Unravelling the Skin Secretion Peptides of the Gliding Leaf Frog, Agalychnis spurrelli (Hylidae)

Frog skin secretions contain medically-valuable molecules, which are useful for the discovery of new biopharmaceuticals. The peptide profile of the skin secretion of Agalychnis spurrelli has not been investigated; therefore, the structural and biological characterization of its compounds signify an inestimable opportunity to acquire new biologically-active chemical scaffolds. In this work, skin secretion from this amphibian was analysed by molecular cloning and tandem mass spectrometry. Although the extent of this work was not exhaustive, eleven skin secretion peptides belonging to five peptide families were identified. Among these, we report the occurrence of two phyllokinins, and one medusin-SP which were previously reported in other related species. In addition, eight novel peptides were identified, including four dermaseptins, DRS-SP2 to DRS-SP5, one phylloseptin-SP1, and three orphan peptides. Phylloseptin-SP1 and dermaseptins-SP2 were identified in HPLC fractions based on their molecular masses determined by MALDI-TOF MS. Among the antimicrobial peptides, dermaseptin-SP2 was the most potent, inhibiting Escherichia coli, Staphylococcus aureus, and ORSA with a minimum inhibitory concentration (MIC) of 2.68 μM, and Candida albicans with an MIC of 10.71 μM, without haemolytic effects. The peptides described in this study represent but a superficial glance at the considerable structural diversity of bioactive peptides produced in the skin secretion of A. spurrelli.


Introduction
The resistance of microorganisms to current chemotherapy has limited successful treatments which stresses the urgent need for more efficient therapeutic strategies [1]. Animal venoms and secretions, such as the skin secretions of frogs, represent a treasure trove of small molecules that offer a promising way to tackle this global challenge [2]. The skin secretions of phyllomedusine hylids are complex molecular libraries composed by alkaloids, biogenic amides, inorganic molecules, opioids, steroids, nucleic acids, and proteins, and are mainly rich in a promising array of diverse peptides [3,4]. and characterize the primary structures of peptides from the skin secretion of Agalychnis spurrelli by integrating proteomic and molecular tools, and subsequently, to evaluate their antimicrobial activity.

Animals and Skin Secretion Samples
Six specimens of Agalychnis spurrelli were employed in this study: two wild adults, collected in northwestern Ecuador and four captive-bred juveniles, provided by Centro Jambatu for Research and Conservation of Amphibians, in Ecuador. Skin secretions were stimulated by massaging the frogs' backs, and then the whitish secretions were washed off the animals with distilled water. Samples were immediately frozen and stored at −20 • C. The wild frogs were returned to their habitat after the extraction and captive frogs were returned to their enclosures. The samples were freeze-dried, and later transported to Queen's University, Belfast, for analysis. Collection and exportation permits were issued by the Ministry of Environment of Ecuador (MAE) as detailed in Acknowledgements.

Molecular Cloning
Five milligrams of lyophilized skin secretions were dissolved in 1 mL of cell lysis/binding buffer, and the polyadenylated mRNA was isolated using magnetic Dynabeads Oligo (dTs), as described by the manufacturer (Dynal Biotec, Oslo, Norway). A cDNA library was constructed and was subjected to 3 -rapid amplification of cDNA by using the SMART-RACE kit (Clontech, Mountain View, CA, USA). In brief, three sets of 3 -RACE reactions were employed, and each one used a nested universal primer (NUP), provided with the kit, and one of the following sense primers: S1: 5 -CAGCACTTTCTGAATTACAAGACCAA-3 , designed based on the signal sequence of phylloseptin-s5 of Phyllomedusa sauvagii; S2: 5 -GACCAAAGATGTCWTTCTTGAAGAAAT-3 , designed based on the opioid peptide of A. dacnicolor; or F2: 5 -TGAAGGGCCCTAACATGTCTT-3 , designed based on the signal sequence of dermaseptin-As1 of A. spurrelli. 3 -RACE products were purified and cloned using a pGEM-T vector system (Promega Corporation), and then sequenced using an ABI 3100 automated sequencer [23,24].

Reverse-Phase, High Performance Liquid Chromatography (HPLC) Fractionation
Five milligrams of freeze-dried skin secretion of A. spurrelli were dissolved in 1.2 mL of buffer A (99.95% H 2 O, 0.05% trifluoroacetic acid) and clarified by centrifugation. Supernatant (1 mL) was subjected to reverse phase HPLC employing a Waters Binary pump HPLC system fitted with an analytical column (Phenomenex Jupiter C-18; 4.6 × 250 mm). Peptides were eluted with a linear gradient formed from 100% buffer A (99.95% H 2 O, 0.05% trifluoroacetic acid) to 100% buffer B (80.00% Acetonitrile, 19.95% H 2 O, 0.05% trifluoroacetic acid) for 240 min at a flow rate of 1 mL/min. Fractions (1 mL) were collected each minute. Absorbance at 214 nm and 280 nm was monitored continuously.

Tandem Mass Spectrometry
Twenty microliters of the remaining skin secretion samples, dissolved in buffer A from the HPLC step, were subjected directly to an analytical column (Phenomenex C-18; 4.6 × 150 mm) connected to an LCQ Fleet ESI ion-trap mass spectrometer (Thermo Fisher, San Jose, CA, USA). The linear elution gradient was formed from 100% buffer A (99.90% H 2 O, 0.1% formic acid) to 100% buffer B (19.9% H 2 O, 80% acetonitrile, 0.1% formic acid) for 135 min at a flow rate of 20 µL/min. Mass analysis was performed in a positive ion mode, and the conditions set for the spectra acquisition were a mass range of m/z 500-2000 and a relative intensity <50%. The parameters for electrospray ionization ion-trap mass spectrometry (ESI/MS) were the following: the spray voltage was +4.5 kV, the drying gas temperature was 320 • C, the drying gas flow was 200 µL/min, and the maximum accumulation time for the ion trap was 350 ms. Each fraction was analysed by two rounds of mass spectrometry (MS/MS) in order to obtain the amino acid sequences of the different peptides. Sequences obtained by MS/MS were compared with the sequence database built by molecular cloning in order to confirm their primary structures. The LCQ was controlled by Xcalibur software (Thermo, San Jose, CA, USA), and data analysis was performed using Proteome Discoverer 1.0 (Thermo, San Jose, CA, USA).

Bioinformatic Analysis
Nucleotide sequences were analysed by MEGA 6.0 and Vector NTI and compared with databases from the National Centre for Biotechnology Information (NCBI) by employing the BLAST tool [29][30][31]. Signal peptides were predicted using the SignalP 4.1 server, and theoretical peptide masses were calculated with peptide mass calculator v3.2 [32,33]. Secondary structure prediction was performed using the SOPMA programme and the physicochemical properties of the peptides were calculated using HeliQuest Compu Parameters and the Peptide property calculator from Bachem [34][35][36]. Finally, structural prediction models were constructed with HeliQuest and SWISS-MODEL online software [37].

Antimicrobial Activities of the Novel Peptides Identified
For the in vitro evaluation of antimicrobial potentials of new biochemical structures identified herein, five synthetic peptides were purchased from a commercial supplier (Biomatik). Approximately 100 mg of each peptide was synthetized by Fmoc technology. The purities of the amidated peptides were greater than 90%. The lyophilized peptides were kept at −20 • C and diluted in dimethyl sulfoxide (DMSO) before the antimicrobial assays, which were performed according to broth microdilution methodology detailed by Proaño et al. [7]. Minimal inhibitory concentration of the synthetic peptides was determined against the following Gram-positive bacteria: Staphylococcus aureus ATCC 25923 and S. aureus oxacillin resistant strain; Gram-negative bacteria: Escherichia coli ATCC 25922 and Klebsiella pneumonia clinical isolate; and the opportunistic pathogenic yeast Candida albicans. Briefly, peptides were dissolved in DMSO at the following concentrations: 512, 256, 128, 64, 32, 16, 8, 4, 2, 1 × 10 2 mg/L. Each bacteria/yeast was cultured until reach log phase and then it was diluted to obtain 1 × 10 6 CFU/mL for bacteria and 1 × 10 5 CFU/mL for the yeast. Assays were prepared in 96 well sterile plates where 198 µL of each microorganism was challenged with 2 µL of the diluted peptides at the serial dilutions mentioned above. Negative control included 2 µL of DMSO instead of peptide and 198 µL of microbial culture where we expected no inhibition. Another control included 200 µL of Muller Hinton broth without microorganisms to be used as blank with no growth. Seven replicates per peptide concentration was employed and experiments were performed in triplicates. Plates were cultured at 37 • C for 18 h and the inhibition of microbial growth was measured by spectrophotometry at 600 nm using a GloMax Multi Detection System (Promega, Madison, WI, USA). MIC was determined as the minimal concentration of peptide required to inhibit the growth of >95% of microbial growth.

The Determination of Toxicity to Red Blood Cells
Aiming to determine the toxicity of frog peptides to red blood cells, in vitro assays were performed with different concentrations of each peptide. The diluted peptides (employing the same concentrations than for the antimicrobial assay described above) were incubated with a suspension of red blood cells (2%) (7.7 × 10 6 ± 0.3 × 10 6 cells/mL) at a ratio of 1:1 (v/v), in a total volume of 400 µL for 2 h at 37 • C. After the incubation period, the cells were centrifuged at 3000 rpm for 5 min and transferred to a 96 well plates. The haemolytic activity was monitored at 550 nm and was performed in triplicate. PBS and 2% (v/v) Triton X-100 were used as negative and positive controls, respectively. The concentrations that produce 100% haemolysis are reported.

Molecular Cloning of the Novel Peptide Precursors Encoding cDNA Sequences, and the HPLC Profile
Eleven nucleotide sequences encoding peptide precursors were successfully cloned from the skin secretion of Agalychnis spurrelli. Among these were two phyllokinins and one medusin that were all reported previously in other species. The other eight mature peptide sequences were novel, including one phylloseptin, four dermaseptins, and three unclassified peptides. Nucleotide sequences were submitted to GenBank (NCBI) under accession numbers: MK532479-MK5232483 and MK766838-MK766843. The C18 HPLC profile of skin secretion of Agalychnis spurrelli revealed the presence of many peaks ( Figure 1). The fractions containing dermaseptin-SP2 and phylloseptin-SP1 are identified with the letters A and B in the chromatogram, respectively. Herein we describe the main findings of each peptide family, with emphasis on novel primary structures of dermaseptins and phylloseptin, identified in this sample.
Biomolecules 2019, 9,  Eleven nucleotide sequences encoding peptide precursors were successfully cloned from the skin secretion of Agalychnis spurrelli. Among these were two phyllokinins and one medusin that were all reported previously in other species. The other eight mature peptide sequences were novel, including one phylloseptin, four dermaseptins, and three unclassified peptides. Nucleotide sequences were submitted to GenBank (NCBI) under accession numbers: MK532479-MK5232483 and MK766838-MK766843. The C18 HPLC profile of skin secretion of Agalychnis spurrelli revealed the presence of many peaks ( Figure 1). The fractions containing dermaseptin-SP2 and phylloseptin-SP1 are identified with the letters A and B in the chromatogram, respectively. Herein we describe the main findings of each peptide family, with emphasis on novel primary structures of dermaseptins and phylloseptin, identified in this sample. The nucleotide sequences of [Ser 6 , Val 10 , Asp 11 ]-phyllokinin and [Thr 6 , Val 10 , Asp 11 ]-phyllokinin were 87%-91% similar to the preprophyllokinin-HA1 of Phyllomedusa hypochondrialis, the Hyp 3 -Thr 6 -BK precursor of A. callidryas, and the Thr 6 -phyllokinin of P. sauvagii (accession numbers AM283482.1, HE967330.1, and AJ549500.1 respectively). However, the comparison of the mature peptide sequences within the NCBI database using BLAST/p yielded different results; i.e., while the amino acid sequence of [Thr 6 , Val 10 , Asp 11 ]-phyllokinin was, as expected, 100% identical to that of Thr 6 -BK of A. dacnicolor and Hyp 3 -Thr 6 -BK of A. callidryas (cannot confirm hydroxy-proline) (accession numbers CCJ67650.1 and CCJ67651.1 respectively), the sequence of [Ser 6 , Val 10 , Asp 11 ]-phyllokinin was, in contrast, 100% identical to the bradykinin-like peptide RD-11 of Ascaphus truei and

Phyllokinins
Two 300 bp novel nucleotide sequences of peptide precursors that encoded phyllokinins were identified (accession numbers: MK766838 and MK766839). The translated open reading frames consisted of 186-189 nucleotides that encoded: (1) a 22 amino acid putative signal peptide; (2) a 29 amino acid acidic spacer that contained two pro-peptide processing sites (KR) and a sequence of four amino acids (ESPD or ESPE) at the C-terminal side of the latter; and (3) an 11 amino acid mature peptide (Supplementary Figure S1). These phyllokinin were named [Ser 6 , Val 10 , Asp 11 ]-phyllokinin and [Thr 6 , Val 10 , Asp 11 ]-phyllokinin to highlight their similar but different amino acid sequences.

Medusin-AS
A nucleotide precursor of 433 pb encoding a medusin was identified in the skin (accession number MK766840). The open reading frame consisted of: (1) a 22 amino acid putative signal peptide; (2) a 27 amino acid acidic spacer that contained two KR pro-peptide processing sites; (3) an 18 amino acid mature medusin sequence; and (4) a glycine amide donor residue (Supplementary Figure S2). This peptide precursor was named medusin-AS to represent its origin in A. spurrelli.

Phylloseptin-AS1
A novel nucleotide sequence encoding a phylloseptin was cloned from the skin secretion (accession number MK532479). The translated open reading frame consisted of: (1) a 22 amino acid putative signal peptide; (2) a 23 amino acid acidic spacer with one pro-peptide processing site; (3) a 22 amino acid mature peptide; and (4) a glycine residue amide donor at the C-terminus ( Figure 2 and Supplementary Figure S3). This peptide was named phylloseptin-SP1-SP for the species name "spurrelli"-in accordance with the nomenclature rules proposed for the antimicrobial peptides of Phyllomedusine frogs [38].

Medusin-AS
A nucleotide precursor of 433 pb encoding a medusin was identified in the skin (accession number MK766840). The open reading frame consisted of: (1) a 22 amino acid putative signal peptide; (2) a 27 amino acid acidic spacer that contained two KR pro-peptide processing sites; (3) an 18 amino acid mature medusin sequence; and (4) a glycine amide donor residue (Supplementary Figure 2). This peptide precursor was named medusin-AS to represent its origin in A. spurrelli.

Orphan Peptides
Three novel precursors were identified and temporarily assigned to the orphan peptide group because their highly divergent sequences did not allow them to be assigned to any of the peptide families described to date (accession numbers: MK766841-MK766843). The first novel nucleotide sequence of peptide unknown-1 was 82%-93% similar to medusin precursors of A. dacnicolor, A. callidryas, P. hypochondrialis, and P. sauvagii (accession numbers HE863818.1, HE863819.1, HE863820.1, and AM903081.1). However, its translated mature sequence was highly divergent and did not produce any significant hits when compared with the databases in NCBI. In addition, the two novel nucleotide sequences of peptides unknown-2 and 3 were 88%-90% similar to tryptophyllin precursors, including tryptophyllin-PdT1 and PdT2 of A. dacnicolor, tryptophyllin-AcT2 of A. callidryas, and tryptophyllin HA5 of P. hypochondrialis. Peptides unknown-1 and 2 could be considered phenylalanine-rich peptides due to their high F content (43%). Nevertheless, their translated mature peptide sequences did not produce any relevant hits when they were compared with NCBI databases using BLAST-P. In addition, DRS-SP2 was identified in HPLC fraction 134. Its monoisotopic molecular mass of 2988.24 Da was identified by MALDI-TOF mass spectrometric analysis. The fraction also contained another peptide of 3005.36 Da. that co-eluted (Table 1 and Supplementary Figure S4).

Orphan Peptides
Three novel precursors were identified and temporarily assigned to the orphan peptide group because their highly divergent sequences did not allow them to be assigned to any of the peptide families described to date (accession numbers: MK766841-MK766843). The first novel nucleotide sequence of peptide unknown-1 was 82%-93% similar to medusin precursors of A. dacnicolor, A. callidryas, P. hypochondrialis, and P. sauvagii (accession numbers HE863818.1, HE863819.1, HE863820.1, and AM903081.1). However, its translated mature sequence was highly divergent and did not produce any significant hits when compared with the databases in NCBI. In addition, the two novel nucleotide sequences of peptides unknown-2 and 3 were 88%-90% similar to tryptophyllin precursors, including tryptophyllin-PdT1 and PdT2 of A. dacnicolor, tryptophyllin-AcT2 of A. callidryas, and tryptophyllin HA5 of P. hypochondrialis. Peptides unknown-1 and 2 could be considered phenylalanine-rich peptides due to their high F content (43%). Nevertheless, their translated mature peptide sequences did not produce any relevant hits when they were compared with NCBI databases using BLAST-P.

Peptide Identification by Tandem Mass Spectrometry
The peptide database created by molecular cloning was compared with the database generated by fragmentation of the peptide ions in the tandem mass spectrometric analysis of the skin secretion of A. spurrelli. As a result, 10 putative mature peptides were 100% identified while one sequence was not found at all (Table 1). In summary, 15 to 103 peptide fragments were sequenced per peptide, which contained between 11 and 31 amino acids and a molecular mass range of between 1273.66 and 2988.62 Da.

The Antimicrobial and Haemolytic Activities of Synthetic Peptides Based on Amino Acid Sequences Identified and Toxicity to Red Blood Cells
The synthetic dermaseptins showed antimicrobial activity against Gram-positive and Gram-negative bacteria, and yeast. The peptides were more active against bacteria than yeast ( Table 2). The dermaseptin-2 was the most potent peptide, inhibiting bacterial growth at 2.68 µM against E. coli, S. aureus, and a S. aureus oxacillin resistant strain [39][40][41][42].
Dermaseptins induced no red blood cell lysis at MIC concentrations. In general, dermaseptins only triggered a significant haemolytic effect at concentrations higher than 512 µM ( Figure 5). The phylloseptin-SP1 showed greater toxicity to red blood cells than did the dermaseptins, also increasing with increasing peptide concentration. ules 2019, 9, x FOR PEER REVIEW ermaseptins induced no red blood cell lysis at MIC concentrations. In general, derm iggered a significant haemolytic effect at concentrations higher than 512 μM (Figu septin-SP1 showed greater toxicity to red blood cells than did the dermaseptins, also i creasing peptide concentration. gure 5. Haemolytic activity of Dermaseptins SP2-SP5 and Phylloseptin SP-1. The total hemo 00%) was determined using Triton X-100.

condary Structures and Physicochemical Properties of Synthetic Peptides
he net charge, hydrophobic moment, and α-helix parameters of synthetic peptides t bacteria and yeasts were determined in silico by use of bioinformatic tools (Suppl 3; Table 4). Interestingly, the most active peptide, dermaseptin-2, possessed a higher n hen compared to the other peptides tested. ioinformatic analyses have revealed the high propensity of peptides to adopt an mation. Dermaseptin-SP2 had a lower α-helix% (Supplementary Table 3; Table 4). In wheel plots illustrate the amphipathic characteristics and properties of alpha heli ed peptides ( Figure 6).   (32)

Secondary Structures and Physicochemical Properties of Synthetic Peptides
The net charge, hydrophobic moment, and α-helix parameters of synthetic peptides evaluated against bacteria and yeasts were determined in silico by use of bioinformatic tools (Supplementary  Table S3; Table S4). Interestingly, the most active peptide, dermaseptin-2, possessed a higher net charge (+4) when compared to the other peptides tested.
Bioinformatic analyses have revealed the high propensity of peptides to adopt an α-helical conformation. Dermaseptin-SP2 had a lower α-helix% (Supplementary Table S3; Table S4). In addition, helical wheel plots illustrate the amphipathic characteristics and properties of alpha helices in the analysed peptides ( Figure 6).

Secondary Structures and Physicochemical Properties of Synthetic Peptides
The net charge, hydrophobic moment, and α-helix parameters of synthetic peptides evaluated against bacteria and yeasts were determined in silico by use of bioinformatic tools (Supplementary Table 3; Table 4). Interestingly, the most active peptide, dermaseptin-2, possessed a higher net charge (+4) when compared to the other peptides tested.
Bioinformatic analyses have revealed the high propensity of peptides to adopt an α-helical conformation. Dermaseptin-SP2 had a lower α-helix% (Supplementary Table 3; Table 4). In addition, helical wheel plots illustrate the amphipathic characteristics and properties of alpha helices in the analysed peptides ( Figure 6).

Discussion
The granular glands of frogs are responsible for the production of diverse peptides, some of which have shown a broad spectrum of action with potential biomedical applications [2]. The HPLC chromatogram of a skin secretion of A. spurrelli confirmed the abundance of peptides present (Figure 1). Other biochemical studies have revealed similar molecular complexity and diversity from skin secretions of frogs [4]. By peptidomic analysis and integrating tandem mass spectrometry with molecular cloning, eleven peptides belonging to five families were identified in A. spurrelli skin secretion and screened for antimicrobial activity. The peptide sequences identified in this work are detailed and discussed in the light of their families.

Phyllokinins
The deduced amino acid sequences revealed the presence of two previously reported bradykinin-like peptides. This was confirmed by BLAST/p comparisons which showed that [Ser 6 , Val 10 , Asp 11 ]-phyllokinin is identical to the bradykinin-like peptide RD-11 of the frog, Ascaphus truei, and vespakinin-T of the hornet, Vespa tropica; and that [Thr 6 , Val 10 , Asp 11 ]-phyllokinin is identical to Hyp 3 -Thr 6 -BK of A. callidryas and Thr 6 -BK of A. dacnicolor. The nucleotide comparison of frog peptides precursors-encoding cDNA, ignoring the vespakinin-T precursor because it is too divergent, revealed that these related phyllokinin precursors have 24 non-synonymous substitutions that encode for 12 amino acids and eight synonymous substitutions in their signal and acidic spacer peptides. In addition, there are eight synonymous substitutions in the region corresponding to the mature peptides between these precursors. The occurrence of the same Thr 6 -phyllokinin in the three species of Agalychnis is not surprising and reflects taxonomic relationships. In contrast, the presence of the bradykinin-like peptide RD-11 of A. truei (also known as vespakinin-T) of the hornet, V. tropica, was unexpected. Ascaphus truei is an ancient basal species and sister-group of all other amphibian species. Therefore, this could show an early evolutionary origin of the ancestral gene of this bradykinin-like peptide, which should be confirmed whenever the precursor sequence of RD-11 becomes available (Supplementary Table S1) [43]. Here, we have named this peptide precursor as [Ser 6 , Val 10 , Asp 11 ]-phyllokinin for the sake of consistency with other bradykinin-related peptides of phyllomedusine frogs. The occurrence of this bradykinin-like peptide (vespakinin-T) in the venom of the hornet, V. tropica, could be due to convergent evolution because its precursor is too divergent to be related to the frog phyllokinin precursors.
A previous study showed that RD-11 is able to induce relaxation of pre-contracted mouse trachea interacting with the bradykinin B2 receptor in the same way as bradykinin, only less potently [43,44]. Although the biological role of these bradykinin-related peptides remains unclear, the observation of antipredator behaviour in some snakes, after the initial taste of some frogs that contain these peptides, indicates a defensive role. In addition, studies that show bradykinin to be a potent stimulator of gastrointestinal smooth muscle and pain sensitivity pathways in mammals, as well as its effect on the vascular system of snakes, strongly suggest an antipredator-defensive role among these peptides [3,45,46].

Medusin-AS
A medusin with an identical amino acid sequence to medusin-AC was identified in this skin secretion. The nucleotide sequences of corresponding precursors are virtually identical, having three non-synonymous substitutions that lead to three different amino acids and four synonymous substitutions in the region corresponding to the signal peptide and acidic spacer. In addition, the mature peptide sequence contains only one synonymous substitution. It is notable that this medusin is highly conserved across species. At the moment, medusin-AC is known to occur in A. callidryas, A. spurrelli, A. lemur (described as Phylloseptin-L1), and in Cruziohyla calcarifer. (Supplementary Table S2). This is the first antimicrobial peptide occurring in four different species of two distinct genera and could be indicative of a secondary role acting on cell receptors. At the moment, this peptide has been found to have antimicrobial activity against S. aureus, C. albicans, and Batrachochytrium dendrobatidis, but has been found to be inactive against E. coli. In addition, it is cytolytic against HepG2 human hepatoma-derived cells and human erythrocytes at similar concentrations (LC 50 45 µM and LC 50 40 µM respectively) [20,22].

Phylloseptin-AS1
A novel phylloseptin was identified in the skin secretion of A. spurrelli. It shares limited similarity with other phylloseptins (63%), although the nucleotide precursor sequences showed higher similarity (82%) with phylloseptins and medusins, which indicates a close relationship between these two peptides families [22]. As shown in our findings, conserved amino acids are concentrated in their N-terminal regions, while the C-terminal end is highly divergent. As is the case for PLS-H3, H6, and S1, PLS-SP1 shares the N-terminal conserved sequence FLSLIP, an H in position seven, and a C-terminal amidated residue that was predicted from the position of the glycine amide donor (Figure 3). These are all the main characteristics of phylloseptins, so the new sequence was named phylloseptin AS-1 in accordance with the nomenclature proposed for antimicrobial peptides of this family [38]. In addition, PLS-SP1 constitutes the longest phylloseptin (with 22 residues) reported to date ( Figure 2).
Additionally, phylloseptin-SP1 was identified in HPLC fractions 153 and 154 ( Figure 1). The observed m/z of 2354.00 in those fractions was determined by MALDI-TOF mass spectrometry and confirmed later by tandem mass spectrometry as 2354.43, which was very close to the calculated mass of 2353.83 for this phylloseptin. (Supplementary Figure S3).
Cationic peptides, such as phylloseptins, usually exhibit antimicrobial activity that disrupts the bacterial cell membrane. The lysis starts by electrostatic interaction of the cationic peptides with the anionic microbial membrane, followed by the rearrangement of the peptides into an amphipathic helix. The alpha helix is inserted into the membrane through interaction of the hydrophobic face of the helix with the lipid acyl chains of the bilayer, causing disruption of the membrane after reaching a critical peptide concentration [40,[47][48][49].
PLS-SP1 has similar physicochemical properties to PLS-S1, including a high hydrophobicity (0.848-0.806 H), hydrophobic moment (0.516-0.551 µM), and a very high alpha helix percentage (100%-94.7%) in its structure. However, PLS-SP1 and PLS-S1 have three main differences in terms of sequence, size, and charge. PLS-SP1 and PLS-S1 share only 42.11% sequence similarity, and the sequence of PLS-SP1 is three amino acids longer than PLS-S1 (22 versus 19 residues respectively). In addition, PLS-SP1 is less cationic than PLS-S1 (+1 versus +2 respectively). PLS-SP1 showed lower antimicrobial effect when compared to PLS-S1 and the other peptides tested, evidencing the essential role of charge to antimicrobial activity ( Table 2). The effect of charge on the differential bactericidal effect was demonstrated in another study with PLS-S3, where the reduced cationicity (+1 instead of +2) was attributed as a main factor for its lack of antimicrobial activity, despite it having similar physicochemical properties to other paralogous antimicrobial phylloseptins (PLS-S1, PLS-S2, and PLS-4) (Supplementary Figure S3). The same effect was evidenced in the synthetic peptide P19(1|E) with a +1 charge that is devoid of antimicrobial activity but shares a similar mean hydrophobicity, amphipathicity, and degree of secondary structure formation to P19(3|E), P19(5), and P19(6) (with +3, +5 and +6 charges respectively). According to this paper, increasing the peptide charge to at least +3 is required for an antimicrobial activity in these peptides: [48].
In summary, PLS-SP1 is a hydrophobic and amphipathic helical peptide, which exhibited low antimicrobial activity at physiological pH, probably owing to its low charge (+1). However, its histidine content PLS-SP1 can convert it into an antimicrobial molecule at acidic pH by increasing cationicity. These hypotheses should be confirmed by appropriate experimental approaches in future studies.

Dermaseptin-SP
The following group of peptides includes four novel dermaseptins identified in the skin secretion of A. spurrelli. The novel peptides were named dermaseptin-SP2 to SP5 in accordance with the proposed nomenclature [38]. The similarity with nucleotides in other dermaseptin precursors was very high (88%-95%), indicating that the novel peptides belong to the dermaseptin superfamily.
The first novel dermaseptin-SP2 was 92% similar to the insulinotropic peptide-1 of A. spurrelli (formerly A. litodryas), and 85% similar to dermaseptin-B4 of P. bicolor. The insulinotropic peptide-1 (FSIP) belongs to the dermaseptin family because it contains a W (Trp) in the three position and the mid-region conserved sequence, but was not recognized as such in the corresponding publication nor in the peptide database of the NCBI. Therefore, hereafter this peptide is called dermaseptin-SP1 (DRS-SP1). The sequence alignment of DRS-SP2 with its closest paralogous peptide DRS-SP1 and the orthologous DRS-B4, showed that there are only three amino acid substitutions in the acidic spacer and six substitutions in the mature peptide region (Figure 4). The physical properties of these peptides are very similar in terms of hydrophobicity (0.317-0.358 H), hydrophobic moment (0.275-0.308), and helicity (78.57%-100%). However, they differ in cationicity, where DRS-SP2 is highly cationic +4, followed by DRS-B4 +3 and DRS-SP1 +1 (Supplementary Figure S4). The effect of cationicity over biological activity was demonstrated with phylloseptin-S3 and the synthetic peptide P19(1|E) [40,48].
In brief, cationicity is directly proportional to antimicrobial activity and cytotoxicity in amphipathic helical peptides.
In this sense, DRS-B4, with +3 charge, showed a potent antimicrobial activity against S. aureus (MIC 3 µM), P. aeruginosa (MIC 4.6-11.6 µM), and E. coli (MIC 0.5-2.3 µM), but it also exhibited cytotoxicity at 12 µM [39]. Continuing this trend, DRS-SP2 was the most potent antimicrobial peptide (MIC 2.68 µM against E. coli. S. aureus, and ORSA (Figure 7). This synthetic molecule shares structural features with others AMPs, such as hydrophobicity and net charge, reflecting the positive amino acids of primary structure [6]. These features are important for molecular mechanism of action of peptides [36]. Several studies have showed the cell membrane as the main target of antibacterial peptides [50,51]. Briefly, cationic and hydrophobic peptides trigger leakage of bacterial content or osmotic shock by opening artificial water channels or membrane destabilization [52]. The variable effect on bacteria and yeast can be a result from a difference in membrane composition [36]. Interestingly, the dermaseptin-2 peptide with higher activity has the higher charge, which can be favour electrostatic interactions with negative elements of microbial membrane. Basically, the killing mechanisms of bacteria induced by cationic peptides is based on membranolytic activity; however, their antifungal properties derive from biochemical and molecular processes more complex, which require the entry of them into the cell in the most cases. The antifungal peptides exert their effects by several mechanisms, such as interaction with intracellular targets; cell wall binding; the inhibition of DNA, RNA, and protein syntheses; the induction of signalling cascades or metabolic pathways; and the production of free radicals [53,54]. The dichotomy between toxicity to human cell and microbial cells is key to development of peptide drugs. In light of this, dermaseptins induced the death of bacteria and yeast without haemolytic effect at MIC concentrations.
The other three novel dermaseptin sequences, DRS-SP3 to SP5, were similar to dermaseptins DRS-A3, DRS-C1, DRS-C3, DRS-H2, and DRS-H3 of A. annae, A. callidryas, and P. hypochondrialis respectively. As expected, the amino acid sequences between the paralogous peptides DRS-SP3, -SP4, and SP-5 are 100% identical in the region corresponding to the signal peptide and acidic spacer, and 96% identical in the mature peptide. There are only two variable positions in the mature sequence of these dermaseptins-in position three (W/R) and position 25 (V/L). In comparison with the closest related orthologous peptide DRS-A3, there are three amino acids substitutions in the signal peptide and seven in the mature peptide (74% similarity). The antimicrobial activity against E. coli of DMS-SP3 to DMS-SP5 was lower than DMS-SP2 (47.50-96.06 µM versus 2.68 µM) but comparable with the MIC of Dermaseptin-S1 (46 µM) of Phyllomedusa sauvagii. Moreover, DMS-SP's MIC was, as excepted, a lot higher the MIC of a conventional antibiotic, such as ampicillin (

Orphan Peptides
The last three novel peptides have similarity in their precursors with medusins and tryptophyllin precursors, but their highly divergent mature sequences do not permit more precise identification. Therefore, they have been temporarily assigned to the orphan peptide family until further information becomes available. This highlights that there is still much to discover in the complexity of amphibian skin secretions.

Orphan Peptides
The last three novel peptides have similarity in their precursors with medusins and tryptophyllin precursors, but their highly divergent mature sequences do not permit more precise identification. Therefore, they have been temporarily assigned to the orphan peptide family until further information becomes available. This highlights that there is still much to discover in the complexity of amphibian skin secretions.

Tandem Mass Sequencing
Ten of the thirteen sequences analysed above were 100% identified by tandem mass spectrometry, which shows the robustness of the combined proteomic/molecular cloning approach in revealing novel peptides sequences in previously unanalysed species for which there is limited information. Notably, two of the three sequences that were unable to be identified by this methodology belong to the tryptophyllins, and this could be attributed to either (1) low expression of these peptides under the detection limit of the instrument and method employed; (2) no expression, implying that these tryptophyllins were transcribed but not translated by the skin glands at the time-point the samples were collected; or (3) that these peptides were subject to rapid degradation.
In conclusion, 11 novel peptides from A. spurrelli were identified and confirmed by molecular cloning and peptidomic approaches, which required a minimum amount of sample without harming the animal source. These peptides belong to five distinct peptide families. Dermaseptins, mainly dersmaseptin-2, showed a promising antimicrobial effect, which can be explored for translation in antibacterial therapies. This study stresses the remarkable variety of skin secretion peptides in Agalychnis spurrelli, and encourages further analysis in this and other unexplored amphibian species.  Table S3: Physicochemical properties of phylloseptin-SP1 and other related phylloseptins. PLS-SP1 of A. spurrelli; PLS-H3, PLS-H6, and PLS-H13 of P. hypochondrialis; and PLS-S1 of P. sauvagii. Table S4