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.     

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 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.     

Albumin evolution in polyploid species of the genusXenopus

Electrophoresis of serum from 21 Xenopus species and subspecies reveals variable numbers of albumin bands. The diploid X. tropicalis has one albumin, while the tetraploid species (laevis, borealis, muelleri, clivii, fraseri, epitropicalis) have two. The octoploid species (amieti, boumbaensis, wittei, vestitus, andrei) have two to three bands, and the dodecaploid X. ruwenzoriensis has three. The molecular weight of the Xenopus albumins varies from 68 kd (in the tropicalis group) to 74 kd. The subspecies of X. laevis possess two albumins of different molecular weights (70 and 74 kd), whereas most species have only 70-kd albumins. Peptide maps have been obtained from albumin electromorphs by limited proteolysis in sodium dodecyl sulfate (SDS) gels, using S. aureus V8 protease. The peptide patterns produced by electromorphs from the same tetraploid Xenopus species generally differ from each other, suggesting that the two albumin genes contain a substantial amount of structural differences. In addition, the peptide maps are diagnostic for most tetraploid species and for some subspecies of X. laevis as well. Proteolysis of albumins from most octoploid and dodecaploid species results in patterns which are very similar to the ones produced by the electromorphs from X. fraseri. The albumins of X. vestitus differ from those of the other octoploid species. X. andrei possesses two fraseri-type and one vestitus-type albumin, which indicates that it probably originated by allopolyploidy.     

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).     

Evolution of the closely related, sex-related genes DM-W and DMRT1 in African clawed frogs (Xenopus)

DM-W is a dominant, female-specific, regulator of sex determination in the African clawed frog Xenopus laevis. This gene is derived from partial duplication of DMRT1, a male-related autosomal gene. We set out to better understand sex determination in Xenopus by studying this pair of genes. We found that DM-W evolved in Xenopus after divergence from the sister genus Silurana but before divergence of X. laevis and X. clivii, and that DM-W arose from partial duplication of DMRT1β, which is one of the two DMRT1 paralogs in the tetraploid ancestor of Xenopus. Using the rate ratio of nonsynonymous to synonymous substitutions per site and multilocus polymorphism data, we show that DM-W evolved non-neutrally. By cloning paralogs and using a pyrosequencing assay, we also demonstrate that DMRT1 underwent phylogenetically biased pseudogenization after polyploidization, and that expression of this gene is regulated by mechanisms that vary through development. One explanation for these observations is that the expression domain of DMRT1β was marginalized, which would explain why this paralog is dispensable in Xenopus polyploids and why DM-W has a narrow expression domain. These findings illustrate how evolution of the genetic control of stable phenotypes is facilitated by redundancy, degeneration, and compartmentalized regulation.     

A mitochondrial DNA phylogeny of African clawed frogs: phylogeography and implications for polyploid evolution

The African clawed frogs (Silurana and Xenopus), model organisms for scientific inquiry, are unusual in that allopolyploidization has occurred on multiple occasions, giving rise to tetraploid, octoploid, and dodecaploid species. To better understand their evolution, here we estimate a mitochondrial DNA phylogeny from all described and some undescribed species. We examine the timing and location of diversification, and test hypotheses concerning the frequency of polyploid speciation and taxonomy. Using a relaxed molecular clock, we estimate that extant clawed frog lineages originated well after the breakup of Gondwana, about 63.7 million years ago, with a 95% confidence interval from 50.4 to 81.3 million years ago. Silurana and two major lineages of Xenopus have overlapping distributions in sub-Saharan Africa, and dispersal-vicariance analysis suggests that clawed frogs originated in central and/or eastern equatorial Africa. Most or all extant species originated before the Pleistocene; recent rainforest refugia probably acted as "lifeboats" that preserved existing species, rather than "species pumps" where many new successful lineages originated. We estimate that polyploidization occurred at least six times in clawed frogs.     

Peptidomic analysis of skin secretions demonstrates that the allopatric populations of Xenopus muelleri (Pipidae) are not conspecific

Mueller's clawed frog Xenopus muelleri (Peters 1844) occupies two non-contiguous ranges in east and west Africa. The phylogenetic relationship between the two populations is unclear and it has been proposed that the western population represents a separate species. Peptidomic analysis of norepinephrine-stimulated skin secretions from X. muelleri from the eastern range resulted in the identification of five antimicrobial peptides structurally related to the magainins (magainin-M1 and -M2), xenopsin-precursor fragments (XPF-M1) and caerulein-precursor fragments (CPF-M1 and -M2) previously found in skin secretions of other Xenopus species. A cyclic peptide (WCPPMIPLCSRF.NH₂) containing the RFamide motif was also isolated that shows limited structural similarity to the tigerinins, previously identified only in frogs of the Dicroglossidae family. The components identified in skin secretions from X. muelleri from the western range comprised one magainin (magainin-MW1), one XPF peptide (XPF-MW1), two peptides glycine-leucine amide (PGLa-MW1 and -MW2), and three CPF peptides (CPF-MW1, -MW2 and -MW3). Comparison of the primary structures of these peptides suggest that western population of X. muelleri is more closely related to X. borealis than to X. muelleri consistent with its proposed designation as a separate species. The CPF peptides showed potent, broad-spectrum activity against reference strains of bacteria (MIC 3-25 μM), but were hemolytic against human erythrocytes.     

Phylogenetic relationships of the pipid frogs Xenopus and Silurana: an integration of ribosomal DNA and morphology

Relationships of the pipid frog genus Silurana (= Xenopus tropicalis group of some authors) are of particular interest to developmental and molecular biologists because of the purported ancestral (i.e., unduplicated) karyotype of S. tropicalis relative to the genus Xenopus. Although most previous studies have assumed that Silurana is the sister group of Xenopus, recent morphological work suggests that Silurana is more closely related both to the South American genus Pipa and to the African genera Hymenochirus and Pseudhymenochirus than it is to Xenopus. We examined 1,486 bp of relatively variable regions of the ribosomal DNA array (including portions of the 18S and 28S genes, as well as part of an internal transcribed spacer) in Hymenochirus, Silurana, and Xenopus, as well as the outgroup genus Spea, in order to test the alternative hypotheses of relationships for Silurana. Maximum-parsimony analysis using bootstrapping and an analysis using Lake's method of invariants both significantly support the sister-group relationship between Xenopus and Silurana rather than the relationship suggested by morphology. Analysis of the combined morphological/molecular data matrix also significantly supports the Xenopus-Silurana relationship. Although our results are not inconsistent with the recognition of the genus Silurana to accommodate the species formerly called X. tropicalis and X. epitropicalis, the proposed relationships do not require the recognition of this genus in order to render Xenopus monophyletic.     

Caerulein precursor fragment (CPF) peptides from the skin secretions of Xenopus laevis and Silurana epitropicalis are potent insulin-releasing agents

Peptidomic analysis of norepinephrine-stimulated skin secretions of the tetraploid clawed frog Xenopus laevis (Pipidae) led to the identification of 10 peptides with the ability to stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line. These peptides were purified to near homogeneity and structural characterization showed that they belong to the magainin (2 peptides), peptide glycine-leucine-amide (PGLa) (1 peptide), xenopsin precursor fragment (1 peptide), and caerulein precursor fragment (CPF) (6 peptides) families. CPF-1, CPF-3, CPF-5 and CPF-6 were the most potent producing a significant (P < 0.05) increase in the rate of insulin release at concentration of 0.03 nM. CPF-7 (GFGSFLGKALKAALKIGANALGGAPQQ) produced the maximum stimulation of insulin release (571 ± 30% of basal rate at 3 μM). In addition, CPF-SE1 (GFLGPLLKLGLKGVAKVIPHLIPSRQQ), previously isolated from skin secretions of the tetraploid frog Silurana epitropicalis, produced a significant (P < 0.05) increase in the rate of insulin release at 0.03 nM with a 514 ± 13% increase over basal rate at 3 μM. No CPF peptide stimulated release of the cytosolic enzyme, lactate dehydrogenase from BRIN-BD11 cells at concentrations up to 3 μM indicating that the integrity of the plasma membrane had been preserved. The mechanism of action of the CPF peptides involves, at least in part, membrane depolarization and an increase in intracellular Ca²⁺ concentration. The CPF peptides show potential for development into agents for the treatment of Type 2 diabetes.     

Peptidomic analysis of skin secretions provides insight into the taxonomic status of the African clawed frogs Xenopus victorianus and Xenopus laevis sudanensis (Pipidae)

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from Xenopus victorianus Ahl, 1924 (also described as the subspecies X. laevis victorianus) and Xenopus laevis sudanensis Perret, 1966 with the previously determined distributions in Xenopus laevis (Daudin, 1802) and Xenopus petersii Bocage, 1895. Peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families were purified by reversed-phase HPLC and characterized by electrospray mass spectrometry. Magainin-P2, PGLa-P1, CPF-P1, CPF-P2, and CPF-P3 previously isolated from X. petersii and structurally different from orthologous peptides from X. laevis, were identified in X. victorianus and X. laevis sudanensis skin secretions whereas the corresponding X. laevis peptides were absent. Magainin-1, identical in X. petersii and X. laevis, was also identified in the secretions. Xenopsin-precursor fragment (XPF) peptides, absent from X. petersii but present in X. laevis skin secretions, were not identified in the X. victorianus and X. laevis sudanensis secretions. The data indicate that X. victorianus and X. laevis sudanensis are more closely related to X. petersii than to X. laevis and support separate species status. The study illustrates the value of analysis of host-defense peptides in the evaluation of taxonomic and phylogenetic relationships between closely related frog species.     

Evidence from peptidomic analysis of skin secretions that allopatric populations of Xenopus gilli (Anura:Pipidae) constitute distinct lineages

The International Union for Conservation of Nature (IUCN) Endangered Cape Platanna Xenopus gilli inhabits disjunct ranges at the tip of Cape Peninsula and near the town of Kleinmond on opposite sides of False Bay in the extreme southwest of Africa. Peptidomic analysis of host-defense peptides in norepinephrine-stimulated skin secretions from frogs from the Cape Peninsula range resulted in the identification of two magainins, two peptide glycine–leucine–amide (PGLa) peptides, two xenopsin-precursor fragment (XPF) peptides, nine caerulein-precursor fragment (CPF) peptides, and a peptide related to peptide glycine–glutamine (PGQ) previously found in an extract of Xenopus laevis stomach. The primary structures of the peptides indicate a close phylogenetic relationship between X. gilli and X. laevis but only magainin-1, PGLa and one CPF peptide are identical in both species. Consistent with previous data, the CPF peptides show the greatest antimicrobial potency but are hemolytic. There are appreciable differences in the expression of host-defense peptide genes in frogs from the population of animals sampled near Kleinmond as peptides corresponding to magainin-G2, XPF-G1, XPF-G2, and four CPF peptides, present in secretions from the Cape Peninsula frogs, were not identified in the skin secretions from Kleinmond frogs. Conversely, PGLa-G3, XPF-G3, and three CPF peptides were identified in the Kleinmond frogs but not in the Cape Peninsula animals. The data support the conclusion from morphometric analyses and comparisons of the nucleotide sequences of mitochondrial genes that the disjunct populations of X. gilli have undergone appreciable genetic, morphological, and phenotypic divergence.     

Novel antimicrobial peptides isolated from the skin secretions of Hainan odorous frog, Odorrana hainanensis

Long time geographical isolation of Hainan Island from the China continent has resulted in appearance of many novel frog species. As one of them, Hainan odorous frog, Odorrana hainanensis possesses some special antimicrobial peptides distinct from those found in other Odorrana. In this study, three antimicrobial peptides have been purified and characterized from the skin secretion of O. hainanensis. With the similarity to the temporin family, two peptides are characterized by amidated C-terminals, so they are named as temporin-HN1 (AILTTLANWARKFL-NH₂) and temporin-HN2 (NILNTIINLAKKIL-NH₂). The third antimicrobial peptide belongs to the brevinin-1 family which is widely distributed in Eurasian ranids, and thus, it is named as brevinin-1HN1 (FLPLIASLAANFVPKIFCKITKKC). Furthermore, after sequencing 68 clones, eight cDNAs encoding antimicrobial peptide precursors were cloned from the skin-derived cDNA library of O. hainanensis. These eight cDNAs can encode seven mature antimicrobial peptides including the above three, as well as brevinin-1V, brevinin-2HS2, odorranain-A6, and odorranain-B1. Twelve different species of microorganisms were chosen, including Gram-positive, Gram-negative and fungi, to test the antimicrobial activities of temporin-HN1, temporin-HN2, brevinin-1HN1, brevinin-1V, and brevinin-2HS2. The result shows that, in addition to their activities against Gram-positive bacteria, temporin-HN1 and temporin-HN2 also possess activities against some Gram-negative bacteria and fungi. However, the two antimicrobial peptides, brevinin-1HN1 and brevinin-1V of the brevinin-1 family have stronger antimicrobial activities than temporin-HN1 and temporin-HN2 of the temporin family. Brevinin-1HN1 possesses activity against Staphylococcus aureus (ATCC25923), Rhodococcus rhodochrous X15, and Slime mould 090223 at the concentration of 1.2 μM.     

Evidence from peptidomic analysis of skin secretions that the red-legged frogs, Rana aurora draytonii and Rana aurora aurora, are distinct species

The northern red-legged frog Rana aurora aurora and the California red-legged frog Rana aurora draytonii are traditionally classified together in the same species group. Ten peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of R. aurora draytonii and purified to near homogeneity. The peptides were identified as belonging to the ranatuerin-2 family (two peptides), brevinin-1 family (four peptides), temporin family (three peptides), and a novel peptide, RV-23 (RIGVLLARLPKLFSLFKLMGKKV) that has limited structural similarity to the bee venom peptide, melittin. This distribution of peptides contrasts with that found previously in skin secretions from R. aurora aurora collected under the same conditions and at the same time of year (one ranatuerin-2 peptide, two brevinin-1 peptides, and one temporin peptide). The variation in amino acid sequences between corresponding R. aurora draytonii and R. aurora aurora peptides is comparable with the variation in sequences of orthologs from other members of the Amerana group of New World ranid frogs (Rana boylii, Rana muscosa, and Rana luteiventris). It is proposed, therefore, that the red-legged frogs should be regarded as separate species (R. aurora and R. draytonii) within the Amerana group rather than conspecific subspecies. The data emphasize that amino acid sequences of antimicrobial peptides in skin secretions may be used to infer taxonomic and phylogenetic relationships between species of ranid frogs.     

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and North American Rana frogs share the same families of skin antimicrobial peptides

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and the North American pickerel frog (Rana palustris) occupy different ecological niches on two different continents with no overlap in geographical distribution. R. palustris skin secretions contain a formidable array of antimicrobial peptides including homologs of brevinin-1, esculentin-1, esculentin-2, ranatuerin-2, a temporin and a family of peptides considered of unique structural attributes when isolated, palustrins 1-3. Here we describe the structures of mature peptides and precursors of eight putative antimicrobial peptides from the skin secretion of the Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis). Each peptide represents a structural homolog of respective peptide families isolated from R. palustris, including two peptides identical in primary structure to palustrin 1c and palustrin 3b. Additionally, two peptides were found to be structural homologs of ranatuerin 2B and ranatuerin 2P from the closely-related North American species, Rana berlandieri (the Rio Grande leopard frog) and Rana pipiens (the Northern leopard frog), respectively. Both palustrins and ranatuerins have hitherto been considered unique to North American ranid frogs. The use of primary structures of amphibian skin antimicrobial peptides is thus questionable as a taxonomic device or alternatively, the micro-evolution and/or ancestry of ranid frogs is more highly complex than previously thought.     

Phylloxin, a novel peptide antibiotic of the dermaseptin family of antimicrobial/opioid peptide precursors.

A novel family of peptide precursors that have very similar N-terminal preprosequences followed by markedly different C-terminal domains has been identified in the skin of hylid frogs belonging to the genus Phyllomedusinae. Biologically active peptides derived from the variable domains include the dermaseptins, 28-34-residue peptides that have a broad-spectrum microbicidal activity, and dermorphin and the deltorphins, D-amino acid containing heptapeptides that are very potent agonists for the micro-opioid and delta-opioid receptors, respectively. This report describes the isolation, synthesis and cloning of phylloxin, a prototypical member of a novel family of antimicrobial peptides derived from the processing of a dermaseptin/dermorphin-like precursor. The structure of phylloxin (GWMSKIASGIGTFLSGIQQ amide) shows no homology to the dermaseptins, but bears some resemblance to the levitide-precursor fragment and the xenopsin-precursor fragment, two antimicrobial peptides isolated from the skin of an evolutionarily distant frog species, Xenopus laevis. Circular dichroism spectra of phylloxin in low polarity medium, which mimics the lipophilicity of the membrane of target microorganisms, indicated 60-70% alpha-helical conformation, and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning residues 1-19. Phylloxin is an addition to the structurally and functionally diverse peptide families encoded by the rapidly evolving C-terminal domains of the dermorphin/dermaseptin group of precursors.     

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.     

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.     

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.     

A novel family of antimicrobial peptides from the skin of Amolops loloensis

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Sichuan region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the rufous-spotted torrent frog, Amolops loloensis. Members of the new peptide family named amolopins are composed of 18 amino acids with a unique sequence, for example, NILSSIVNGINRALSFFG. By BLAST search, amolopins did no show similarity to any known peptides. Among the tested microorganisms, native and synthetic peptides only showed antimicrobial activities against Staphylococcus aureus ATCC2592 and Bacillus pumilus, no effects on other microorganisms. The CD spectroscopy showed that it adopted a structure of random combined with beta-sheet in water, Tris-HCl or Tris-HCl-SDS. Several cDNAs encoding amolopins were cloned from the skin cDNA library of A. loloensis. The precursors of amolopin are composed of 62 amino acid residues including predicted signal peptides, acidic propieces, and mature antimicrobial peptides. The preproregion of amolopin 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 amolopins 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.