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
]. Nevertheless, scientists found that HDPs are more inclined to induce the immune response mechanisms under physiological conditions to protect hosts from infection of pathogens, than to be directly involved in killing bacteria [4
With the ever-increasing problems of antibiotic resistance and the emergence of superbacteria, the current clinical antibiotics cannot meet the demand of patients [6
]. 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
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
], 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.
Dermaseptins, which belong to an important antimicrobial peptide family, have a wide spectrum of antimicrobial activity against various microorganisms, including bacteria, fungi, enveloped viruses, protozoa, and mollicutes [18
]. The first dermaseptin peptide named Dermaseptin S1 was discovered by Mor from frog, Phyllomedusa sauvagii
]. This novel 34 amino acid residue peptide displayed lethal activity against pathogenic microorganisms, including filamentous fungi, which is the main reason for opportunistic infections of immunocompromised patients. In this study, we isolated a novel dermaseptin peptide, namely Dermaseptin-PH, from the skin secretion of the South American orange-legged leaf frog, Pithecopus hypochondrialis
. Compared to the alignments of the amino acid sequences of the preprodermaseptins, Dermaseptin-PH shows similar structural characteristics to other members with a conserved signal peptide region, which constitutes 22 amino acids as canonical architecture. However, the variations of signal peptide region of dermaseptin peptide from same species were observed [20
], which might be explained by the mutation occurring due to the different distribution of specimen. Additionally, the canonical structure in the mature peptide comprises an identical Trp residue in position three and a conserved –AA(A/G)KAAL(G/N)A– amphipathic domain. Besides, the C-terminal amide could influence the activities of AMPs because of 1+ net positive charge of amide group, and resistance to natural degradation [23
]. Moreover, a Val–Gly amino acid pairing exists in the middle region of Dermaseptin B2, which can divide the amphipathic helix into N-terminal domain and C-terminal domain, and this may promote the penetration into the membrane of bacteria [24
]. However, the Val–Gly amino acid pairing is absent in Dermaseptin-PH, which may decrease the antimicrobial activity.
AMPs derived from amphibian skins, as the initial defence protecting themselves from invasion of pathogens, have a potent and broad-spectrum antimicrobial activity. These low molecular weight peptides are regarded as a potential source for developing new antibiotics to solve the drug resistance caused by conventional antibiotics [25
]. Dermaseptin-PH demonstrated a broad-spectrum antimicrobial activity against Gram-negative, Gram-positive bacteria, and yeast. The antimicrobial mechanism of dermaseptins has been reported to bind to lipid bilayers by coil-to-helix transition, and induce the permeation/disruption of the lipid plasma membrane through the carpet model [18
]. It could explain the weaker antimicrobial potency of Dermaseptin-PH than Dermaseptin S9 as Dermaseptin S9 has a larger proportion of the α
-helical domain than Dermaseptin-PH [26
]. Dermaseptin-PH displayed more effective permeabilisation activity on E. coli
than on P. aeruginosa
may also owe to the more resistant cellular envelop structure of P. aeruginosa
. Unexpectedly, Dermaseptin-PH exhibited permeation on MRSA though it was not able to inhibit growth of MRSA in a suitable growing environment. It is speculated that Dermaseptin-PH form the transmembrane pores rapidly, but this crisis in the membrane might be relieved by some kind of “self-fixing” strategy or enzymatic degradation of peptides. This special phenomenon might be due to that incident mutations of MRSA promote the adaption of harsh environment [27
]. On the other hand, the acting pattern of Dermaseptin-PH on MRSA indicated that it may permeabilise cell membranes through other pore formation models instead of the carpet model. Although Dermaseptin-PH can interact with cell membranes and cause permeabilisation, its antimicrobial activity was significantly decreased when encountering more complex bacteria strains and propagation circumstances, such as biofilm-sessile bacteria. It is widely accepted that high cationic charge may play a more important role in antimicrobial activity of the most antimicrobial peptides. The published literature revealed that the most common approach to enhance antimicrobial activity is to introduce basic amino acids in the amino acids sequence, like lysine. Whereas antimicrobial results of Dermaseptin-PH may illustrate it exerts antimicrobial functions more dependent on amphipathicity, than positively charged features, upon interaction with the cell membrane.
Many AMPs display anticancer activity so that they attract much attention of researchers on anticancer drug discovery [28
]. The mechanisms of antimicrobial peptides exhibiting anticancer activity such as disrupting cell membranes, inhibiting the growth of cancer cells, or killing cancer cells, have not yet been studied thoroughly. Some literature declared that some AMPs can kill tumour cells specifically. One reason is that the negatively-charged substances, like heparin sulphates and O
-glycosylated mucins on the surface of cancer cells, could favour the interaction between AMPs and tumour cells [29
]. Meanwhile, some scientists deduced that electrostatic force is not the decisive factor of the interaction between AMPs and cancer cells [34
]. Although studies showed that some AMPs can induce cell apoptosis on eukaryote by reactive oxygen species (ROS) production, caspase activation, cytochrome c
release, DNA fragmentation, and mitochondrial membrane depolarisation [36
], Dermaseptin B2 was reported to induce the cell necrosis by the membrane disruption of cancer cells [38
]. Here, considering Dermaseptin-PH contains less cationic charges and low degree of α
-helicity, a further study of the permeabilisation of Dermaseptin-PH on cancer cells was carried out through a time course assay. By observation of rapid membrane permeabilisation activity on cancer cells, it was speculated that Dermaseptin-PH possesses similar permeation/disruption mechanisms for both cancer and bacteria cells. Interestingly, the interaction between Dermaseptin-PH and cancer cell membrane entered a slow growth period, after the initial rapid rise duration. This phenomenon may be explained by the long-lasting light irradiation from the light source inside the plate reader [39
], and the lack of appropriate growth conditions. It might cause cell cytological instability, which makes cancer cells more sensitive to Dermaseptin-PH.
In summary, we reported the identification and characterisation of a novel dermaseptin peptide, named Dermaseptin-PH, from the skin secretion of the South America orange-legged leaf frog, Pithecopus hypochondrialis. Dermaseptin-PH showed a broad-spectrum of antimicrobial and anticancer activities. The cell membrane permeability against bacteria and cancer cells indicated that Dermaseptin-PH stands a good chance of a permeation/disruption dependent peptide. However, concerning the diverse structures of dermaseptins, the bioactivities may also be relevant to their structures. Hence, whether functions of dermaseptins have a strong connection with their structures still need to be investigated intensively. The discovery of AMPs with multiple functions brought a new possibility for the treatment of human disease. Although there are shortcomings for the clinical application of AMPs, there is still a strong confidence for the prospect of their therapeutic applications.
4. Materials and Methods
4.1. Acquisition of Pithecopus hypochondrialis Skin Secretion
Specimens of the South American orange-legged leaf frog, Pithecopus hypochondrialis
were obtained as captive-bred adults, and were settled into their new surroundings for least three months prior to experimentation. All specimens were housed separately in purpose designed terraria under 12 h/12 h light/dark cycles, and were fed multivitamin-loaded crickets three times per week. Skin secretions were obtained by transdermal electrical stimulation after the method of Tyler et al. and these were washed from the skin using de-ionised water, snap-frozen in liquid nitrogen, lyophilised, and stored at 20 °C prior to analysis [40
]. The study was performed according to the guidelines in the UK Animal (Scientific Procedures) Act 1986, project license PPL 2694, issued by the Department of Health, Social Services and Public Safety, Northern Ireland. Procedures had been vetted by the IACUC of Queen’s University Belfast, and approved on 1 March 2011.
4.2. “Shotgun” Cloning of Dermaseptin-PH Precursor-Encoding cDNA from a Skin Secretion-Derived cDNA Library of Pithecopus hypochondrialis
Five milligrams of lyophilised skin secretion were dissolved in 1 mL lysis/binding buffer (Dynal Biotech, Merseyside, UK). Polyadenylated mRNA was isolated by using a magnetized Dynabeads® mRNA DirectTM Kit (Dynal Biotech, Merseyside, UK) from the decanted supernatant. Subsequently, the first-strand cDNA was synthesised, and the rapid amplification of cDNA ends PCR (RACE-PCR) was performed to obtain the full length of prepropeptide nucleic acid sequence using a SMART-RACE Kit (Clontech, Palo Alto, CA, USA). The nested universal primer supplied with the kit was used by combination of the degenerate sense primer (S1; 5′-ACTTTCYGAWTTRYAAGMCCAAABATG-3′) (Y = C/T; W = A/T; R = A/G; M = A/C; B = T/C/G) that was designed based on the highly conserved signal peptide regions of dermaseptin peptides published previously from subfamily Phyllomedusinae. Purification of PCR products were carried out using a ConcertTM Rapid PCR Purification System (Life Technologies, Warrington, UK) and the products were cloned using a pGEM®-T Easy Vector system (Promega Corporation, Southampton, UK). And an ABI 3100 automated capillary sequencer (Applied Biosystems, Foster City, CA, USA) was used to analyse the nucleotide sequence of selected cloned products.
4.3. Identification and Structural Characterization of the Putative Dermaseptin-PH from the Skin Secretion of Pithecopus hypochondrialis
Five mg of lyophilised skin secretion from Pithecopus hypochondrialis were dissolved in 1 mL of trifluoracetic acid (TFA)/water (0.05/99.95, v/v) and centrifuged at 5000× g for 20 min to clarify. The supernatant was injected into an RP-HPLC system (Waters, Miford, MA, USA) fitted with a column (Jupiter C-5, 5 μM, 4.6 mm × 250 mm, Phenomenex, Macclesfield, Cheshire, UK) and eluted with a linear gradient mobile phase from TFA/waster (0.05/99.95, v/v) to TFA/water/acetonitrile (0.05/19.95/80.00, v/v/v) at a flow rate of 1 mL/min in 240 min, detected by the ultraviolet absorbance at 214 nm. Each fraction was collected automatically at 1 min intervals, and then analysed by a MALDI-TOF-MS (Voyager DE, Perspective Biosystems, Foster City, CA, USA) in positive detection mode, using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix. The system was calibrated by standards with precision reaching ± 0.1%. Fractions containing identical molecular masses correspond with the deduced mature peptide from the cloned cDNA were injected into to an LCQ-Fleet electrospray ion-trap mass spectrometer (Thermo Fisher Scientific, San Francisco, CA, USA) to analysis sequence structure by MS/MS fragmentation sequencing technique.
4.4. Solid-Phase Peptide Synthesis
Peptide was chemically-synthesised by solid-phase Fmoc Chemistry using a Tribute® PS4 automated solid-phase peptide synthesizer (Protein Technologies, Tucson, AZ, USA) for bioactive assessment after the primary structure of the peptide was confirmed. Purity result of the synthesised peptide was identified by RP-HPLC and MALDI-TOF-MS reaching up to 95%.
4.5. Determination of Dermaseptin-PH Second Structure Using Circular Dichroism Spectroscopy
The secondary structure of Dermaseptin-PH was determined using a JASCO J-815 CD spectrometer (Jasco, Essex, UK). Chemically-synthesised peptide was dissolved in 10 mM ammonium acetate and 10 mM ammonium acetate with 50% TFE, respectively, and was prepared at 50 μM in a 1 mm precision quartz cell (Hellma Analytics, Essex, UK). CD spectra were recorded at a wavelength ranging from 190 nm to 260 nm with a 200 nm/min scan speed. The parameters were set as 1 nm bandwidth and 0.5 nm data pitch and the results were analysed by the DichroWeb webserver to estimate α
-helical content [17
4.6. Antimicrobial Assays
Antimicrobial activity of synthetic Dermaseptin-PH was assessed by determination of MIC and MBC assays using six standard bacteria, including Gram-negative bacteria Escherichia coli (NCTC 10418), and Pseudomonas aeruginosa (ATCC 27853); Gram-positive bacteria Staphylococcus aureus (NCTC 10788), methicillin-resistant Staphylococcus aureus (MRSA) (NCTC 12493), and Enterococcus faecalis (NCTC 12697); and pathogenic yeast Candida albicans (NCYC 1467). Each microorganism was inoculated in Mueller–Hinton broth (MHB) (Sigma-Aldrich, St. Louis, MO, USA) for 16–18 h, and then subcultured until reaching the logarithmic growth phase, by measuring optical density (OD) of the cultures at wavelength 550 nm. Then, the cultures were diluted to obtain 1 × 106 colony forming units (cfu)/mL for the bacteria, and 1 × 105 cfu/mL for the yeast, and incubated with various concentrations of Dermaseptin-PH, with final concentration range from 1 to 512 μM in a 96 well microtiter cell culture plate. Plates were incubated for 18 h at 37 °C in a humid atmosphere, and the growth of bacteria/yeast was determined by means of measuring absorbance values at wavelength 550 nm using a Synergy HT plate reader (Biotech, Minneapolis, MN, USA). The MIC results were defined as the lowest concentrations of peptide at which no distinct growth of the microorganisms was detectable. For the MBC assay, the culture in each well was spotted onto a Mueller–Hinton agar (MHA) (Sigma-Aldrich, St. Louis, MO, USA) plate, and incubated at 37 °C in a humid atmosphere for 18 h. The MBC results were defined as the lowest concentrations of peptide at which no obvious colony was observed.
4.7. Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC) Assays
Gram-negative bacterium Escherichia coli
(NCTC 10418) was inoculated in Luria–Bertani (LB) broth (Sigma-Aldrich, St. Louis, MO, USA); Gram-positive bacteria Staphylococcus aureus
(NCTC 10788) was inoculated in tryptic soy broth (TSB) (Sigma-Aldrich, St. Louis, MO, USA) for 16–18 h, and then subcultured until reaching the logarithmic growth phase by measuring OD of the cultures at wavelength 550 nm. For the MBIC assay, the cultures were diluted to obtain 1 × 106
cfu/mL, and incubated with various concentrations of Dermaseptin-PH with final concentration range from 1 to 512 μM in a 96 well flat-bottomed microtiter cell culture plate for 16 to 20 h at 37 °C in a shaking incubator at different revolutions per minute (rpm), according to different bacteria. After, plates were washed twice by phosphate buffered saline (PBS) and fixed with methanol for 10 min. For the MBEC assay, 100 μL of the cultures were placed in a 96 well flat-bottomed microtiter cell culture plate with a diluted colony concentration of 1 × 106
cfu/mL, and incubated at 37 °C for 16–20 h in a shaking incubator at different rpm, on the basis of different bacteria. After, plates were washed twice by PBS and incubated with 100 μL of Dermaseptin-PH dissolved in relevant culture medium, in a range of concentrations from 1 to 512 μM. Sixteen to twenty hours after exposure, plates were washed twice by PBS, and fixed with methanol for 10 min. Both MBIC and MBEC plates were stained with 150 μL 0.1% (g
) crystal violet solutions for 30 min, and washed by PBS until no apparent stains were observed. Evaporating crystal violet stains were dissolved by 100 μL 30% (v
) acetic acid, and the absorbance values were measured at wavelength 595 nm using a Synergy HT plate reader (Biotech, Minneapolis, MN, USA). The minimum concentration that inhibits the formation of biofilm is defined when compared to the negative control group as MBIC i.e., ≥90% (for MBIC90
) and MBIC i.e., ≥50% (for MBIC50
). The minimum concentration that eradicates the biofilm is defined when compared to the negative control group as MBEC i.e., ≥90% (for MBEC90
) and MBEC i.e., ≥50% (for MBEC50
4.8. Bacteria Cell Membrane Permeability Assay of Dermaseptin-PH
Bacteria were inoculated in TSB (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 16 to 18 h, and then subcultured until reaching the logarithmic growth phase, by measuring OD of the cultures at wavelength 550 nm. Then, bacterial cells were collected, centrifuged at 1000× g for 10 min at 4 °C, where after, cells were washed twice with 5% TSB in 0.85% NaCl solution. The pellet was resuspended with 5% TSB in 0.85% NaCl solution to obtain a cell concentration of 1 × 108 cfu/mL by measuring optical density (OD) at wavelength 590 nm. The cells were incubated with Dermaseptin-PH in a range of concentrations based on the MIC results in a 96 well black plate at 37 °C. Two hours after exposure, the cells were stained with SYTOXTM green nucleic acid stain (Life technologies) dissolved in 5% TSB in 0.85% NaCl to a final concentration of 5 μM, and incubated for 5min in the dark at 37 °C. The fluorescent intensity was measured with a Synergy HT plate reader (Biotech, Minneapolis, MN, USA) by an excitation and emission wavelength of 485 and 528 nm, respectively. Bacteria cells treated with 70% (v/v) isopropanol for 1 h were used as positive control.
4.9. Haemolysis Assay
The defibrinated horse blood (TCS Biosciences Ltd., Botolph Clayton, UK) was collected, centrifuged at 1000× g for 5 min, where after, cells were washed with PBS until the supernatant was clean. The cells were resuspended with PBS to obtain a cell concentration of 4% (v/v). The pellet was incubated with Dermaseptin-PH in a range of final concentration from 1 to 512 μM at 37 °C. Two hours after exposure, supernatant was collected, centrifuged at 1000× g for 5 min, and placed into a 96 well plate. The absorbance was measured at wavelength 570 nm using a Synergy HT plate reader (Biotech, Minneapolis, MN, USA). Cells treated with 1% (v/v) Triton-X 100 for 2 h were used as positive control.
4.10. MTT Cell Proliferation Assay
MCF-7 (ATCC-HTB-22), MDA-MB-435S (ATCC-HTB-129), and U251MG (ECACC-09063001) were cultured in DMEM (Invitrogen, Paisley, UK) supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 1% (v/v) penicillin–streptomycin (Invitrogen, Paisley, UK). PC-3 (ATCC-CRL-1435) and H157 (ATCC-CRL-5802) were cultured in RPMI-1640 (Invitrogen, Paisley, UK) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin–streptomycin. HMEC-1 (ATCC-CRL-3243) was cultured in MCDB131 (Gibco, Paisley, UK) supplemented with 10% (v/v) FBS, 1% (v/v) penicillin–streptomycin, 10 ng/mL epidermal growth factor (EGF), 10 mM l-glutamine, and 1 μg/mL hydrocortisone. Cells were grown in a 96 well plate (5000 cells/well/100 μL) for 24 h, and treated with relevant serum-free medium for 12 h. After, cells were treated with Dermaseptin-PH dissolved in relevant serum-free medium in a range of concentrations, from 10−9 to 10−4 M. Twenty-four hours after exposure, the cells were incubated with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphehyltetrazolium Bromide) dissolved in PBS to a final concentration of 0.5 mg/mL at 37 °C for 4 to 6 h. After, medium in each well was removed and 100 μL dimethyl sulfoxide (DMSO) was added. Cell viability was measured at wavelength 570 nm using a Synergy HT plate reader (Biotech, Minneapolis, MN, USA).
4.11. Cancer Cell Membrane Permeability Assay of Dermaseptin-PH
The permeability of Dermaseptin-PH on MCF-7 cell was determined using SYTOXTM green nucleic acid stain (Life technologies). MCF-7 cells were grown in a sterile black 96 well plate (5000 cells/well/100 μL) for 24 h, and treated with relevant serum-free medium for 12 h. After, cells were incubated with SYTOXTM green stain (1 μM) for 15 min and subsequently were treated with various concentrations of Dermaseptin-PH with final concentrations range from 10−6 to 10−4 M. The fluorescence was measured every 15 min for 75min with a Synergy HT plate reader (Biotech, Minneapolis, MN, USA) by an excitation and emission wavelength of 485 and 528 nm, respectively. Cells treated with 0.5% Triton X-100 were used as positive control.