Purification, conformational analysis and cytotoxic activities of host-defense peptides from the Tungara frog Engystomops pustulosus (Leptodactylidae; Leiuperinae).

The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited  regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol-water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.

Antimicrobial, cytotoxic, and insulin-releasing activities of the amphibian host-defense peptide ocellatin-3N and its L-lysine-substituted analogs.

The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2 ), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp4 →Lys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 µM) compared with the naturally occurring peptide. The substitution Ala18 →Lys and the double substitution Asp4 →Lys and Ala18 →Lys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC50 values in the range of 12-20 µM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC50 = 20 µM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 µM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca2+ ] in BRIN-BD11 cells when incubated at a concentration of 1 µM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.

Peptidomic analysis in the discovery of therapeutically valuable peptides in amphibian skin secretions.

Introduction: The emergence of multidrug-resistant microorganisms has necessitated a search for new antimicrobial agents. Skin secretions of many frog species contain peptides that possess potent, broad-spectrum antibacterial and antifungal activities and so show promise for development into anti-infective agents. Several such peptides also possess cytokine-mediated anti-inflammatory properties and a range of anti-diabetic activities.Areas covered: A peptidomic approach, involving reversed-phase HPLC and MALDI mass spectrometry, to the comprehensive identification of peptides of potential therapeutic importance in frog skin secretions is described and its advantages over analyses involving bioassays discussed. Peptidomic studies relating to the characterization of peptides with demonstrated antimicrobial, anti-inflammatory and anti-diabetic properties in skin secretions of frogs belonging to the extensive Pipidae and Ranidae families are reviewed.Expert commentary: The initial promise of frog skin peptides as agents to treat infections produced by drug-resistant microorganisms has not been fulfilled although topical applications to treat skin diseases and a role in promoting wound healing remains a possibility. Future directions are more likely to involve the application of such peptides in the treatment of patients with sepsis and related inflammatory conditions and as a component of a therapeutic regime for Type 2 diabetes.

Immunomodulatory, insulinotropic, and cytotoxic activities of phylloseptins and plasticin-TR from the Trinidanian leaf frog Phyllomedusa trinitatis.

The aim of the study was to determine the in vitro immunomodulatory, cytotoxic, and insulin-releasing activities of seven phylloseptin-TR peptides and plasticin-TR, first isolated from the frog Phyllomedusa trinitatis. The most cationic peptides, phylloseptin-1.1TR and phylloseptin-3.1TR, showed greatest cytotoxic potency against A549, MDA-MB231, and HT-29 human tumor-derived cells and against mouse erythrocytes. Phylloseptin-4TR was the most hydrophobic and the most effective peptide at inhibiting production of the proinflammatory cytokines TNF-α and IL-1ß by mouse peritoneal cells but was without effect on production of the antiinflammatory cytokine IL-10. Phylloseptin-2.1TR and phylloseptin-3.3TR were the most effective at stimulating the production of IL-10. The noncytotoxic peptide, plasticin-TR, inhibited production of TNF-α and IL-1ß but was without effect on IL-10 production. The results of CD spectroscopy suggest that the different properties of plasticin-TR compared with the immunostimulatory activities of the previously characterized plasticin-L1 from Leptodactylus laticeps may arise from greater ability of plasticin-TR to oligomerize and adopt a stable helical conformation in a membrane-mimetic environment. All peptides stimulated release of insulin from BRIN-BD11 rat clonal ß cells with phylloseptin-3.2TR being the most potent and effective and phylloseptin-2.1TR the least effective suggesting that insulinotropic potency correlates inversely with helicity. The study has provided insight into structure-activity relationships among the phylloseptins. The combination of immunomodulatory and insulinotropic activities together with low cytotoxicity suggests that phylloseptin-3.3TR and plasticin-TR may represent templates for the development of agents for use in antiinflammatory and type 2 diabetes therapies.

Purification, Conformational Analysis, and Properties of a Family of Tigerinin Peptides from Skin Secretions of the Crowned Bullfrog Hoplobatrachus occipitalis.

Four host-defense peptides belonging to the tigerinin family (tigerinin-1O: RICTPIPFPMCY; tigerinin-2O: RTCIPIPLVMC; tigerinin-3O: RICTAIPLPMCL; and tigerinin-4O: RTCIPIPPVCF) were isolated from skin secretions of the African crowned bullfrog Hoplobatrachus occipitalis. In aqueous solution at pH 4.8, the cyclic domain of tigerinin-2O adopts a rigid amphipathic conformation that incorporates a flexible N-terminal tail. The tigerinins lacked antimicrobial (MIC > 100 µM) and hemolytic (LC50 > 500 µM) activities but, at a concentration of 20 µg/mL, significantly (P < 0.05) inhibited production of interferon-γ (IFN-γ) by peritoneal cells from C57BL/6 mice without affecting production of IL-10 and IL-17. Tigerinin-2O and -4O inhibited IFN-γ production at concentrations as low as 1 µg/mL. The tigerinins significantly (P ≤ 0.05) stimulated the rate of insulin release from BRIN-BD11 clonal ß-cells without compromising the integrity of the plasma membrane. Tigerinin-1O was the most potent (threshold concentration 1 nM) and the most effective (395% increase over basal rate at a concentration of 1 µM). Tigerinin-4O was the most potent and effective peptide in stimulating the rate of glucagon-like peptide-1 release from GLUTag enteroendocrine cells (threshold concentration 10 nM; 289% increase over basal rate at 1 µM). Tigerinin peptides have potential for development into agents for the treatment of patients with type 2 diabetes.

Multifunctional host-defense peptides isolated from skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae).

Five host-defense peptides (figainin 2PL, hylin PL, raniseptin PL, plasticin PL, and peptide YL) were isolated from norepinephrine-stimulated skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae) collected in Trinidad. Raniseptin PL (GVFDTVKKIGKAVGKFALGVAKNYLNS.NH2) and figainin 2PL (FLGTVLKLGKAIAKTVVPMLTNAMQPKQ. NH2) showed potent and rapid bactericidal activity against a range of clinically relevant Gram-positive and Gram-negative ESKAPE + pathogens and Clostridioides difficile. The peptides also showed potent cytotoxic activity (LC50 values < 30 µM) against A549, MDA-MB-231 and HT29 human tumor-derived cell lines but appreciably lower hemolytic activity against mouse erythrocytes (LC50 = 262 ± 14 µM for raniseptin PL and 157 ± 16 µM for figainin 2PL). Hylin PL (FLGLIPALAGAIGNLIK.NH2) showed relatively weak activity against microorganisms but was more hemolytic. The glycine-leucine-rich peptide with structural similarity to the plasticins (GLLSTVGGLVGGLLNNLGL.NH2) and the non-cytotoxic peptide YL (YVPGVIESLL.NH2) lacked antimicrobial and cytotoxic activities. Hylin PL, raniseptinPL and peptide YL stimulated the rate of release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥100 nM. Peptide YL was the most effective (2.3-fold increase compared with basal rate at 1 µM concentration) and may represent a template for the design of a new class of incretin-based anti-diabetic drugs.

Transformation of the naturally occurring frog skin peptide, alyteserin-2a into a potent, non-toxic anti-cancer agent.

Alyteserin-2a (ILGKLLSTAAGLLSNL.NH(2)) is a cationic, amphipathic α-helical cell-penetrating peptide, first isolated from skin secretions of the midwife toad Alytes obstetricans. Structure-activity relationships were investigated by synthesizing analogs of alyteserin-2a in which amino acids on the hydrophobic face of the helix were replaced by L-tryptophan and amino acids on the hydrophilic face were replaced by one or more L-lysine or D-lysine residues. The Trp-containing peptides display increased cytotoxic activity against non-small cell lung adenocarcinoma A549 cells (up to 11-fold), but hemolytic activity against human erythrocytes increases in parallel. The potency of the N15K analog against A549 cells (LC(50) = 13 µM) increases sixfold relative to alyteserin-2a and the therapeutic index (ratio of LC(50) for erythrocytes and tumor cells) increases twofold. Incorporation of a D-Lys(11) residue into the N15K analog generates a peptide that retains potency against A549 cells (LC(50) = 15 µM) but whose therapeutic index is 13-fold elevated relative to the native peptide. [G11k, N15K] alyteserin-2a is also active against human hepatocarcinoma HepG2 cells (LC(50) = 26 µM), breast adenocarcinoma MDA-MB-231 cells (LC(50) = 20 µM), and colorectal adenocarcinoma HT-29 cells (LC(50) = 28 µM). [G11k, N15K] alyteserin-2a, in concentrations as low as 1 µg/mL, significantly (P < 0.05) inhibits the release of the immune-suppressive cytokines IL-10 and TGF-ß from unstimulated and concanavalin A-stimulated peripheral blood mononuclear cells. The data suggest a strategy of increasing the cationicity while reducing the helicity of naturally occurring amphipathic α-helical peptides to generate analogs with improved cytotoxicity against tumor cells but decreased activity against non-neoplastic cells.

Evaluation of the skin peptide defenses of the Oregon spotted frog Rana pretiosa against infection by the chytrid fungus Batrachochytrium dendrobatidis.

Population declines due to amphibian chytridiomycosis among selected species of ranid frogs from western North America have been severe, but there is evidence that the Oregon spotted frog, Rana pretiosa Baird and Girard, 1853, displays resistance to the disease. Norepinephrine-stimulated skin secretions were collected from a non-declining population of R. pretiosa that had been exposed to the causative agent Batrachochytrium dendrobatidis. Peptidomic analysis led to identification and isolation, in pure form, of a total of 18 host-defense peptides that were characterized structurally. Brevinin-1PRa, -1PRb, -1PRc, and -1PRd, esculentin-2PRa and -PRb, ranatuerin-2PRa, -2PRb, -2PRc, and -2PRe, temporin-PRb and -PRc were identified in an earlier study of skin secretions of frogs from a different population of R. pretiosa known to be declining. Ranatuerin-2PRf, -2PRg, -2PRh, temporin-PRd, -PRe, and -PRf were not identified in skin secretions from frogs from the declining population, whereas temporin-PRa and ranatuerin-2PRd, present in skin secretions from the declining population, were not detected in the current study. All purified peptides inhibited the growth of B. dendrobatidis zoospores. Peptides of the brevinin-1 and esculentin-2 families displayed the highest potency (minimum inhibitory concentration = 6.25-12.5 µM). The study provides support for the hypothesis that the multiplicity and diversity of the antimicrobial peptide repertoire in R. pretiosa and the high growth-inhibitory potency of certain peptides against B. dendrobatidis are important in conferring a measure of resistance to fatal chytridiomycosis.

Purification, Conformational Analysis and Cytotoxic Activities of Host-Defense Peptides from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae).

Frogs from the extensive amphibian family Hylidae are a rich source of peptides with therapeutic potential. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae) collected in Trinidad led to the isolation and structural characterization of five host-defense peptides with limited structural similarity to figainin 2 and picturin peptides from other frog species belonging to the genus Boana. In addition, the skin secretions contained high concentrations of tryptophyllin-BN (WRPFPFL) in both C-terminally α-amidated and non-amidated forms. Figainin 2BN (FLGVALKLGKVLG KALLPLASSLLHSQ) and picturin 1BN (GIFKDTLKKVVAAVLTTVADNIHPK) adopt α-helical conformations in trifluroethanol-water mixtures and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). The CD data also indicate contributions from turn structures. Both peptides and picturin 2BN (GLMDMLKKVGKVALT VAKSALLP) inhibited the growth of clinically relevant Gram-negative and Gram-positive bacteria with MIC values in the range 7.8-62.5 µM. Figainin 2BN was potently cytotoxic to A549, MDA-MB-231 and HT-29 human tumor-derived cells (LC50 = 7-14 µM) but displayed comparable potency against non-neoplastic HUVEC cells (LC50 = 15 µM) indicative of lack of selectivity for cancer cells.

Peptidomic analysis of the host-defense peptides in skin secretions of the Amazon River frog Lithobates palmipes (Ranidae).

Skin secretions of certain frog species represent a source of host-defense peptides (HDPs) with therapeutic potential and their primary structures provide insight into taxonomic and phylogenetic relationships. Peptidomic analysis was used to characterize the HDPs in norepinephrine-stimulated skin secretions from the Amazon River frog Lithobates palmipes (Ranidae) collected in Trinidad. A total of ten peptides were purified and identified on the basis of amino acid similarity as belonging to the ranatuerin-2 family (ranatuerin-2PMa, -2PMb, -2PMc, and-2PMd), the brevinin-1 family (brevinin-1PMa, -1PMb, -1PMc and des(8-14)brevinin-1PMa) and the temporin family (temporin-PMa in C-terminally amidated and non-amidated forms). Deletion of the sequence VAAKVLP from brevinin-1PMa (FLPLIAGVAAKVLPKIFCAISKKC) in des[(8-14)brevinin-1PMa resulted in a 10-fold decrease in potency against Staphylococcus aureus (MIC = 31 µM compared with 3 µM) and a > 50-fold decrease in hemolytic activity but potency against Echerichia coli was maintained (MIC = 62.5 µM compared with 50 µM). Temporin-PMa (FLPFLGKLLSGIF.NH2) inhibited growth of S. aureus (MIC = 16 µM) but the non-amidated form of the peptide lacked antimicrobial activity. Cladistic analysis based upon the primary structures of ranaturerin-2 peptides supports the division of New World frogs of the family Ranidae into the genera Lithobates and Rana. A sister-group relationship between L. palmipes and Warszewitsch's frog Lithobates warszewitschii is indicated within a clade that includes the Tarahumara frog Lithobates tarahumarae. The study has provided further evidence that peptidomic analysis of HDPs in frog skin secretions is a valuable approach to elucidation of the evolutionary history of species within a particular genus.

Identification of an Antimicrobial Peptide from the Venom of the Trinidad Thick-Tailed Scorpion Tityus trinitatis with Potent Activity against ESKAPE Pathogens and Clostridioides difficile.

Envenomation by the Trinidad thick-tailed scorpion Tityus trinitatis may result in fatal myocarditis and there is a high incidence of acute pancreatitis among survivors. Peptidomic analysis (reversed-phase HPLC followed by MALDI-TOF mass spectrometry and automated Edman degradation) of T. trinitatis venom led to the isolation and characterization of three peptides with antimicrobial activity. Their primary structures were established asTtAP-1 (FLGSLFSIGSKLLPGVFKLFSRKKQ.NH2), TtAP-2 (IFGMIPGLIGGLISAFK.NH2) and TtAP-3 (FFSLIPSLIGGLVSAIK.NH2). In addition, potassium channel and sodium channel toxins, present in the venom in high abundance, were identified by CID-MS/MS sequence analysis. TtAP-1 was the most potent against a range of clinically relevant Gram-positive and Gram-negative aerobes and against the anaerobe Clostridioides difficile (MIC = 3.1-12.5 µg/mL). At a concentration of 1× MIC, TtAP-1 produced rapid cell death (<15 min against Acinetobacter baumannii and Staphylococcus aureus). The therapeutic potential of TtAP-1 as an anti-infective agent is limited by its high hemolytic activity (LC50 = 18 µg/mL against mouse erythrocytes) but the peptide constitutes a template for the design of analogs that maintain the high bactericidal activity against ESKAPE pathogens but are less toxic to human cells. It is suggested that the antimicrobial peptides in the scorpion venom facilitate the action of the neurotoxins by increasing the membrane permeability of cells from either prey or predator.

Analogues of the frog skin peptide alyteserin-2a with enhanced antimicrobial activities against Gram-negative bacteria.

The emergence of strains of multidrug-resistant Gram-negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin-2a (ILGKLLSTAAGLLSNL.NH2) is a cationic, α-helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin-2a while maintaining amphipathicity by the substitution Gly¹¹ → Lys enhanced the potency against both Gram-negative and Gram-positive bacteria by between fourfold and 16-fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC50=24 µM). Antimicrobial potency was increased further by the additional substitution Ser7 →Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC50=38 µM). However, the peptide containing D-lysine at positions 7 and 11 showed high potency against a range of Gram-negative bacteria, including multidrug-resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 µM) but appreciably lower haemolytic activity (LC50=185 µM) and cytotoxicity against A549 human alveolar basal epithelial cells (LC50=65 µM). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics.

Host-defense peptides in skin secretions of African clawed frogs (Xenopodinae, Pipidae).

African clawed frogs of the Xenopodinae (Xenopus+Silurana) constitute a well-defined system in which to study the evolutionary trajectory of duplicated genes and are a source of antimicrobial peptides with therapeutic potential. Allopolyploidization events within the Xenopodinae have given rise to tetraploid, octoploid, and dodecaploid species. The primary structures and distributions of host-defense peptides from the tetraploid frogs Xenopus borealis, Xenopus clivii, Xenopus laevis, Xenopus muelleri, "X. muelleri West", and Xenopus petersii may be compared with those from the octoploid frogs Xenopus amieti and X. andrei. Similarly, components in skin secretions from the diploid frog Silurana tropicalis may be compared with those from the tetraploid frog Silurana paratropicalis. All Xenopus antimicrobial peptides may be classified in the magainin, peptide glycine-leucine-amide (PGLa), caerulein-precursor fragment (CPF), and xenopsin-precursor fragment (XPF) families. However, the numbers of paralogs from the octoploid frogs were not significantly greater than the corresponding numbers from the tetraploid frogs. Magainins were not identified in skin secretions of Silurana frogs and the multiplicity of the PGLa, CPF, and XPF peptides from S. paratropicalis was not greater than that of S. tropicalis. The data indicate, therefore, that nonfunctionalization (gene silencing) has been the most common fate of antimicrobial peptide genes following polyploidization. While some duplicated gene products retain high antimicrobial potency (subfunctionalization), the very low activity of others suggests that they may be evolving towards a new biological role (neofunctionalization). CPF-AM1 and PGLa-AM1 from X. amieti show potential for development into anti-infective agents for use against antibiotic-resistant gram-negative bacteria.

Caerulein-and xenopsin-related peptides with insulin-releasing activities from skin secretions of the clawed frogs, Xenopus borealis and Xenopus amieti (Pipidae).

Caerulein-related peptides were identified in norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus borealis and the octoploid frog Xenopus amieti using negative ion electrospray mass spectrometry and their primary structures determined by positive ion tandem (MS/MS) mass spectrometry. X. borealis caerulein-B1 (pGlu-Gln-Asp-Tyr(SO(3))-Gly-Thr-Gly-Trp-Met-Asp-Phe.NH2) contains an additional Gly(5) residue compared with X. laevis caerulein and caerulein-B2 (pGlu-Asp-Tyr(SO(3))-Thr-Gly-Trp-Met-Asp-Phe.NH2) contains a Gln(2) deletion. X. amieti caerulein was identical to the X. laevis peptide. In addition, xenopsin, identical to the peptide from X. laevis, together with xenopsin-AM2 (pGlu-Gly-Arg-Arg-Pro-Trp-Ile- Leu) that contains the substitution Lys(3)→Arg were isolated from X. amieti secretions. X. borealis caerulein-B1, and X. amieti xenopsin and xenopsin-AM2 produced significant (P<0.05) and concentration-dependent stimulations of insulin release from the rat BRIN-BD11 clonal ß cell line at concentrations ⩾30nM. The peptides did not stimulate the release of lactate dehydrogenase at concentrations up to 3µM demonstrating that the integrity of the plasma membrane had been preserved. While their precise biological role is unclear, the caerulein- and xenopsin-related peptides may constitute a component of the animal's chemical defenses against predators.

Cholinergic stimulation of the immune system protects against lethal infection by Salmonella enterica serovar Typhimurium.

SUMMARY: The cholinergic nervous system has been demonstrated to attenuate the inflammatory response during sepsis via the inhibitory action of acetylcholine (ACh) on macrophages. These findings were largely based on experimental sepsis models using endotoxin as the inducing agent. Herein, however, we report that the specific inhibition of acetylcholinesterase (AChE) renders animals more resistant to infection by a virulent strain of Salmonella enterica serovar Typhimurium, a Gram-negative enteric pathogen. Inhibition of AChE was induced by a subchronic exposure to paraoxon, a potent anti-cholinesterase metabolite of the organophosphorous compound parathion. Our findings indicate that inhibition of AChE enhanced survival of infected mice in a dose-dependent fashion and this correlated with efficient control of bacterial proliferation in target organs. Immunologically, inhibition of AChE enabled the animals to mount a more effective inflammatory anti-microbial response, and to secrete higher levels of interleukin-12, a key T helper type 1-promoting cytokine. The ACh-induced enhancement in resistance to infection was abrogated by co-administration of an oxime which can reactivate AChE. Hence, in a model of Gram-negative bacterial infection, cholinergic stimulation is shown to enhance the anti-microbial immune response leading to effective control of bacterial proliferation and enhanced animal survival.

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.

Selection of antimicrobial frog peptides and temporin-1DRa analogues for treatment of bacterial infections based on their cytotoxicity and differential activity against pathogens.

Cationic, amphipathic, α-helical host-defense peptides (HDPs) that are naturally secreted by certain species of frogs (Anura) possess potent broad-spectrum antimicrobial activity and show therapeutic potential as alternatives to treat infections by multidrug-resistant pathogens. Fourteen amphibian skin peptides and twelve analogues of temporin-1DRa were studied for their antimicrobial activities against clinically relevant human or animal skin infection-associated pathogens. For comparison, antimicrobial potencies of frog skin peptides against a range of probiotic lactobacilli were determined. We used the VITEK 2 system to define a profile of antibiotic susceptibility for the bacterial panel. The minimal inhibitory concentration (MIC) values of the naturally occurring temporin-1DRa, CPF-AM1, alyteserin-1c, hymenochirin-2B, and hymenochirin-4B for pathogenic bacteria were threefold to ninefold lower than the values for the tested probiotic strains. Similarly, temporin-1DRa and its [Lys4 ], [Lys5 ], and [Aib8 ] analogues showed fivefold to 6.5-fold greater potency against the pathogens. In the case of PGLa-AM1, XT-7, temporin-1DRa and its [D-Lys8 ] and [Aib13 ] analogues, no apoptosis or necrosis was detected in human peripheral blood mononuclear cells at concentrations below or above the MIC. Given the differential activity against commensal bacteria and pathogens, some of these peptides are promising candidates for further development into therapeutics for topical treatment of skin infections.

Modulation of macrophage proinflammatory functions by cytokine-expressing Salmonella vectors.

We previously reported that the intraperitoneal administration of recombinant strains of Salmonella enterica serovar Typhimurium, engineered to express murine IL-2 (designated GIDIL2) or IFN-gamma (GIDIFNgamma), induced a cytokine-specific modulation of the host innate immune response. Interestingly, the bacteria-expressed cytokines were not secreted, but instead were associated with the bacterial cytosol. To understand the mechanism by which these two transfectants influence immune cells, we investigated their effect on two macrophage populations, J774A.1 cell line and ex vivo isolated peritoneal macrophages (PM). The parental, cytokine-negative, Salmonella strain (designated BRD509E), was used as a control. The capacity of the bacterial strains to activate macrophages was assessed by modulation of surface expression of costimulatory molecules CD40, CD80 (B7-1) and CD86 (B7-2) and activation marker Ly-6A/E, and by induction of cytokine production. Our data revealed that GIDIFNgamma was the only strain capable of upregulating the expression of cell-surface markers. Moreover, infection of macrophages with GIDIFNgamma induced a stronger cytokine response in comparison with BRD509E or GIDIL2 strain, as demonstrated by the production of TNF-alpha, IL-6, IL-12/IL23p40 and NO. The ability of GIDIL2 and GIDIFNgamma strains to activate macrophages was not due to enhanced invasiveness, as their cellular invasion rates were 2-fold lower than the parental strain. Further investigation of cytokine expression by GIDIL2 and GIDIFNgamma strains showed that while the cytokines were not secreted, they were expressed on the bacterial surface suggesting that their effect on macrophages could be through a direct interaction with their receptors on target cells. This was confirmed by showing that cytochalasin D-treated macrophages, a treatment which effectively inhibited bacterial invasion, could be induced to secrete high levels of cytokines by GIDIFNgamma organisms. Our data demonstrate that cytokine-expressing bacteria modulate macrophage activation independently of their entry into cells and may explain the rapid action of these bacterial strains when injected systemically into susceptible mice.

Peptidomic analysis of the host-defense peptides in skin secretions of Rana graeca provides insight into phylogenetic relationships among Eurasian Rana species.

Peptidomic analysis of norepinephrine-stimulated skin secretions from the Greek stream frog Rana graeca Boulenger, 1891 led to the identification and structural characterization of a range of host-defense peptides. These comprised brevinin-1GRa, brevinin-1GRb and an N-terminally extended form of brevinin-1GRb, ranatuerin-2GR together with its oxidized form and (11-28) fragment, temporin-GRa, temporin-GRb and its non-amidated form, and a melittin-related peptide, MRP-GR and its (1-18) fragment. The most abundant peptide, MRP-GR significantly (P < 0.001) stimulated insulin release from BRIN-BD11 clonal ß-cells at concentrations ≥0.1 nM. Rana graeca (formerly Rana graeca graeca) and the morphologically similar Italian stream frog Rana italica Dubois, 1987 (formerly Rana graeca italica) were originally regarded as sub-species. However, the primary structures of the host defense peptides from both frogs support the claim based upon comparisons of the nucleotide sequences of S1 satellite DNA that R. graeca and R. italica are separate species. Cladistic analyses based upon the primary structures of the brevinin-1 and ranatuerin-2 peptides from Eurasian frogs indicate a close phylogenetic relationship between R. graeca and Rana latastei whereas R. italica is most closely related to Rana dalmatina.

Peptides from frog skin with potential for development into agents for Type 2 diabetes therapy.

Several frog skin peptides, first identified as result of their antimicrobial or immunomodulatory activities, have subsequently been shown to stimulate insulin release both in vitro and in vivo and so show potential for development into incretin-based drugs for treatment of patients with Type 2 diabetes mellitus. However, their therapeutic potential as anti-diabetic agents is not confined to this activity as certain frog skin-derived peptides, such as magainin-AM2 and CPF-SE1 and analogs of hymenochirin-1B, tigerinin-1R, and esculentin-2CHa, have been shown to increase insulin sensitivity, promote ß-cell proliferation, suppress pancreatic and circulating glucagon concentrations, improve the lipid profile, and selectively alter expression of genes involved in insulin secretion and action in mice with diet-induced obesity, insulin resistance and impaired glucose tolerance. This review assesses the therapeutic possibilities of peptides from frogs belonging to the Pipidae, Dicroglossidae, and Ranidae families, focusing upon work that has been carried out since 2014.