Secretion 2.2. Identification StructuralCharacterization Characterization of of Dermaseptin-PH Dermaseptin-PH ininSkin Secretion of of 2.2. Identification and Structural Characterization of Dermaseptin-PH in Skin Secretion of Pithecopus hypochondrialis Pithecopus hypochondrialis Pithecopus hypochondrialis TheThe fractions from thethe lyophilised secretionofofPithecopus Pithecopus hypochondrialis were eluted fractions from lyophilisedcrude crude skin skin secretion hypochondrialis were eluted The fractionsand from the lyophilised crude skinnatural secretion of Pithecopus hypochondrialis were eluted by RP-HPLC fractionscontaining containingdifferent different natural components collected every minute by RP-HPLC and fractions componentswere were collected every minute by RP-HPLC and fractions containing different natural components were collected every minute (Figure 4). Each fraction wasanalysed analysedby by matrix-assisted matrix-assisted laser ionization time-of-flight (Figure 4). Each fraction was laserdesorption desorption ionization time-of-flight (Figure 4).spectrometer Each fraction was analysed byand matrix-assisted laser desorption ionization time-of-flight mass (MALDI-TOF-MS), the fraction with a mass coincident with that of the mass spectrometer (MALDI-TOF-MS), and the fraction with a mass coincident with that of the masspredicted spectrometer (MALDI-TOF-MS), and thegenomic fractionstudy, with ais mass coincident with that of the predicted mature peptide identified by the indicated by an arrow. The amino acidacid predicted mature peptide identified by the genomic study, is indicated by an arrow. The amino sequence of identified mature peptide, Dermaseptin-PH, was further confirmed by MS/MS fragmentation mature peptide by the genomic study, is indicated an arrow. amino acid sequence sequence of mature peptide, Dermaseptin-PH, was further by confirmed byThe MS/MS fragmentation sequencing (Figure 5). The amidated C-terminal post-translational modification with the Gly residue of mature peptide, was further confirmed by MS/MS fragmentation sequencing sequencing (FigureDermaseptin-PH, 5). The amidated C-terminal post-translational modification with the Gly residue following the C-terminal amino acid residue of the mature peptide domain was also verified in the the (Figure 5). The amidated C-terminal post-translational modification with the Gly residue following following the C-terminal amino acid residue of the mature peptide domain was also verified in the sequencing result. C-terminal sequencingamino result.acid residue of the mature peptide domain was also verified in the sequencing result.
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 ofof Figure 4.4. Reverse-phase high performance liquid chromatography (RP-HPLC) chromatogram Figure Reverse-phase performance liquid chromatography (RP-HPLC) chromatogram absorbance set at λof = Pithecopus 214 nm. The X-axis indicatesThe the arrow retention time in the minutes and the Y-axis lyophilised skin secretion hypochondrialis. the retention retention time/elution lyophilised skinwas secretion of Pithecopus hypochondrialis. indicates time/elution indicates thefraction relevantcontaining absorbance in arbitrary with units. a mass coincident with that of Dermaseptin-PH. position of the a peptide
position of the fraction containing a peptide with a mass coincident with that of Dermaseptin-PH. The The absorbance = 214 nm. The X-axis indicates retention time minutesand andthe theY-axis Y-axis absorbance waswas set set at λat=λ214 nm. The X-axis indicates thethe retention time ininminutes indicates indicatesthe therelevant relevantabsorbance absorbance in in arbitrary arbitrary units.
Molecules 2017, 22, 1805
5 of 18
Molecules 2017, 22, 1805
5 of 18
Molecules 2017, 22, 1805
5 of 18
Figure 5.5.MS/MS MS/MS fragmentation datasets ions coincident with the mass of Figure fragmentation datasets derivedderived from ionsfrom coincident with the mass of DermaseptinDermaseptin-PH. Predicted singly and doubly charged b-ions andfrom y-ions arising from MS/MS PH. Predicted singly and doubly charged b-ions and y-ions arising MS/MS fragmentation of fragmentation of Ions Dermaseptin-PH. Ions detected by MS/MS fragmentation areblue. indicated in red Dermaseptin-PH. detected by MS/MS fragmentation are indicated in red and Figure 5. MS/MS fragmentation datasets derived from ions coincident with the mass of Dermaseptinand blue. PH. Predicted singly and doubly charged b-ions and y-ions arising from MS/MS fragmentation of
2.3. Circular Dichroism Spectroscopy Dermaseptin-PH. Ions detected by MS/MS fragmentation are indicated in red and blue. 2.3. Circular Dichroism Spectroscopy The secondary structure of Dermaseptin-PH was successfully examined by circular dichroism 2.3. Circular Dichroism Spectroscopy The secondary structure of and Dermaseptin-PH was successfully examined dichroism spectroscopy in both aqueous membrane-mimic environment (Figure by 6).circular Dermaseptin-PH secondary structure of membrane-mimic Dermaseptin-PH was successfully(Figure examined by circular dichroism spectroscopy in both and environment 6).while Dermaseptin-PH showed aThe random coilaqueous conformation in ammonium acetate/water solution, it exhibited ashowed typical spectroscopy in both aqueous and membrane-mimic environment (Figure 6). Dermaseptin-PH a randomstructure coil conformation in ammonium acetate/water solution, while it acetate/water exhibited a typical α-helical α-helical in 50% 2,2,2-trifluoroethanol (TFE)/10 mM ammonium solution. The showed a random coil conformation in ammonium acetate/water solution, while it exhibited a typical structure 50% 2,2,2-trifluoroethanol (TFE)/10 mM solution. The helicity helicity ofin Dermaseptin-PH was calculated as 35% byammonium DichroWebacetate/water webserver [17]. α-helical structure in 50% 2,2,2-trifluoroethanol (TFE)/10 mM ammonium acetate/water solution. The of Dermaseptin-PH was calculated as 35% by DichroWeb webserver [17]. helicity of Dermaseptin-PH was calculated as 35% by DichroWeb webserver [17]. ammonium acetate
40
20 CD(mdeg)
CD(mdeg)
30
10 0
ammonium acetate 50%TFE/ammonium acetate
40
50%TFE/ammonium acetate
30 20 10 0
-10 -10 -20 -20
200
200
220
220
240 240
260
260
Wavelength(nm) Wavelength(nm)
Figure dichroism (CD) spectra of Dermaseptin-PH (50 µM) in 10 mM ammonium Figure 6.Circular Circular dichroism (CD) spectra Dermaseptin-PH (50 inin 10 mM ammonium acetate/water Figure 6.6.Circular dichroism (CD) spectra ofofDermaseptin-PH (50μM) μM) 10 mM ammonium acetate/water acetate/water solution line) and in 50% 2,2,2-trifluoroethanol (TFE)/10 mM solution ammonium solution line) in 50% 2,2,2-trifluoroethanol (TFE)/10 acetate/water solution (blue(blue line) andand in(blue 50% 2,2,2-trifluoroethanol (TFE)/10mM mMammonium ammonium acetate/water solution acetate/water solution (red line). (red line). (red line).
2.4. Antimicrobial Activity and HaemolyticActivity Activity of 2.4. Antimicrobial Antimicrobial Activity and Haemolytic of Dermaseptin-PH Dermaseptin-PH 2.4. Activity and Haemolytic Activity of Dermaseptin-PH Dermaseptin-PH was demonstrated to have a broad-spectrum of antimicrobial activity against
Dermaseptin-PH was demonstrated demonstrated to to have have a broad-spectrum broad-spectrum of of antimicrobial antimicrobial activity activity against against Dermaseptin-PH the Gram-negative was bacterium, Escherichia coli; thea Gram-positive bacterium, Staphylococcus aureus; the Gram-negative Gram-negative bacterium, Escherichia coli; theminimum Gram-positive bacterium, Staphylococcus aureus; the bacterium, Escherichia coli; the Gram-positive bacterium, Staphylococcus aureus; and the pathogenic yeast, Candida albicans, with inhibitory concentration (MIC) results and the pathogenic yeast, Candida albicans, with minimum inhibitory concentration (MIC) results
Molecules 2017, 22, 1805
6 of 18
and the pathogenic yeast, Candida albicans, with minimum inhibitory concentration (MIC) results Molecules 2017, of 18 16 to ranging from 1622,to1805 32 µM and minimum bactericidal concentration (MBC) results ranging 6from 64 µM (Table 1). The antimicrobial activity of Dermaseptin-PH against the tested microorganisms ranging from 16 to 32 μM and minimum bactericidal concentration (MBC) results ranging from 16 to was 64 showed in Figure Dermaseptin-PH more effective potency to E.microorganisms coli and C. albicans μM (Table 1). The7.antimicrobial activityshowed of Dermaseptin-PH against the tested at the concentration of 16 µM, than to S. aureus with 32 µM. It is also important to note was showed in Figure 7. Dermaseptin-PH showed more effective potency to E. coli and C. albicans at that Dermaseptin-PH exhibited effective activity againsttoE.note colithat (16Dermaseptin-PH µM) than ampicillin the concentration of 16 μM,more than to S. aureusantimicrobial with 32 μM. It is also important exhibited more effective antimicrobial activity against E. coli lower (16 μM)efficiency than ampicillin (36.6 μM). (Amp) (36.6 µM). However, Dermaseptin-PH displayed than (Amp) norfloxacin (Nor) on However, Dermaseptin-PH displayed lower efficiency than norfloxacin (Nor) on both MIC and MBC both MIC and MBC results against the six tested microorganisms. In addition, it is worthy of note that results against showed the six tested microorganisms. In addition, it is worthy of note thateffective Dermaseptin-PH Dermaseptin-PH low cytotoxicity to mammalian red blood cells at its concentration showed low cytotoxicity to mammalian red blood cells at its effective concentration against against microorganisms in a range of concentrations from 16 to 64 µM (Figure 8). microorganisms in a range of concentrations from 16 to 64 μM (Figure 8).
Table 1. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) Table 1. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations of Dermaseptin-PH, ampicillin (Amp) and norfloxacin (Nor) against tested microbes. (MBCs) of Dermaseptin-PH, ampicillin (Amp) and norfloxacin (Nor) against tested microbes.
MIC (μM) MIC (µM) Dermaseptin-PH Amp Dermaseptin-PH Amp Escherichia coli 16 36.6 Escherichia coli 16 36.6 Pseudomonas aeruginosa 64 Pseudomonas aeruginosa 64 Staphylococcus aureus 32 0.3 Staphylococcus aureus 32 0.3 MRSA MRSA >512>512 - Enterococcus faecalis 32 Enterococcus faecalis 32 4.8 4.8 Candida albicans Candida albicans 16 16 - Strains Strains
Nor Nor 0.6 0.6 2.5 2.5 1.3 1.3 2.5 2.5 5.2 5.2 1.3 1.3
MBC (μM) MBC (µM) Dermaseptin-PH Dermaseptin-PH Amp Amp 16 36.6 16 36.6 >512 >512 64 0.3 64 0.3 >512 ->512 64 4.8 64 4.8 -32
(a)
(b)
(c)
(d) Figure 7. Cont.
Nor Nor 0.6 0.6 5.2 5.2 2.5 2.5 5.2 5.2 5.2 5.2 2.5 2.5
Molecules 2017, 22, 1805 Molecules 2017, 22, 1805
7 of 18 7 of 18
Molecules 2017, 22, 1805
7 of 18
(e)(e)
(f) (f)
Figure 7.7. The antimicrobial ofof Dermaseptin-PH against growth of E. (a) E.(a)coli; Figure antimicrobial activities of Dermaseptin-PH against the growth ofcoli; coli; Figure 7.The The antimicrobialactivities activities Dermaseptin-PH against thethe growth of (a) (b)E. (b) aeruginosa; (c) S.S.aureus; MRSA; (e)E. E. faecalis; and C.C.albicans in in a range from from P. (c) S. aureus; (d)(d) MRSA; (e) faecalis; and(f) (f) albicans a range of concentrations (b)aeruginosa; P.P.aeruginosa; (c) aureus; (d) MRSA; (e) E. faecalis; and (f) C. albicans inofa concentrations range of concentrations toto 512 μM. Data represent means stand error the (SEM) of (SEM) 5ofreplicates. 1from μM1to 512 μM. Data represent means ±±stand oferror themean mean (SEM) 5 replicates. 1μM µM 512 µM. Data represent means ± error standof of the mean 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 8. The haemolytic activity of Dermaseptin-PH was tested on horse red blood cells. The positive
Figure The haemolytic activity of Dermaseptin-PH tested on horse red blood cells. The positive control8.group was treated with 1% Triton X-100 lysiswas buffer. 2.5. Anti-biofilm Activity of Dermaseptin-PH control group was treated with 1% Triton X-100 lysis buffer.
Dermaseptin-PH exhibited moderate antibiofilm activities against both E. coli and S. aureus 2.5. Anti-Biofilm Activity of Dermaseptin-PH 2.5. Anti-biofilm of Dermaseptin-PH (Figure 9). ItActivity processed the same potency on the inhibition of biofilm of both strains, but showed 2Dermaseptin-PH exhibited against both E. fold more effective activity whenmoderate eradicatingantibiofilm 90% matureactivities biofilm of S. aureus (Table 2). coli and S. aureus Dermaseptin-PH moderate activities against E. coli S. aureus (Figure 9). It processedexhibited the same potency onantibiofilm the inhibition of biofilm of bothboth strains, but and showed 2-fold (Figure 9). It processed the same potency on the inhibition of biofilm of both strains, but showed 2Table 2.when The antibiofilm activity of mature Dermaseptin-PH E. coli and S. aureus. more effective activity eradicating 90% biofilmagainst of S. aureus (Table 2). fold more effective activity when 90%90mature S. aureusMBEC (Table 2). Strains MBIC50eradicating (μM) MBIC (μM) biofilm MBECof 50 (μM) 90 (μM) Table against E. coli 2. The antibiofilm 32 activity of Dermaseptin-PH 64 64 E. coli and S. aureus. 256 Table 2. The antibiofilm activity of Dermaseptin-PH against S. aureus 32 64 64 E. coli and S. aureus. 128 Strains MBIC 5050 (μM) MBIC 90 (μM) MBEC (μM) MBEC 90 (μM) Strains MBIC (µM) MBIC90 (µM)minimum MBEC (µM) MBEC MBIC, minimum biofilm inhibitory concentration; MBEC, biofilm eradication concentration; 50 50 90 (µM) 50, minimum biofilm inhibitory concentration of biofilm; MBIC E. coli 3232 64required to inhibit the 256 E. coli 64 6464formation of 50%256 MBIC minimum biofilm inhibitory concentration of biofilm; S. aureus 3232 64required to inhibit the 128 S.90,aureus 64 6464formation of 90%128 MBEC50, minimum biofilm eradication concentration required to eradicate 50% of the formed biofilm; MBIC, biofilminhibitory inhibitory concentration; MBEC, minimum biofilm eradication concentration; MBIC, minimum minimum biofilm concentration; MBEC, minimum biofilm eradication concentration; MBIC50 , 90, minimum biofilm eradication concentration required to eradicate 90% of the formed biofilm. MBECbiofilm minimum inhibitory concentration required to inhibit the formation of 50% of biofilm; MBIC , minimum
MBIC50, minimum biofilm inhibitory concentration required to inhibit the formation of 50%90of biofilm; biofilm inhibitory concentration required to inhibit the formation of 90% of biofilm; MBEC50 , minimum biofilm MBIC 90, minimum biofilm inhibitory concentration required to inhibit the formation of 90% of biofilm; eradication concentration required to eradicate 50% of the formed biofilm; MBEC90 , minimum biofilm eradication concentration required to eradicate 90% of the formed biofilm. 50, minimum biofilm eradication concentration required to eradicate 50% of the formed biofilm; MBEC MBEC90, minimum biofilm eradication concentration required to eradicate 90% of the formed biofilm.
Molecules 2017, 22, 1805
8 of 18
Molecules 2017, 22, 1805
8 of 18
(a)
(b)
(c)
(d)
Figure9.9. The formation inhibitory activityactivity of Dermaseptin-PH against (a) E.against coli and (a) (b) S. Figure Thebiofilm biofilm formation inhibitory of Dermaseptin-PH E.aureus, coli and the biofilm eradication activity of Dermaseptin-PH against (c) E.against coli and(c) (d)E.S.coli aureus. Negative (b)and S. aureus, and the biofilm eradication activity of Dermaseptin-PH and (d) S. aureus. control was obtained relevant culture medium.culture Data represent means ± SEM of Negative control wasfollowing obtainedincubation followingwith incubation with relevant medium. Data represent 5 replicates. means ± SEM of 5 replicates.
2.6. Bacteria Cell Membrane Permeabilisation Activities of Dermaseptin-PH 2.6. Bacteria Cell Membrane Permeabilisation Activities of Dermaseptin-PH The cell membrane permeability of Dermaseptin-PH was tested in a range of concentrations The cell membrane permeability of Dermaseptin-PH was tested in a range of concentrations from from 8 μM to 128 μM (Figure 10). Dermaseptin-PH showed more effective permeable activity on E. coli, 8 µM to 128 µM (Figure 10). Dermaseptin-PH showed more effective permeable activity on E. coli, S. aureus, MRSA, and C. albicans at concentration of 8 μM. The efficient permeabilisation concentrations S. aureus, MRSA, and C. albicans at concentration of 8 µM. The efficient permeabilisation concentrations of Dermaseptin-PH on P. aeruginosa and E. faecalis were two times and four times higher, at 16 and of32 Dermaseptin-PH onAtP. the aeruginosa and E.the faecalis were two times and four higher, rate at 16ofand μM, respectively. MICs against respective microorganisms, thetimes permeability 32Dermaseptin-PH µM, respectively. At the MICs against the respective microorganisms, the permeability rate of on E. coli, P. aeruginosa, S. aureus, E. faecalis, and C. albicans was 46.4%, 131.5%, Dermaseptin-PH on E. coli, P. aeruginosa, S. aureus, E. faecalis, and C. albicans was 46.4%, 131.5%, 103.6%, 103.6%, 44.3%, and 22.9%. And, the permeability rate of Dermaseptin-PH on E. coli, S. aureus, 44.3%, and and 22.9%. And, the rate of Dermaseptin-PH on E. coli, MBCs. S. aureus, faecalis, E. faecalis, C. albicans waspermeability 46.4%, 110.8%, 47.8%, and 37.4% at the respective EachE.assay and C. albicans was 46.4%, 110.8%, 47.8%, and 37.4% at the respective MBCs. Each assay was carried was carried out over at least three individual experiments, with 5 replicates in each. out over at least three individual experiments, with 5 replicates in each. 2.7. Cell Proliferation Assay of Dermaseptin-PH 2.7. Cell Proliferation Assay of Dermaseptin-PH Synthetic peptide Dermaseptin-PH exhibited a broad-spectrum antiproliferative activity against Synthetic Dermaseptin-PH a broad-spectrum activityfrom against MCF-7, H157,peptide U251MG, MDA-MB-435S, exhibited and PC-3 human cell lines in aantiproliferative range of concentrations −9 −4 MCF-7, and PC-3 human cell lines in a range 10 to H157, 10 M U251MG, (Figure 11).MDA-MB-435S, The most obvious proliferation inhibitions observed wereofonconcentrations the human 9 to lines cancer MCF-7 with ICThe 50 value 0.69 μM. Besides, Dermaseptin-PH showed similar from 10−cell 10−4 M (Figure 11). mostofobvious proliferation inhibitions observed were on the inhibitory activities against H157, and cellDermaseptin-PH lines, with IC50 values of 2.01, human cancer cell lines MCF-7 withU251MG, IC50 value of MDA-MB-435S 0.69 µM. Besides, showed similar 2.36, and 9.94 μM, respectively, andU251MG, weak anticancer activity againstcell PC-3lines, cell lines, IC50 value inhibitory activities against H157, and MDA-MB-435S withwith IC50an values of 2.01, of 11.8 addition, it is worth that Dermaseptin-PH showed slight on IC human 2.36, andμM. 9.94In µM, respectively, andnoting weak anticancer activity against PC-3 cellcytotoxicity lines, with an 50 value microvascular cells, HMEC-1, with an IC50 value of 4.85 μM. ofdermal 11.8 µM. In addition, endothelium it is worth noting that Dermaseptin-PH showed slight cytotoxicity on human dermal microvascular endothelium cells, HMEC-1, with an IC50 value of 4.85 µM.
Molecules 2017, 22, 1805 Molecules 2017, 22, 1805
9 of 18 9 of 18
(a)
(b)
(c)
(d)
(e)
(f)
Figure 10. 10. Cell Cellmembrane membranepermeability permeability effects Dermaseptin-PH E. coli; (b)aeruginosa; P. aeruginosa; Figure effects of of Dermaseptin-PH on on (a) (a) E. coli; (b) P. (c) (c)aureus; S. aureus; MRSA; faecalis; and albicans,bybyusing usingSYTOX SYTOXGreen Green(Life (Life technologies, technologies, S. (d) (d) MRSA; (e) (e) E. E. faecalis; and (f)(f) C.C.albicans, Carlsbad, CA, CA, USA) corresponding to MIC results. Positive control was Carlsbad, USA) assay assayatatpeptide peptideconcentrations concentrations corresponding to MIC results. Positive control obtained following incubation with 70% isopropyl alcohol. Negative control (NC) was obtained was obtained following incubation with 70% isopropyl alcohol. Negative control (NC) was obtained following incubation incubation with with 5% 5% tryptic tryptic soy soy broth Data represent represent following broth (TSB) (TSB) in in 0.85% 0.85% NaCl NaCl solution. solution. Data means ±±SEM SEMofof55replicates. replicates.The Thelevels levelsofofsignificance significanceare: are:* *pp
