Purification and characterization of novel antimicrobial peptides from the skin secretion of Hylarana guentheri

Linear, amphipathic and cationic antimicrobial peptides have been previously reported from a wide range of amphibian species especially frogs of the genus Rana. Such antimicrobial peptides are attracting increasing attention in pharmacological applications because they mainly act by permeabilizing and disrupting the target cell or virion membranes with a low degree of resistance. The Guenther's frog, Hylarana guentheri, is a Chinese frog of the genus Rana that is widely distributed in Southern China. It is commonly the dominant amphibian species even where the amphibian population is declining. In this study, we describe the isolation, purification, structural and biological characterization of five novel peptides from H. guentheri frog skin secretions that possess antimicrobial activity, including brevinin-2GHa, brevinin-2GHb, brevinin-2GHc, temporin-GH and a novel antimicrobial peptide named guentherin. The cDNAs encoding two novel members of the brevinin-2 family, brevinin-2GHb and brevinin-2GHc were also subsequently cloned and sequenced.     

Purification and characterization of antimicrobial peptides from the skin of the North American green frog Rana clamitans

Ten peptides with differential growth-inhibitory activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli, and the yeast Candida albicans were isolated from an extract of the skin of a North American frog, the green frog Rana clamitans. Ranatuerin-1C (SMLSVLKNLGKVGLGLVACKINKQC), ranalexin-1Ca (FLGGLMKAFPALICAVTKKC), ranalexin-1Cb (FLGGLMKAFPAIICAVTKKC), ranatuerin-2Ca (GLFLDTLKGAAKDVAGKLLEGLKCKIAGC KP), and ranatuerin-2Cb (GLFLDTLKGLAGKLLQGLKCIKAGCKP), are members of three previously characterized families of antimicrobial peptides, first identified in the North American bullfrog Rana catesbeiana. In addition, five structurally related peptides (temporin-1Ca, -1Cb, -1Cc, -1Cd, and -1Ce), comprising 13 amino acid residues and containing a C-terminally alpha-amidated residue, belong to the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of Asian and European Ranid frogs, were not identified in the extract. The data support the hypothesis that the distribution and amino acid sequences of the skin antimicrobial peptides are valuable tools in the identification and classification of Ranid frogs.     

Purification and characterization of antimicrobial peptides from the skin secretions of the mink frog (Rana septentrionalis)

Skin secretions were obtained from male, female, and juvenile specimens of the mink frog (Rana septentrionalis) by electric stimulation and shown to contain 10 peptides that differentially inhibited the growth of microorganisms. The elution profiles of secretions from the three groups following reverse-phase HPLC were almost identical indicating that there were no major sexual or developmental differences in chemical composition. Four peptides of the brevinin-1 family, with potent antimicrobial activity and strong hemolytic activity, two members of ranatuerin-2 family and three members of the temporin family, were purified and characterized structurally. A 21-amino-acid C-terminally alpha-amidated peptide (GIWDTIKSMGKVFAGKILQNL.NH(2)) with broad-spectrum antimicrobial activity was also isolated from the skin secretions. This peptide shows limited structural similarity with the N-terminal region of brevinin-2 peptides previously isolated from R. temporaria skin but lacks the C-terminal cyclic heptapeptide domain associated with this family. Molecular and morphological data support the placement of R. septentrionalis in the R. catesbeiana species group, but analysis based upon the distribution of the molecular forms of the antimicrobial peptides is indicative of a closer phylogenetic relationship between R. septentrionalis and the frogs of the R. pipiens and R. boylii groups.     

Purification and characterization of bioactive peptides from skin extracts of Rana esculenta

The peptide fraction extracted by methanol from the skin of Rana esculenta, a species widely distributed in Western Europe, was investigated. The pharmacological activity found in the extract is attributable to the presence of authentic bradykinin, together with a shorter, partially active version of this molecule, des-Arg9-bradykinin. Also the bradykinin fragment 1-7 has been isolated, but it was inactive in our bioassay system. Moreover, a family of hydrophobic peptides has been purified and characterized, which appeared devoid of pharmacological activities when tested on smooth muscle preparations, but were provided with hemolytic activities.     

Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio

Eight peptides with differential growth–inhibitory activity against the gram-positive bacterium Staphylococcus aureus, the gram-negative bacterium Escherichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary structures of these antimicrobial peptides were different from previously characterized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previously characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranalexin families, first identified in the North American bullfrog, Rana catesbeiana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of other species of Ranid frogs, were not identified in the extracts. The ranatuerin-1 and ranalexin peptides showed broadest spectrum of antimicrobial activity whereas the temporins were active only against S. aureus. Synthetic replicates of temporin-1Gb (SILPTIVSFLSKFL.NH₂) and temporin-1Gd (FILPLIASFLSKFL.NH₂) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC₅₀=2.4±0.1 μM for temporin-1Gb and 2.3±0.2 μM for temporin-1Gd). The antimicrobial peptides that were isolated in extracts of the skin R. grylio were present in the same molecular forms in electrically-stimulated skin secretions of the animal demonstrating that the peptides are stored in the granular glands of the skin in their fully processed forms.     

Purification, molecular cloning, and antimicrobial activity of peptides from the skin secretion of the black-spotted frog, Rana nigromaculata

Antimicrobial peptides from a wide range of amphibian species, especially frogs of the genus Rana, have been characterised and are potential therapeutic agents. Here we describe the isolation, purification, and structural and biological characterisation of three novel antimicrobial peptides from the skin secretions of the black spotted frog, Rana nigromaculata, from Northeastern China. The peptides were identified as belonging to two known families: the temporin, which was first identified in R. nigromaculata from China, and the brevinin-2. Temporin-1RNa and temporin-1RNb both containing three positive charges and have a high potency against microorganisms (MIC: 3.13–8.3 μM against Gram-positive bacteria, 12.5–25.0 μM against Gram-negative bacteria, and 6.25–12.5 μM against Candida albicans) and a high haemolytic activity against human erythrocytes (HC₅₀: 100–150 μM). Brevinin-2RNa contains a single intra-disulphide bridge at the C-terminus that is active towards the tested Gram-positive bacteria but is not active against E. coli and P. aeruginosa. The cDNAs encoding three novel peptide precursors were also subsequently cloned from an R. nigromaculata skin cDNA library and sequenced. The precursors contain 58–72 amino acid residues, which include a conserved signal peptide, acidic propeptide, and the mature temporin-1RNa, temporin-1RNb and brevinin-2RNa. The CD spectra of temporin-1RNa and temporin-1RNb in water, 30 mM SDS and 50 % trifluoroethanol (TFE) indicated that both peptides adopted an aperiodic structure in water and an organised structure with an α-helical conformation in TFE and SDS solution. The conformational transition induced by TFE or SDS reflects the potential ability of temporin-1RNa and temporin-1RNb to interact with anionic membranes.     

Purification and characterization of antimicrobial peptides from the skin secretions of the carpenter frog Rana virgatipes (Ranidae, Aquarana)

The members of the Aquarana (or Rana catesbeiana species group) form a well-supported monophyletic clade but phylogenetic relationships between species within the group are incompletely understood. Peptides that differentially inhibited the growth of bacteria were purified from electrically stimulated skin secretions of the carpenter frog Rana virgatipes. Structural characterization identified members of the ranatuerin-2 (3 peptides) and temporin (3-peptides) families, previously found in the skins of R. catesbeiana, R. clamitans, R. grylio and R. septentrionalis. Ranalexin, a peptide previously found only in the Aquarana, was isolated together with a variant (FFGLHNLVPSMLCVVRKKC) that lacks the propensity to adopt an alpha-helical conformation and so was devoid of antimicrobial activity. Two C-terminally alpha-amidated peptides belonging to the brevinin-2 family were isolated from the skin secretions that, like an ortholog from R. septentrionalis, lacked the C-terminal cyclic heptapeptide domain associated with members of this family. Ranatuerin-1, previously isolated from R. catesbeiana, R. clamitans and R. grylio but absent from R. septentrionalis, was also not identified in R. virgatipes. Synthetic replicates of temporin-1Va (FLSSIGKILGNLL.NH2), temporin-IVb (FLSIIAKVLGSLF.NH2) and temporin-1Vc (FLPLVTMLLGKLF.NH2) potently inhibited growth of Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus). Temporin-1Va was also active against Gram-negative bacteria and the opportunistic yeast pathogen Candida albicans and had relatively weak hemolytic activity (LD50=120 microM) and may therefore represent a candidate for drug development. Our data support the placement of R. virgatipes in the Aquarana and indicate a closer phylogenetic relationship of R. virgatipes with R. septentrionalis than with R. catesbeiana, R. clamitans and R. grylio.     

Molecular cloning and functional characterization of novel antimicrobial peptides from the skin of brown frog, Rana zhenhaiensis

Rana zhenhaiensis, a species of brown frog, is widely distributed in central and south China. In the present study, a total of 14 cDNA sequences encoding eight novel antimicrobial peptides (AMPs) were cloned from the synthesized cDNAs of R. zhenhaiensis skin. The eight novel AMPs belong to four families: brevinin-1 (four peptides), brevinin-2 (one peptide), ranatuerin-2 (one peptide) and chensinin-1 (two peptides), five AMPs from the four families (brevinin-1ZHa, brevinin-1ZHb, brevinin-2ZHa, ranatuerin-2ZHa and chensinin-1ZHa) were chemically synthesized, their antimicrobial and hemolytic activities were examined. The results indicated that the five AMPs possess different antimicrobial and hemolytic activities. Of these, brevinin-2ZHa exhibited the strongest and most broad-spectrum antimicrobial activity. Furthermore, scanning electron microscopy (SEM) experiment was carried out to investigate the potential antimicrobial mechanism of chensinin-1ZHa. The result indicated that chensinin-1ZHa may exert its function through disruption of the bacterial membrane.     

Two novel antimicrobial peptides from skin secretions of the frog,Rana nigrovittata

Two novel antimicrobial peptides with similarity to brevinin‐2 family are purified and characterized from the skin secretions of the frog, Rana nigrovittata. Their amino acid sequences were determined as GAFGNFLKGVAKKAGLKILSIAQCKLSGTC (brevinin‐2‐RN1) and GAFGNFLKGVAKKAGLKILSIAQCKLFGTC (brevinin‐2‐RN2), respectively, by Edman degradation. Different from brevinin‐2, which is composed of 33 amino acid residues (aa), both brevinin‐2‐RN1 and ‐RN2 contain 30 aa. Five cDNA sequences (Genbank accession numbers, EU136465‐9) encoding precursors of brevinin‐2‐RN1 and ‐RN2 were screened from the skin cDNA library of R. nigrovittata. These precursors are composed of 72 aa including a predicted signal peptide, an acidic spacer peptide, and a mature brevinin‐2‐RN. Both brevinin‐2‐RN1 and ‐RN2 showed strong antimicrobial activities against gram‐positive and gram‐negative bacteria and fungi. The current work identified and characterized two novel antimicrobial peptides with unique primary structure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A new family of antimicrobial peptides from skin secretions of Rana pleuraden

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Guizhou region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the Yunnan frog, Rana pleuraden. Members of the new peptide family named pleurain-As are composed of 26 amino acids with a unique N-terminal sequence (SIIT) and a disulfide-bridged heptapeptide sequence (CRLYNTC). By BLAST search, pleurain-As had no significant similarity to any known peptides. Native and synthetic peptides showed antimicrobial activities against tested microorganisms including Gram-negative and Gram-positive bacteria and fungi. Twenty different cDNAs encoding pleurain-As were cloned from the skin cDNA library of R. pleuraden. The precursors of pleurain-As are composed of 69 amino acid residues including predicted signal peptides, acidic propieces, and cationic mature antimicrobial peptides. The preproregion of pleurain-A precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature pleurain-As are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. Furthermore, pleurain-As could exert antimicrobial capability against Helicobacter pylori. This is the first report of naturally occurring peptides with anti-H. pylori activity from Rana amphibians.     

Structure-activity relationships of antimicrobial peptides from the skin of Rana esculenta inhabiting in Korea

The anuran (frogs and toads) skin is a rich source of antimicrobial peptides that can be developed therapeutically. We searched the skin secretions of Korean Rana esculenta for antimicrobial peptides, and isolated two cationic peptides with antimicrobial activity and little hemolytic activity: a 46-residue peptide of the esculentin-1 family and a 24-residue peptide of the brevinin-1 family. Their sequences showed some differences from the esculentins-1 and brevinins-1 of European Rana esculenta, indicating that sequence diversification of anuran skin antimicrobial peptides can arise from differences in habitat as well as from species differences. The 46-residue peptide named esculentin-1c had broad antimicrobial activity, while the 24-residue peptide named brevinin-1Ed exhibited limited activity. The solution structure of brevinin-1Ed was in good agreement with that of other brevinin-1-like peptides, with an amphipathic alpha-helix spanning residues 3-20, stabilized in membrane-mimetic environments. The weak bioactivity of brevinin-1Ed was attributable to the unusual presence of an anionic amino acid in the middle of the helical hydrophilic face. This report contributes to world-wide investigations of the structure-activity relationships and evolutional diversification of anuran-skin antimicrobial peptides.     

黑眶蟾蜍皮肤抗菌肽的制备及其性质

收集黑眶蟾蜍皮肤分泌物,经Sephadex G-25去除大分子蛋白后,利用微量测定法进行抗菌活性分析。结果发现:黑眶蟾蜍皮肤分泌物对革兰氏阳性菌——金黄色葡萄球菌、枯草芽孢杆菌的抑制作用较强,对革兰氏阴性菌中的嗜水气单细胞菌也表现出较强的抑制作用,对溶藻弧菌、副溶血弧菌、河流弧菌、大肠杆菌的抑制相对较弱。利用胰蛋白酶对黑眶蟾蜍皮肤抗菌肽水解后,其抗菌活性消失。将黑眶蟾蜍皮肤抗菌肽在37~95℃和pH 2.5~5.0下保温,发现其抗菌活性成分对热及酸耐受性较强。黑眶蟾蜍皮肤分泌物在低浓度无溶血活性。     

Histamine-releasing and antimicrobial peptides from the skin secretions of the dusky gopher frog, Rana sevosa

Seven novel peptides were isolated from the skin secretions of the North American dusky gopher frog, Rana sevosa, on the basis of antimicrobial activity and histamine release from rat peritoneal mast cells. The peptides were purified to homogeneity using HPLC and characterized by electrospray ion-trap mass spectrometry, MALDI-TOF mass spectrometry and Edman sequencing. Bioinformatic analysis of primary structures revealed that the novel peptides could be assigned to four established families of ranid frog antimicrobial peptides, namely esculentin-1, esculentin-2, brevinin-1 and ranatuerin-2. The peptides were named in accordance with accepted terminology as ranatuerin 2SEa, etc., reflecting the peptide family name, the species of origin (SE for sevosa) and the isotype (a). Of major interest was the fact that brevinin 1SE displayed significant structural similarity to ponericin W5, an antibacterial venom peptide from the ant, Pachyconyla goeldii. This is a further example of amphibian skin defensive peptides showing striking structural similarities to peptides from insects. These data may shed some light on the functional biological relevance of defensive peptides that possess both antimicrobial and histamine-releasing activities.     

Solution structure of antimicrobial peptide esculentin-1c from skin secretion of Rana esculenta

Granular glands in the skins of frogs synthesize and secrete a remarkably diverse range of peptides capable of antimicrobial activity. These anuran skin antimicrobial peptides are commonly hydrophobic, cationic and form an amphipathic α-helix in a membrane mimetic solution. Recently, they have been considered as useful target molecules for developing new antibiotics drugs. Esculentin-1c is a 46-amino acid residue peptide isolated from skin secretions of the European frog, Rana esculenta. It displays the most potent antimicrobial activity among bioactive molecules. Esculentin-1c has the longest amino acids among all antimicrobial peptides. The present study solved the solution structure of esculentin-1c in TFE/water by NMR, for the first time. We conclude that this peptide is comprised of three α-helices with each helix showing amphipathic characteristics, which seems to be a key part for permeating into bacterial membranes, thus presenting antimicrobial activity.     

Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio

Eight peptides with differential growth–inhibitory activity against the gram-positive bacterium Staphylococcus aureus, the gram-negative bacterium Escherichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary structures of these antimicrobial peptides were different from previously characterized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previously characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranalexin families, first identified in the North American bullfrog, Rana catesbeiana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of other species of Ranid frogs, were not identified in the extracts. The ranatuerin-1 and ranalexin peptides showed broadest spectrum of antimicrobial activity whereas the temporins were active only against S. aureus. Synthetic replicates of temporin-1Gb (SILPTIVSFLSKFL.NH₂) and temporin-1Gd (FILPLIASFLSKFL.NH₂) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC₅₀=2.4±0.1 μM for temporin-1Gb and 2.3±0.2 μM for temporin-1Gd). The antimicrobial peptides that were isolated in extracts of the skin R. grylio were present in the same molecular forms in electrically-stimulated skin secretions of the animal demonstrating that the peptides are stored in the granular glands of the skin in their fully processed forms.     

Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae

The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus.     

Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedusa hypochondrialis

Amphibian antimicrobial peptides have been known for many decades and several of them have been isolated from anuran species. Dermaseptins are among the most studied antimicrobial peptides and are found in the skin secretion of tree frogs from the Phyllomedusinae subfamily. These peptides exert a lytic action on bacteria, protozoa, yeast, and filamentous fungi at micromolar concentrations, but unlike polylysines, present little hemolytic activity. In this work, two antimicrobial peptides were isolated from the crude skin secretion of Phyllomedusa hypochondrialis and tested against Gram-positive and Gram-negative bacteria, presenting no hemolytic activity at the tested concentrations. One of them was identified with the recently reported peptide PS-7 belonging to the phylloseptin family, and another was a novel peptide, named DPh-1, which was fully purified, sequenced by ‘de novo’ mass spectrometry and grouped into Dermaseptins (DPh-1).     

Tigerinins: novel antimicrobial peptides from the Indian frog Rana tigerina

Four broad-spectrum, 11 and 12 residue, novel antimicrobial peptides have been isolated from the adrenaline-stimulated skin secretions of the Indian frog Rana tigerina. Sequences of these peptides have been determined by automated Edman degradation, by mass spectral analysis and confirmed by chemical synthesis. These peptides, which we have named as tigerinins, are characterized by an intramolecular disulfide bridge between two cysteine residues forming a nonapeptide ring. This feature is not found in other amphibian peptides. Conformational analysis indicate that the peptides tend to form beta-turn structures. The peptides are cationic and exert their activity by permeabilizing bacterial membranes. Tigerinins represent the smallest, nonhelical, cationic antimicrobial peptides from amphibians.     

Evaluation of antimicrobial and anticancer activities of three peptides identified from the skin secretion of Hylarana latouchii

The skins of frogs of the family Ranidae are particularly rich sources of biologically active pep-tides,among which antimicrobial peptides (AMPs) constitute the...