Peptidomic Analysis of Skin Secretions of the Caribbean Frogs Leptodactylus insularum and Leptodactylus nesiotus (Leptodactylidae) Identifies an Ocellatin with Broad Spectrum Antimicrobial Activity

Ocellatins are peptides produced in the skins of frogs belonging to the genus Leptodactylus that generally display weak antimicrobial activity against Gram-negative bacteria only. Peptidomic analysis of norepinephrine-stimulated skin secretions from Leptodactylus insularum Barbour 1906 and Leptodactylus nesiotus Heyer 1994, collected in the Icacos Peninsula, Trinidad, led to the purification and structural characterization of five ocellatin-related peptides from L. insularum (ocellatin-1I together with its (1–16) fragment, ocellatin-2I and its (1–16) fragment, and ocellatin-3I) and four ocellatins from L. nesiotus (ocellatin-1N, -2N, -3N, and -4N). While ocellatins-1I, -2I, and -1N showed a typically low antimicrobial potency against Gram-negative bacteria, ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2) was active against an antibiotic-resistant strain of Klebsiella pneumoniae and reference strains of Escherichia coli, K. pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium (minimum inhibitory concentrations (MICs) in the range 31.25–62.5 μM), and was the only peptide active against Gram-positive Staphylococcus aureus (MIC = 31.25 μM) and Enterococcus faecium (MIC = 62.5 μM). The therapeutic potential of ocellatin-3N is limited by its moderate hemolytic activity (LC50 = 98 μM) against mouse erythrocytes. The peptide represents a template for the design of long-acting, non-toxic, and broad-spectrum antimicrobial agents for targeting multidrug-resistant pathogens.


Introduction
In an ongoing effort to address the global problem of antimicrobial resistance (AMR) and to promote the research and development of new antibiotics against emerging multidrug-resistant

Purification of the Peptides from L. nesiotus
The pooled skin secretions from L. nesiotus were subjected to the same chromatographic procedures used to purify peptides from L. insularum. The elution profile on a Vydac C-18 semipreparative column is shown in Figure 2 and the peptides of major abundance at peaks 1-4 were collected. Subsequent structural analysis revealed that peak 1 contained ocellatin-1N, peak 2 ocellatin-2N, and peak 3 and 4 ocellatin-3N and ocellatin-4N. The final purity of the peptides was estimated to be >98%.

Structural Characterization
The amino acid sequences of the ocellatins isolated from L. insularum and L. nesiotus skin secretions were established without ambiguity by automated Edman degradation ( Figure 3).

Structural Characterization
The amino acid sequences of the ocellatins isolated from L. insularum and L. nesiotus skin secretions were established without ambiguity by automated Edman degradation ( Figure 3).  The molecular masses of the peptides, determined by MALDI-TOF mass spectrometry, were consistent with the proposed structures and demonstrated that ocellatin-1I and -2I from L. insularum and all ocellatin peptides from L. nesiotus were C-terminally α-amidated. Ocellatin-1I and -2I differ by three amino acid residues (positions 5, 6, and 12). In addition, (1-16) fragments of both ocellatin-1I and -2I were purified from the L. insularum secretions. Ocellatin-1N and -2N differ by two amino acid residues at positions 9 and 10, whereas ocellatin-3N and -4N differ only by the substitution of Ile by Leu at position 2. The physicochemical properties of the ocellatin-related peptides isolated in this study are shown in Table 1. The Grand Average of Hydropathy (GRAVY) was determined using The molecular masses of the peptides, determined by MALDI-TOF mass spectrometry, were consistent with the proposed structures and demonstrated that ocellatin-1I and -2I from L. insularum and all ocellatin peptides from L. nesiotus were C-terminally α-amidated. Ocellatin-1I and -2I differ by Antibiotics 2020, 9, 718 5 of 15 three amino acid residues (positions 5, 6, and 12). In addition, (1-16) fragments of both ocellatin-1I and -2I were purified from the L. insularum secretions. Ocellatin-1N and -2N differ by two amino acid residues at positions 9 and 10, whereas ocellatin-3N and -4N differ only by the substitution of Ile by Leu at position 2. The physicochemical properties of the ocellatin-related peptides isolated in this study are shown in Table 1. The Grand Average of Hydropathy (GRAVY) was determined using the hydrophobicity scales of Kyte and Doolittle [37]. The presence of helical domains in the peptides was predicted using the AGADIR program [38]. The isoelectric point (pI) was calculated using the following web-site: http://www.bachem.com/service-support/peptide-calculator/.

Antimicrobial and Hemolytic Activities
The minimum inhibitory concentration (MIC) values for synthetic ocellatin-1I and -2I and ocellatin-1N and -3N against strains of Gram-positive bacteria Staphylococcus aureus, Enterococcus faecium, and Enterococcus faecalis and Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium, as well as ampicillin-resistant S. aureus and antibiotic-resistant K. pneumoniae, are shown in Table 2. Ocellatin-1I, -2I, and -1N exhibited relatively weak antimicrobial activities (MICs in the range of 62.5-250 µM) against the Gram-negative bacteria only. Ocellatin-3N from L. nesiotus displayed the greatest activity against all Gram-positive and Gram-negative microorganisms tested, including the multidrug-resistant strains (MICs in the range of 31.25-62.5 µM). However, this peptide was 4-8 times less potent against the Gram-positive bacterium E. faecalis (MIC = 250 µM).

Bacteria
Ocellatin  Figure 4 shows the primary structures of 27 ocellatins isolated from 10 Leptodactylus species. These amino acid sequences were used to construct the optimal phylogenetic tree shown in Figure 5. It can be seen that evolutionary pressure was added, primarily to conserve the charged residues (Lys and Asp) in this family of peptides. The tree was drawn to scale, with branch lengths being given in the same units as those of the evolutionary distances used to infer the phylogenetic tree. . Sequence alignment of the ocellatin peptides from L. insularum and L. nesiotus with the orthologous peptides from other Leptodactylus species isolated to-date. Gaps denoted by (*) are introduced into certain sequences in order to maximize the structural similarity of the peptides. Shading is used to indicate the amino acid residues that have been very strongly conserved. a Denotes C-terminal α-amidation.

Discussion
This study has described the purification of five peptides from norepinephrine-stimulated skin secretions of L. insularum and four peptides from skin secretions of L. nesiotus. Determination of the primary structures of the peptides revealed that they represent orthologues of the ocellatin frog skin peptides named after the first member to be identified from a skin secretion of L. ocellatus [19]. The peptidomic approach (reversed-phase HPLC combined with MALDI-TOF mass spectrometry and automated Edman degradation) allowed the characterization of all of the peptides that were present in major abundance in the secretions. The advantage of the peptidomic approach is that those peptides with low or no antimicrobial activity would have been easily missed if the more traditional bioassay-guided method was utilized for identification of the HDPs [9].
Skin secretions of L. insularum contained two truncated (1-16) fragments of ocellatin-1I and -2I ( Figure 3). The samples from this species were collected, handled, and processed in the author's laboratory at the same time as those from L. nesiotus, but no such fragments were detected in the secretions from the latter. The presence of shorter ocellatin-related peptides appears to be a common feature for many of the Leptodactylus frogs studied to-date, irrespective of the method used for collection of the secretions [20,22,28,29]. It is unclear whether the ocellatin fragments originate from the proteolytic processing of full-length ocellatins by the peptidases that are present in skin secretions or are products of different genes.
Frog skin HDPs are associated with extreme structural hypervariability and several studies (reviewed in [15]) have shown that comparisons of their amino acid sequences have proved to be of value in elucidating the evolutionary history and phylogenetic relationships of frogs within a particular genus. Such analyses can be used in conjunction with those based upon comparisons of nucleotide sequences of orthologous genes and morphological criteria. Cladistic analysis based upon the primary structures of the ocellatins from Leptodactylus frogs supports the conclusion based upon morphological Antibiotics 2020, 9, 718 8 of 15 criteria [18] that L. nesiotus and L. validus are closely related phylogenetically, whereas L. insularum is most closely related to L. ocellatus.
It is well-documented that most of the ocellatins isolated to-date show low hemolytic activity and display only weak antimicrobial activity targeting preferentially Gram-negative bacteria [21,22,24,26,32]. However, ocellatin 4, isolated from L. ocellatus, displayed broad-spectrum antimicrobial activity by inhibiting both E. coli and S. aureus (MICs = 64 µM) [20] and it was also strongly hemolytic (LC 50 = 14.3 µM). Ocellatin-related fragments may contribute to the general protection exerted by the full-length peptides, as some of them have been documented to possess various degrees of antimicrobial and hemolytic activities [27,33]. The in vitro antimicrobial and hemolytic activities of synthetic replicates of the ocellatins -1I, -2I, -1N, and -3N (selected on the basis of their higher hydrophobicities) were investigated using a range of reference bacteria, including drug-resistant bacterial strains, and mouse erythrocytes ( Table 2). Ocellatin-3N isolated from skin secretions of L. nesiotus was the only peptide that displayed broad-spectrum antimicrobial activity (MICs = 31.25-62.5 µM) against Gram-positive and Gram-negative bacteria, including drug-resistant strains. The hemolytic activity of ocellatin-3N (LC 50 = 98 µM) was appreciably greater than the corresponding activities of fallaxin (LC 50 > 200 µM) [22], pentadactylin (LC 50 > 400 µM) [23], and laticeptin (LC 50 > 400 µM) [25]. However, the peptide, while displaying comparable activity against a range of Gram-positive and Gram-negative bacteria to that of ocellatin 4 [20], was nearly seven-fold less hemolytic. The cytotoxicity of ocellatin-3N against cell lines derived from human tissues, such as A549 alveolar basal epithelial cells and HUVEC umbilical vein endothelial cells, will be addressed in future studies. In addition, the activity of the peptide against reference strains and clinical isolates of opportunist yeast pathogens, such as Candida albicans and Candida parapsilosis, will be investigated.
The factors that determine the activity of any antimicrobial peptide are the helicity, amphipathicity, hydrophobicity, and charge [39][40][41][42]. These parameters are strongly interrelated, which makes predictions based on the amino acid sequence of the peptide antimicrobial activity and mode of action difficult. Studies with model α-helical peptides have shown that to exert activity against Gram-positive bacteria, the peptides need to adopt stabilized amphipathic conformation, while that is not always needed in the case of Gram-negative bacteria [39]. Similarly, it has been demonstrated that the more amphipathic a peptide is, the more hemolytic it becomes for human erythrocytes [43]. As is the case for the vast majority of frog skin antimicrobial peptides, the ocellatins from L. insularum and L. nesiotus are cationic (charge at pH 7 between +1 and +3). In particular, ocellatin-3N has an overall cationic charge of +2, which could facilitate electrostatic interactions with the negatively charged bacterial cell membranes. Two regions of helicity were predicted for this peptide between amino acid residues 4-11 and 13-18 (Table 1). A Schiffer and Edmundson [44] wheel projectionof ocellatin-3N ( Figure 6) demonstrated that the amphipathicity of the α-helix is high, with the polar Asp 4 , Lys 7 , and Lys 11 residues aligning on one face of the helix and the hydrophobic Ile 2 , Leu 6 , Leu 9 , Val 13 , and Leu 17 residues aligning on the opposite face. In addition, ocellatin-3N was the most hydrophobic of peptides tested in this study, with a GRAVY value of 0.911. It is suggested, therefore, that the observed potency of ocellatin-3N against Gram-positive bacteria and the hemolytic activity are a consequence of the combination of a high degree of amphipathicity and hydrophobicity of the peptide. Although ocellatin-1N is even more cationic than ocellatin-3N, its hydrophobicity is low. Similarly, Figure 6 demonstrates that the amphipathicities of ocellatin-1I and -2I are low, which accounts for the weak antimicrobial activity of these three peptides.
most hydrophobic of peptides tested in this study, with a GRAVY value of 0.911. It is suggested, therefore, that the observed potency of ocellatin-3N against Gram-positive bacteria and the hemolytic activity are a consequence of the combination of a high degree of amphipathicity and hydrophobicity of the peptide. Although ocellatin-1N is even more cationic than ocellatin-3N, its hydrophobicity is low. Similarly, Figure 6 demonstrates that the amphipathicities of ocellatin-1I and -2I are low, which accounts for the weak antimicrobial activity of these three peptides. Bessa et al. [45] reported that ocellatins isolated from L. pustulatus acted preferentially on clinical isolates of multidrug-resistant P. aeruginosa (MICs 16-256 μg/mL) compared with reference strains (MICs > 520 μg/mL). In particular, ocellatin-PT3 displayed an ability to inhibit biofilm formation (concentrations 4-8 times higher than the MIC) and showed synergistic effects with the antibiotics ciprofloxacin and ceftazidime, so the peptide was proposed as a promising lead molecule for the design and development of novel therapeutic agents against drug-resistant P. aeruginosa biofilms.
However, the clinical utility of antimicrobial peptides is limited by their short half-life in circulation and their toxicities against human cells. Several strategies have been employed to increase the stability of HDPs, including the substitution of amino acid residues by D-isomers and unnatural amino acids; modification of the peptide termini; dimerization and multimerization of the peptide; cyclization; conjugation with polymers, sugars, and albumin; and the use of peptidase inhibitors [46]. Bessa et al. [45] reported that ocellatins isolated from L. pustulatus acted preferentially on clinical isolates of multidrug-resistant P. aeruginosa (MICs 16-256 µg/mL) compared with reference strains (MICs > 520 µg/mL). In particular, ocellatin-PT3 displayed an ability to inhibit biofilm formation (concentrations 4-8 times higher than the MIC) and showed synergistic effects with the antibiotics ciprofloxacin and ceftazidime, so the peptide was proposed as a promising lead molecule for the design and development of novel therapeutic agents against drug-resistant P. aeruginosa biofilms.
However, the clinical utility of antimicrobial peptides is limited by their short half-life in circulation and their toxicities against human cells. Several strategies have been employed to increase the stability of HDPs, including the substitution of amino acid residues by D-isomers and unnatural amino acids; modification of the peptide termini; dimerization and multimerization of the peptide; cyclization; conjugation with polymers, sugars, and albumin; and the use of peptidase inhibitors [46]. Similarly, the cationicity, hydrophobicity, and amphipathicity of HDPs may be selectively manipulated to increase their antimicrobial potency, while simultaneously reducing their cytotoxicity to mammalian cells [41]. Moreover, the use of suitable carriers and optimized delivery systems, such as inorganic materials, polymers, and self-assembly lipid-based and nanomaterial-based structures, has been reported for both the systemic and local application of AMPs (reviewed in [47,48]). Such approaches would be expected to increase AMP stability, controlled release, and reduced toxicity and simultaneously minimize the potential side effects and/or overcome undesired host immune responses, thus alleviating many of the challenges that AMPs face as therapeutic agents. In this light, we propose future structure-activity studies that will involve the synthesis of long-acting analogs of ocellatin-3N with increased broad-spectrum activity against multidrug-resistant clinical isolates of pathogenic microorganisms and the ability to inhibit biofilm formation.

Collection of Skin Secretions
Relevant permits approving the collection and sampling of live animals were granted by the Wildlife Section, Forestry Division, Trinidad (Special Game License with nationwide validity was issued on 21 June 2016) and by the University of the West Indies (UWI) Campus Ethics Committee (CEC234/07/16). Adult and sub-adult L. insularum frogs (n = 9; SVL 55-85 mm; weight 13.7-74.9 g; sex not determined) and sub-adult specimens of L. nesiotus (n = 7; SVL 21-28 mm; weight 1.1-1.9 g; sex not determined) were collected at 10 • 4 49" N, 61 • 53 25" W in Cedros (Icacos, Trinidad) in May 2019. Species identification was based on audible calls and visual characteristics [35]. The animals were taken to a nearby base for the collection of skin secretions, which was carried out by authorized investigators. The L. insularum frogs were injected via the dorsal lymph sac with norepinephrine hydrochloride (NE; 40 nmol/g body weight), as previously described [49]. The much smaller L. nesiotus frogs were immersed in distilled water (130 mL) containing 40 nmol/g body weight NE for 15 min, as described [49]. The collection solutions were acidified by the addition of concentrated hydrochloric acid (final concentration 1%, v/v) and immediately frozen for transfer to Ulster University. The frogs were monitored closely over a period of a few hours for any signs of distress and were subsequently released unharmed at the site of collection.

Purification of the Peptides
The solutions containing the secretions from each animal were pooled separately and concentrated by passage at a flow rate of approximately 2 mL/min through nine (L. insularum) and three (L. nesiotus) Sep-Pak C-18 cartridges (Waters Associates, Milford, MA, USA) connected in series. Bound material was eluted with acetonitrile/water/trifluoroacetic acid (TFA) (70.0:29.9:0.1, v/v/v) and freeze-dried. The material was redissolved in 0.1% (v/v) TFA/water (2 mL) and injected onto a semipreparative (1.0 cm × 25 cm) Vydac 218TP510 (C-18) reversed-phase HPLC column (Grace, Deerfield, IL, USA) equilibrated with 0.1% (v/v) TFA/water at a flow rate of 2 mL/min. The following linear gradients were used to elute the peptides: 0% to 21% (v/v) acetonitrile over 10 min, followed by 21% to 63% (v/v) over 60 min. Absorbance was monitored at 214 nm and fractions (1 min) were collected using a BioRad 2110 fraction collector.

Structural Characterization
The monoisotopic molecular masses of the purified ocellatins were determined by MALDI-TOF mass spectrometry using an UltrafleXtreme instrument (Bruker Daltonik, Bremen, Germany). Full details of the procedure, including calibration of the instrument with peptides of a known molecular mass in the 1-4 kDa range, have been provided [50,51]. The accuracy of mass determinations was <0.02%. The primary structures of the purified peptides were determined by automated Edman degradation using an Applied Biosystems model 494 Procise sequenator (Foster City, CA, USA).

Synthetic Peptides
The four ocellatin peptides used in this study for an evaluation of antimicrobial and hemolytic activities were supplied in crude form by EZBiolab Inc. (Carmel, IN, USA). The peptides were purified by reversed-phase HPLC on a (2.2 cm × 25 cm) Vydac 218TP1022 (C-18) column equilibrated with acetonitrile/water/TFA (35.0/64.9.9/0.1, v/v/v) at a flow rate of 6 mL/min. The concentration of acetonitrile was raised to 63% (v/v) over 60 min using a linear gradient. Absorbance was measured at 214 nm and the major peak in the chromatogram was collected by hand. The identities of the peptides were confirmed by electrospray mass spectrometry and their final purities were estimated to be >98%.

Antimicrobial Assays
Minimum inhibitory concentrations (MICs) were determined in duplicate by a standard double dilution method according to CSLI guidelines [52] using 96-well microtiter cell-culture plates, as previously described [49]. All reference strain bacteria, including S. aureus (ATCC 12600), E. faecium and ciprofloxacin for the sensitive K. pneumoniae strain, P. aeruginosa, and S. typhimurium), in order to monitor the validity and reproducibility of the assays. The published antibiotic sensitivity/resistance profiles for all bacterial strains were confirmed in the authors' laboratory prior to setting up the MIC experiments.

Hemolysis Assay
All procedures involving mice were approved by Ulster University (UU) Animal Ethics Review Committee and were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and EU Directive 2010/63EU for animal experiments. Hemolytic activity against erythrocytes from NIH Swiss female mice was determined as previously described [22]. Control incubations were carried out in parallel in Krebs Ringer Bicarbonate buffer only or with 1% v/v Triton-X100, in order to determine the absorbance associated with 0% and 100% hemolysis, respectively. The LC 50 value was taken as the mean concentration of peptide producing 50% hemolysis in three independent experiments.

Cladistic Analysis
The optimum phylogenetic tree was constructed using the neighbor-joining method [53]. The evolutionary distances were computed using the Poisson correction method [54] and are given as the number of amino acid substitutions per site. All positions containing alignment gaps and missing amino acid residues were only eliminated in pairwise sequence comparisons (pairwise deletion option). Phylogenetic analyses were conducted in MEGA X [55].

Conclusions
In contrast to the majority of previously isolated ocellatin-related peptides with well-documented weak antimicrobial activity towards Gram-negative bacteria only, ocellatin-3N from skin secretions of the Caribbean frog L. nesiotus displays broad spectrum activity against a range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains. Consequently, this peptide could serve as a template for development into an antimicrobial agent with an improved potency and reduced cytotoxicity for red blood cells. In addition, this study has contributed to our understanding of amphibian biodiversity in Trinidad and the primary structures of the newly characterized ocellatin-related peptides provide insight into the phylogenetic relationships amongst Leptodactylid frogs.