Peptidomic analysis of skin secretions supports separate species status for the tailed frogs,Ascaphus truei and Ascaphus montanus

The tailed frog Ascaphus truei Stejneger, 1899 is the most primitive extant anuran and the sister taxon to the clade of all other living frogs. The species occupies two disjunct ranges in the Northwest region of North America: the Cascade Mountains and coastal area from British Columbia to Northern California, and an inland range in the northern Rocky Mountains and the Blue and Wallowa mountains. A previous study led to the isolation of eight peptides with antimicrobial activity (termed the ascaphins) from skin secretions of A. truei from the coastal range. The present study has used peptidomic analysis to identify the products of orthologous ascaphin genes in electrically-stimulated skin secretions from inland range specimens. Structural characterization of the peptides demonstrated that ascaphins from the inland range contained the following amino acid substitutions compared with orthologs from the coastal range frogs: ascaphin-1 (Ala¹² → Glu), ascaphin-3 (Asp⁴ → Glu), ascaphin-4 (Ala¹⁹ → Ser), ascaphin-5 (Lys¹² → Thr), and ascaphin-7 (Gly⁸ → Ser and Ser²⁰ → Asn). Orthologs of ascaphins-2, -6, and -8 were not identified but a paralog of ascaphin-5, identical to ascaphin-5 from coastal range frogs, was found. The data support the claims, derived from analysis of the nucleotide sequences of mitochondrial genes, that the inland populations of the tailed frog should be recognized as a distinct species, the Rocky Mountain tailed frog Ascaphus montanus and that the divergence of the species from A. truei probably occurred in the late Miocene (approximately 10 Mya).     

Kassinatuerin-1: a peptide with broad-spectrum antimicrobial activity isolated from the skin of the hyperoliid frog, Kassina senegalensis

Kassinatuerin-1 (GFMKYIGPLI(10)PHAVKAISDL(20)I.NH(2)) was isolated in high yield (75 nmol/g) from an extract of the skin of a Hyperoliid frog, the African running frog Kassina senegalensis and its sequence was confirmed by total synthesis. The peptide inhibited growth of the gram-negative bacterium Escherichia coli (minimum inhibitory concentration, MIC = 4 microM), the gram-positive bacterium Staphylococcus aureus (MIC = 8 microM), and the yeast Candida albicans (MIC = 70 microM). A structurally related peptide, kassinatuerin-2 (FIQYLAPLI(10)PHAVKAISDL(20)I.NH(2)) was also isolated in high yield (96 nmol/g) from the extract but was devoid of antimicrobial activity against these microrganisms. Kassinatuerin-1 may be classified with other linear, cationic antimicrobial peptides that can potentially adopt an amphipathic alpha-helical conformation but it contains almost no amino acid sequence identity with previously characterized bioactive peptides from frog skin.     

An antimicrobial peptide from the skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae)

A 25 amino-acid-residue, C-terminally alpha-amidated peptide with antimicrobial activity, which has been termed fallaxin, was isolated in high yield from the norepinephrine-stimulated skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae). The amino acid sequence of the peptide (Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Ile-Ala-Gly-His-Leu-Ala-Ser-Lys-Val-Met-Asn-Lys-Leu.NH2) shows structural similarity with members of the ranatuerin-2 family previously isolated from the skins of frogs of the genus Rana that are only distantly related to the Leptodactylidae. This observation is consistent with the hypothesis that many frog skin antimicrobial peptides are related evolutionarily, having arisen from multiple duplications of an ancestral gene that existed before the radiation of the different families. Fallaxin inhibited the growth of reference strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) but with relatively low potency (MIC> or =20 microM) and was inactive against the Gram-positive bacterium (Staphylococcus aureus) and the yeast Candida albicans. The hemolytic activity of fallaxin was very low (HC50>200 microM). A second peptide, comprising residues (1-22) of fallaxin, was also isolated from the skin secretions but this component was inactive against the microorganisms tested.     

Purification and properties of antimicrobial peptides from skin secretions of the Eritrea clawed frog Xenopus clivii (Pipidae)

Five peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus clivii Peracca, 1898 (Pipidae). Characterization of the peptides demonstrated that they are structurally similar to magainins (2 peptides), caerulein-precursor fragments, CPF (2 peptides), and xenopsin-precursor fragments, XPF (1 peptide) that have been previously isolated from other species of the genus Xenopus. The magainins and the XPF peptide were active only against the Gram-negative microorganism Escherichia coli whereas the CPF peptides were also active against the Gram-positive Staphylococcus aureus. The most abundant antimicrobial peptide in the secretions, CPF-C1 (GFGSLLGKALRLG ANVL.NH(2)) inhibited the growth of the Gram-negative bacteria Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa (MIC≤25μM) suggesting potential for development into an anti-infective agent for use against these emerging antibiotic-resistant pathogens.     

Host-defense peptides from skin secretions of the tetraploid frogs Xenopus petersii and Xenopus pygmaeus, and the octoploid frog Xenopus lenduensis (Pipidae)

Peptidomic analysis of norepinephrine-stimulated skin secretions led to the identification of host-defense peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families from the tetraploid frogs, Xenopus petersii (Peters' clawed frog) and Xenopus pygmaeus (Bouchia clawed frog), and the octoploid frog Xenopus lenduensis (Lendu Plateau clawed frog). Xenopsin-precursor fragment (XPF) peptides were not detected. The primary structures of the antimicrobial peptides from X. petersii demonstrate a close, but not conspecific relationship, with Xenopus laevis whereas the X. pygmaeus peptides show appreciable variation from previously characterized orthologs from other Xenopus species. Polyploidization events within the Xenopodinae (Silurana+Xenopus) are associated with extensive gene silencing (nonfunctionization) but unexpectedly the full complement of four PGLa paralogs were isolated from X. lenduendis secretions. Consistent with previous data, the CPF peptides showed the highest growth-inhibitory activity against bacteria with CPF-PG1 (GFGSLLGKALKIGTNLL.NH(2)) from X. pygmaeus combining high antimicrobial potency against Staphylococcus aureus (MIC=6 μM) with relatively low hemolytic activity (LC(50)=145 μM).     

Purification and characterization of antimicrobial peptides from the Caribbean frog, Leptodactylus validus (Anura: Leptodactylidae)

Peptidomic analysis of norepinephrine-stimulated skin secretions from the Caribbean frog Leptodactylus validus Garman, 1888 led to the identification of three peptides with previously undescribed sequences that were structurally similar to those of antimicrobial peptides isolated from other species of leptodactylid frogs. These paralogs have been termed ocellatin-V1 (GVVDILKGAGKDLLAHALSKLSEKV.NH(2)), ocellatin-V2 (GVLDILKGAGKDLLAHALSKISEKV.NH(2)), and ocellatin-V3 (GVLDILTGAGKDLLAHALSKLSEKV.NH(2)). The very low antimicrobial potency (MIC>200microM) against Escherichia coli and Staphylococcus aureus associated with the peptides is probably a consequence of their lack of amphipathicity and reduced cationicity compared with active members of the ocellatin family from related species.     

The hymenochirins: a family of host-defense peptides from the Congo dwarf clawed frog Hymenochirus boettgeri (Pipidae)

Skin secretions of frogs from the subfamily Xenopodinae (Xenopus+Silurana) within the family Pipidae are a rich source of antimicrobial peptides with therapeutic potential but species from the sister taxon Hymenochirus in the subfamily Pipinae (Hymenochirus+Pseudhymenochirus+Pipa) have not been investigated. Peptidomic analysis of norepinephrine-stimulated skin secretions from two distinct populations of the Congo dwarf clawed frog Hymenochirus boettgeri (Tornier, 1896) has led to identification of five structurally related peptides with broad-spectrum antimicrobial activity. Hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH(2)) is C-terminally α-amidated whereas hymenochirins-2B-5B have the general structure XKIPX(2)VKDTLKKVAKGX(2)SX(2)AGAX(3).COOH. Hymenochirin-3B (IKIPAVVKDTLKKVAKGVLSAVAGALTQ) was the most abundant peptide in the secretions. The hymenochirins show very low structural similarity with the antimicrobial peptides isolated from skin secretions of Silurana tropicalis and Xenopus laevis consistent with the proposed ancient divergence of the Pipinae and Xenopodinae. Synthetic replicates of hymenochirin-1B-4B inhibit the growth of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus (MIC in the range 10-40 μM) and Candida albicans (MIC=80 μM). The peptides display relatively weak hemolytic activity against human erythrocytes (LC(50) in the range 160 to >300 μM).     

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 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.     

Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica

Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an alpha-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH(2); temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 microM; japonicin-2, 12 microM; and temporin-1Ja > 100 microM. The MICs against S. aureus were japonicin-1, > 100 microM; japonicin-2, 20 microM; and temporin-1Ja, > 100 microM.     

Brevinin-1 and -2, unique antimicrobial peptides from the skin of the frog, Rana brevipoda porsa.

Two unique antimicrobial peptides named brevinin-1 and -2 were isolated from the skin of the frog, Rana brevipoda porsa. Both of the peptides did not have any structural homology with bombinin nor magainin; the frog skin derived-antimicrobial peptides isolated from Bombina and Xenopus, nor even with other known antimicrobial peptides of non-amphibian origin. The minimum inhibitory concentration of brevinin-1 against the growth of St. aureus and E. coli was determined to be 8 micrograms/ml and 34 micrograms/ml while that of brevinin-2 was 8 micrograms/ml and 4 micrograms/ml, respectively, indicating the difference of the two peptides in the antimicrobial selectively on Gram-positive and Gram-negative bacteria.     

Host-defense peptides in skin secretions of the tetraploid frog Silurana epitropicalis with potent activity against methicillin-resistant Staphylococcus aureus (MRSA)

A putative genome duplication event within the Silurana lineage has given rise to the tetraploid Cameroon clawed frog Silurana epitropicalis (Fischberg, Colombelli, and Picard, 1982). Peptidomic analysis of norepinephrine-stimulated skin secretions of S. epitropicalis led to identification of 10 peptides with varying degrees of growth-inhibitory activity against Escherichia coli and Staphylococcus aureus. Structural characterization identified the peptides as belonging to the magainin family (magainin-SE1), the caerulein-precursor fragment family (CPF-SE1, -SE2 and -SE3), the xenopsin-precursor fragment family (XPF-SE1, SE-2, SE-3 and -SE4), and the peptide glycine-leucine-amide family (PGLa-SE1 and -SE2). In addition, peptide phenylalanine-glutamine-amide (FLGALLGPLMNLLQ·NH(2)) was isolated from the secretions that lacked antimicrobial activity. Comparison of the multiplicity of orthologous peptides in S. epitropicalis and the diploid Silurana tropicalis indicates that extensive nonfunctionalization (deletion or silencing) of antimicrobial peptide genes has occurred after polyploidization in the Silurana lineage, as in the Xenopus lineage. CPF-SE2 (GFLGPLLKLGLKGAAKLLPQLLPSRQQ; MIC=2.5μM) and CPF-SE3 (GFLGSLLKTGLKVGSNLL·NH(2); MIC=5μM) showed potent growth-inhibitory activity against a range of clinical isolates of methicillin-resistant S. aureus (MRSA). Their utility as systemic anti-infective drugs is limited by significant hemolytic activity against human erythrocytes (LC(50)=50μM for CPF-SE2 and 220μM for CPF-SE3) but the peptides may find application as topical agents in treatment of MRSA skin infections and decolonization of MRSA carriers.     

Peptides with antimicrobial activity of the brevinin-1 family isolated from skin secretions of the southern leopard frog, Rana sphenocephala.

Three peptides with growth-inhibitory activity towards the gram-negative bacterium Eschericia coli were isolated from electrically stimulated secretions from the skin of the southern leopard frog, Rana sphenocephala. Structural characterization demonstrated that the peptides [brevinin-1Sa, minimum inhibitory concentration (MIC) = 55 microM; brevinin-1Sb, MIC = 17 microM; brevinin-1Sc, MIC = 14 microM] represent new members of the brevinin-1 family of antimicrobial peptides, previously isolated from several other species of frogs of the genus Rana. Their high concentration in skin secretions and extreme variability in amino acid sequence suggest that the brevinin family of peptides may be of value as molecular markers for the identification and taxonomic classification of Ranid frogs.     

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.     

Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae).

Seven peptides (XT-1-XT-7) with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of the diploid clawed frog, Xenopus tropicalis. Structural characterization of the peptides demonstrated that amino acid sequence similarity to antimicrobial peptides previously isolated from Xenopus laevis was low, suggesting that the species are not closely related phylogenetically. Peptides XT-5 and XT-3 are probably the orthologs of X. laevis peptide glycine-leucine amide (PGL(a)) and the N-terminal spacer region of prolevitide, respectively. XT-1, XT-6 and XT-7 show limited structural similarity to the spacer region of X. laevis procaeruleins and the paralogs XT-2 and XT-4 are similar to corresponding regions of proxenopsin. Orthologs of the magainins were not identified. The C-terminally alpha-amidated peptide XT-7 (GLLGPLLKIAAKVGSNLL.NH2) showed the lowest minimum inhibitory concentrations against reference microorganisms (Staphylococcus aureus 5 microM, Escherichia coli 5 microM, and Candida albicans 40 microM) and was also active against clinical isolates of methicillin-resistant S. aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus group C, Shigella sonnei, Pseudomonas aeruginosa and Enterobacter cloacae. The peptide was, however, hemolytic against human erythrocytes (50% lysis at 70 microM). Circular dichroism studies showed that XT-7 has a random structure in aqueous solution, pH 7.0 but adopts an alpha-helical conformation in the presence of 50% trifluoroethanol. Decreasing the cationicity of XT-7 either by replacement of the C-terminal CONH2 group by COOH or by deletion of the Lys(8) residue produced analogs with greatly (>10-fold) decreased antimicrobial potencies.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.     

A family of brevinin-2 peptides with potent activity against Pseudomonas aeruginosa from the skin of the Hokkaido frog, Rana pirica

Nine peptides displaying varying degrees of antimicrobial activity were extracted from the skin of the Hokkaido frog, Rana pirica. Five structurally related peptides were identified as members of the brevinin-2 family. These peptides were active against reference strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) and Gram-positive (Staphlococcus aureus) bacteria but displayed relatively low hemolytic activity. The most abundant peptide, brevinin-2PRa (680 nmol/g weight of dry skin) showed high potency [minimal inhibitory concentration (MIC) values between 6 and 12 microM] against a range of clinical isolates of P. aeruginosa. In addition, activity was unaffected by NaCl concentrations up to 200 mM. Cladistic analysis based on the primary structures of brevinin-2 peptides supports a close phylogenetic relationship between R. pirica and Japanese mountain brown frog Rana ornativentris. One peptide of the ranatuerin-2 family and one strongly hemolytic peptide of the brevinin-1 family were also isolated from the extract along with two members of the temporin family, temporin-1PRa (ILPILGNLLNGLL.NH(2)) and temporin-1PRb (ILPILGNLLNSLL.NH(2)) that atypically lacked basic amino acid residues and showed only very weak antimicrobial and hemolytic activity.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris.

Six peptides with antimicrobial activity were isolated from an extract of freeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin-10a FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-10b FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-10c FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-10d FLPLLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic alpha-helical conformation but it is significant that temporin-10d, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 microM compared with 2 microM for temporin-10a). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity.     

Antimicrobial peptides from the skin of the Tsushima brown frog Rana tsushimensis

The Tsushima brown frog Rana tsushimensis Stejneger, 1907 exists in reproductive isolation on the island of Tsushima, Japan. Six peptides with antimicrobial activity were isolated in pure form from an extract of the skin of this species and their amino acid sequences identified them as members of the brevinin-1 (one peptide), brevinin-2 (one peptide) and temporin (four peptides) families. The C-terminally alpha-amidated brevinin-1 peptide (FLGSIVGALASALPSLISKIRN.NH2) lacks the cyclic heptapeptide domain Cys18-(Xaa)4-Lys-Cys24 at the COOH-terminus of the molecule that characterizes other members of that family. A structurally similar brevinin-1 peptide, also lacking the cyclic domain, was previously isolated from the skin of the Ryukyu brown frog Rana okinavana, indicative of a close phylogenetic relationship between the species. Brevinin-2TSa (GIMSLFKGVLKTAGKHVAGSLVDQLKCKITGGC) showed broad-spectrum growth inhibitory activity against a range of Gram-negative and Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus) (minimum inhibitory concentrations< or =25 microM) and relatively low hemolytic activity against human erythrocytes (LD50=100 microM). The peptide therefore represents a candidate for drug development.     

A family of acyclic brevinin-1 peptides from the skin of the Ryukyu brown frog Rana okinavana

The 24 amino-acid residue antimicrobial peptide, brevinin-1 is synthesized in the skins of a wide range of species of Eurasian and North American frogs belonging to the genus Rana. All previously characterized brevinin-1 peptides contain the cyclic heptapeptide domain Cys18-(Xaa)4-Lys-Cys24 at the COOH-terminus of the molecule. Four structurally related peptides were isolated from an extract of the skin of the Ryukyu brown frog Rana okinavana. The amino acid sequences of the peptides [Phe-(Xaa)4-Ile-(Xaa)2-Leu-Ala-Lys-Gly-Leu-Pro-Ser-Leu-Ile-Xaa-Leu-Xaa-Lys-Lys.NH2] identified them as members of the brevinin-1 family that lacked the COOH-terminal cyclic domain but contained a C-terminally alpha-amidated residue. It is suggested, as one possibility, that the Cys18 in the brevinin-1 consensus sequence has been deleted and the Cys24 residue has mutated to a glycine that acts as substrate for peptidyl-glycine alpha-amidating monooxygenase. The peptides potently inhibited the growth of Escherichia coli and Staphylococcus aureus confirming that a cyclic domain is not necessary for antimicrobial activity. A fifth peptide (SFLNFFKGAA10KNLLAAGLDK20LKCKISGTQC30), that also displayed broad-spectrum antimicrobial activity, was isolated from the skin extract and showed structural similarity with members of the ranatuerin-2 family previously isolated from the skin of North American ranid frogs.