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.

Pseudin-2: an antimicrobial peptide with low hemolytic activity from the skin of the paradoxical frog.

Four structurally related peptides (pseudins 1-4) with antimicrobial activity were isolated from an extract of the skin of the paradoxical frog Pseudis paradoxa (Pseudidae). Pseudin-2 (GLNALKKVFQGIHEAIKLINNHVQ) was the most abundant peptide (22 nmol/g tissue) and also the most potent (minimum inhibitory concentrations, MIC = 2.5 microM against Escherichia coli, 80 microM against Staphylococcus aureus, and 130 microM against Candida albicans). The concentration of pseudin-2 producing 50% hemolysis of human erythrocytes was >300 microM. Circular dichroism studies showed that the pseudins belong to the class of cationic, amphipathic alpha-helical antimicrobial peptides but their amino acid sequences are not similar to any previously characterized peptides from frog skin. The pseudins do, however, show sequence similarity with a region at the C-terminus of DEFT, a death effector domain-containing protein expressed in mammalian testicular germ cells that is involved in the regulation of apoptosis.

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.

Multiple antimicrobial peptides and peptides related to bradykinin and neuromedin N isolated from skin secretions of the pickerel frog, Rana palustris.

The skin secretions of the North American pickerel frog Rana palustris are toxic to both microorganisms and predators. A total of 22 peptides with differential growth-inhibitory activity towards bacteria and yeast were isolated from the electrostimulated secretions of R. palustris skin and were characterized structurally. Thirteen of the antimicrobial peptides belong to five of the known families previously identified in the skins of other species of Ranid frogs: brevinin-1 (3 peptides), esculentin-1 (2 peptides), esculentin-2 (1 peptide), ranatuerin-2 (6 peptides), and temporin (1 peptide). Nine peptides show little structural similarity towards other known antimicrobial peptides and so are classified in new families: palustrin-1 (4 peptides) with 27-28 amino acid residues and a cystine-bridged heptapeptide ring; palustrin-2 (3 peptides) with 31 amino acids and a cyclic heptapeptide region and palustrin-3 (2 peptides) with 48 amino acids and a cyclic hexapeptide region. Peptides belonging to the esculentin-1, esculentin-2 and palustrin-3 families are the most potent (minimal inhibitory concentrations approximately 1 microM against Escherichia coli) whereas peptides of the brevinin-1 and esculentin-2 families show the broadest spectrum of activity. As well as bradykinin that is identical to the human peptide, a further 4 peptides structurally related to [Leu(8)]bradykinin and two peptides related to neuromedin-N (the hexapeptide KKPYIL and a larger, cystine-containing form HLRRCGKKPYILMACS) were purified from the skin secretions.

Induction of synthesis of an antimicrobial peptide in the skin of the freeze-tolerant frog, Rana sylvatica, in response to environmental stimuli.

An extract of skin taken from specimens of the freeze-tolerant wood frog, Rana sylvatica, that were collected from cold (<7 degrees C) ponds and maintained at 5 degrees C lacked detectable antimicrobial activity. In contrast, an extract of skin taken from specimens maintained at 30 degrees C for 3 weeks under laboratory conditions contained a high concentration (approximately 4 nmol/g) of a single antimicrobial peptide of the brevinin-1 family (FLPVVAGLAAKVLPSIICAVTKKC). The peptide inhibited growth of Escherichia coli (minimum inhibitory concentration 45 microM) and Staphylococcus aureus (minimum inhibitory concentration 7 microM). The data suggest that synthesis of the peptide is induced when the animal is in an environment that promotes the growth of microorganisms consistent with a role in the animal's defense strategy.

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.NH(2)) and temporin-1Gd (FILPLIASFLSKFL.NH(2)) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC(50) = 2.4+/-0.1 microM for temporin-1Gb and 2.3+/-0.2 microM 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 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.

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.

Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens.

The skins of frogs of the genus Rana synthesize a complex array of antimicrobial peptides that may be grouped into eight families on the basis of structural similarity. A total of 24 peptides with differential growth-inhibitory activity towards the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli and the yeast Candida albicans were isolated from extracts of the skins of three closely related North American frogs, Rana luteiventris (spotted frog), Rana berlandieri (Rio Grande leopard frog) and Rana pipiens (Northern leopard frog). Structural characterization of the antimicrobial peptides demonstrated that they belonged to four of the known families: the brevinin-1 family, first identified in skin of the Asian frog Rana porosa brevipoda; the esculentin-2 family, first identified in the European frog Rana esculenta; the ranatuerin-2 family, first identified in the North American bullfrog Rana catesbeiana; and the temporin family, first identified in the European frog Rana temporaria. Peptides belonging to the brevinin-2, ranalexin, esculentin-1 and ranatuerin-1 families were not identified in the extracts. Despite the close phylogenetic relationship between the various species of Ranid frogs, the distribution and amino-acid sequences of the antimicrobial peptides produced by each species are highly variable and species-specific, suggesting that they may be valuable in taxonomic classification and molecular phylogenetic analysis.

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.