Identification and structure-activity relationship of an antimicrobial peptide of the palustrin-2 family isolated from the skin of the endangered frog Odorrana ishikawae

Recently, we identified nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorrana ishikawae, to assess its innate immune system. In this study an additional antimicrobial peptide was initially isolated based on antimicrobial activity against Escherichia coli. The new antimicrobial peptide belonging to the palustrin-2 family was named palustrin-2ISb. It consists of 36 amino acid residues including 7 amino acids C-terminal to the cyclic heptapeptide Rana box domain. The peptide's primary structure suggests a close relationship with the Chinese odorous frog, Odorrana grahami. The cloned cDNA encoding the precursor protein contained a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and the C-terminal precursor antimicrobial peptide. It also contained 3 amino acid residues at the C-terminus not found in the mature peptide. Finally, the antimicrobial activities against four microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus and Candida albicans) were investigated using several synthetic peptides. A 29 amino acid truncated form of the peptide, lacking the 7 amino acids C-terminal to the Rana box, possessed greater antimicrobial activities than the native structure.     

Molecular cloning and characterization of antimicrobial peptides from skin of the broad-folded frog, Hylarana latouchii

Seven cDNA sequences encoding antimicrobial peptide (AMP) precursors were cloned by screening the skin-derived cDNA library of the broad-folded frog, Hylarana latouchii. Seven of the deduced peptides are highly similar to AMPs in five families of brevinin-2 (brevinin-2LTa, brevinin-2LTb, and brevinin-2LTc), esculentin-1 (esculentin-1LTa), esculentin-2 (esculentin-2LTa), palustrin-2 (palustrin-2LTa), and temporin (temporin-LTe). The actual sequences and characteristics of mature AMPs were analyzed by RP-HPLC and LC–MS/MS-based proteomics approaches in combination of four different protein digestion processes and by LTQ XL in combination of gas-phase fractionation (GPF) analysis. Moreover, most of the peptides found in this study hardly display hemolytic activity in vitro, suggesting they are promising antimicrobial drug candidates.     

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.     

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

We recently reported the primary structures, antimicrobial activities and cDNA precursors of nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorranaishikawae. Their cDNA clones revealed a highly conserved approximately 60 bp region upstream of the start codon. This conserved region was used in the “shotgun” cDNA cloning method to reveal additional cDNAs encoding novel antimicrobial peptides of O.ishikawae. After sequencing 344 clones, we identified novel 13 cDNAs encoding dermal peptides in addition to the previously identified nine antimicrobial peptides. These 13 unique cDNAs encoded precursor proteins each containing a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg/Lys processing site and a dermal peptide at the C-terminus. The dermal peptides were members of the palustrin-2 (two peptides; termed palustrin-2ISc and palustrin-2ISd), nigrocin-2 (one peptide; nigrocin-2ISc), brevinin-1 (one peptide; brevinin-1ISa), odorranain-M (one peptide; odorranain-MISa) and entirely novel peptides (eight peptides; ishikawain-1-8). Although palustrin-2ISd and odorranain-MISa had few antimicrobial activities, palustrin-2ISc and nigrocin-2ISc possessed a broad-spectrum of growth inhibition against bacteria. Brevinin-1ISa had the most potent antimicrobial activities against the Gram-positive bacteria and the fungus but not the Gram-negative bacterium, Escherichiacoli. However, eight novel peptides showed no growth inhibition against these microorganisms.     

Identification and characterization of antimicrobial peptides from skin of Amolops ricketti (Anura: Ranidae)

As one of large amphibian group, there are a total of 45 species of Amolops in the world. However, the antimicrobial peptides (AMPs) existing in this genus has not been extensively studied. In this study, cDNAs encoding five novel AMP precursors were cloned by screening the skin-derived cDNA library of Amolops ricketti, a frog species that exists in southern and western parts of China. Protein sequence analysis led to the identification of five deduced peptides, three belonging to the brevinin-1 family and two belonging to the brevinin-2 family of amphibian AMPs. Thus, they were named as brevinin-1RTa (FLPLLAGVVANFLPQIICKIARKC), brevinin-1RTb (FLGSLLGLVGKVVPTLFCKISKKC), brevinin-1RTc (FLGSLLGLVGKIVPTLICKISKKC), brevinin-2RTa (GLMSTLKDFGKTAAKEIAQSLLSTASCKLAKTC), and brevinin-2RTb (GILDTLKEFGKTAAKGIAQSLLSTASCKLAKTC), respectively. The purification of brevinin-1RTa, brevinin-1RTb, and brevinin-2RTb was carried out by RP-HPLC, and confirmed by the LC-MS/MS-based proteomics approach. All of the peptides displayed different antimicrobial potency against a variety of microorganisms. In addition, brevinin-2RTa and brevinin-2RTb were found to have relatively low hemolytic activity (>400μg/ml) against mammalian red blood cells in vitro, which could potentially be as candidates for developing novel anti-infection agents.     

Characterization of diverse antimicrobial peptides in skin secretions of Chungan torrent frog Amolops chunganensis

We have cloned, synthesized, and characterized 11 novel antimicrobial peptides from a skin derived cDNA library of the Chungan torrent frog, Amolops chunganensis. Seven of the 11 antimicrobial peptides were present in authentic A. chunganensis skin secretions. Sequence analysis indicated that the 11 peptides belonged to the temporin, esculentin-2, palustrin-2, brevinin-1, and brevinin-2 families. The peptides displayed potent antimicrobial activities against several strains of microorganisms. One peptide, brevinin-1CG5, demonstrated antimicrobial activity against all tested Gram-positive and Gram-negative bacteria and fungi, and showed high antimicrobial potency (MIC = 0.6 μM) against Gram-positive bacterium Rhodococcus rhodochrous. Some peptides also demonstrated weak hemolytic activity against human erythrocytes in vitro. Phylogenetic analysis based on the amino acid sequences of brevinin-1, brevinin-2, and esculentin-2 peptides from family Ranidae confirmed that the current taxonomic status of A. chunganensis is correct.     

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 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.     

An atypical member of the brevinin-1 family of antimicrobial peptides isolated from the skin of the European frog Rana dalmatina

A single peptide with antimicrobial activity was extracted from the skin of the European agile frog (R. dalmatina). The primary structure of this 17 amino-acid-residue peptide (ILPLLLGKVVCAITKKC) does not immediately suggest membership of any of the previously described families of antimicrobial peptides from ranid frogs. However, if it is assumed that the peptide has undergone several residue deletions during the course of speciation, it shows sequence similarity with peptides belonging to the widely distributed brevinin-1 family, particularly those isolated from the related species Rana temporaria. The minimum inhibitory concentration of the peptide, termed brevinin-1 Da, against the Gram-positive bacterium Staphylococcus aureus was 7 microM and against the Gram-negative bacterium Escherichia coli was 30 microM.     

The ascaphins: a family of antimicrobial peptides from the skin secretions of the most primitive extant frog, Ascaphus truei

The tailed frog Ascaphus truei occupies a unique position in phylogeny as the most primitive extant anuran and is regarded as the sister taxon to the clade of all other living frogs. Eight structurally related peptides, termed ascaphins 1-8, were isolated from norepinephrine-stimulated skin secretions of A. truei and were shown to possess differential growth inhibitory activity against Escherichia coli and Staphylococcus aureus. Ascaphins 2-7 may be represented by the consensus amino acid sequence GX2DX2KGAAKX3KTVAX2IANX.COOH whereas ascaphin-1 (GFRDVLKGAAKAFVKTVAGHIAN.NH2) and ascaphin-8 (GFKDLLKGAAKALVKTVLF.NH2) contain a C-terminally alpha-amidated residue. The ascaphins show no appreciable structural similarity with other families of antimicrobial peptides from frog skin but display limited sequence identity with the cationic, amphipathic alpha-helical peptides pandinin 1 and opistoporin 1, isolated from the venoms of African scorpions. Ascaphin-8 shows the highest potency against a range of pathogenic microorganisms but has the greatest haemolytic activity. The data indicate that the host defence strategy of using antimicrobial peptides in skin secretions arose early in the evolution of anurans.     

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).     

Battle and balance at mucosal surfaces - The story of Shigella and antimicrobial peptides

Shigella is a major cause of morbidity and mortality for children in many developing countries. Emergence of antibiotic-resistance among Shigella demands the development of effective medicines. Antimicrobial peptides (AMPs) are expressed in phagocytes and at epithelial surfaces and are important effector molecules of innate immunity. We have found that pathogens are able to turn off the endogenous expression of AMPs, resulting in serious infections such as shigellosis. A therapeutic rationale to prevent microbial invasion would be to strengthen the epithelial line of defence through enhancing AMP expression. We have identified several inducers of AMP-production, including butyrate, phenylbutyrate and vitamin D, which have been investigated in animal models of shigellosis as well as in clinical trials. We believe that the conceptual framework presented here can be applied to additional clinical entities and that this novel approach can be an alternative or complement to traditional antibiotics in the future.     

Bombinins, antimicrobial peptides from Bombina species

The skin secretions of Bombina species contain peptides and small proteins with interesting biological properties. These include bombesin, thyrotropin releasing hormone, BSTI and Bv8. In this review, the biosynthesis and antimicrobial activity of two groups of peptides, bombinins and bombinins H, are described. To date, these have only been found in Bombina skin. They are derived from common precursors containing one or two bombinin copies at the amino and a single bombinin H at the carboxyl end. Bombinins are active against Gram-positive and Gram-negative bacteria and fungi but virtually inactive in haemolysis assays. Conversely, bombinins H have lower bactericidal activities but lyse erythrocytes. In the skin secretions, bombinins H are present in two sizes with either 20 or 17 amino acids. Moreover, they occur as epimers with either an l- or a d-amino acid at position 2. An enzyme catalyzing this inversion of chirality of an amino acid in peptide linkage has been isolated from Bombina skin secretions. In different tests, also with different stages of the life cycle of Leishmania parasites, the d-forms were found to be more active. Biophysical studies have yielded some insight into the different behaviours of the epimers in model membranes.     

Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulin-releasing activity

Using a combination of reversed-phase HPLC and electrospray mass spectrometry, peptidomic analysis of norepinephrine-stimulated skin secretions of the American bullfrog Lithobates catesbeianus Shaw, 1802 led to the identification and characterization of five newly described peptides (ranatuerin-1CBb, ranatuerin-2CBc, and -CBd, palustrin-2CBa, and temporin-CBf) together with seven peptides previously isolated on the basis of their antimicrobial activity (ranatuerin-1CBa, ranatuerin-2CBa, brevinin-1CBa, and -1CBb, temporin-CBa, -CBb, and -CBd). The abilities of the most abundant of the purified peptides to stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line were evaluated. Ranatuerin-2CBd (GFLDIIKNLGKTFAGHMLDKIRCTIGTCPPSP) was the most potent peptide producing a significant stimulation of insulin release (119% of basal rate, P < 0.01) from BRIN-BD11 cells at a concentration of 30 nM, with a maximum response (236% of basal rate, P < 0.001) at a concentration of 3 μM. Ranatuerin-2CBd did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3 μM, indicating that the integrity of the plasma membrane had been preserved. Brevinin-1CBb (FLPFIARLAAKVFPSIICSVTKKC) produced the maximum stimulation of insulin release (285% of basal rate, P < 0.001 at 3 μM) but the peptide was cytotoxic at this concentration.     

Antimicrobial function of the GAPDH-related antimicrobial peptide in the skin of skipjack tuna,Katsuwonus pelamis

A 3.4 kDa of antimicrobial peptide was purified from an acidified skin extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea–polyacrylamide gel electrophoresis and C₁₈ reversed-phase HPLC. A comparison of the N-terminal amino acid sequence of the purified peptide with that of other known polypeptides revealed high sequence homology with the YFGAP (Yellowfin tuna Glyceraldehyde-3-phosphate dehydrogenase-related Antimicrobial Peptide); thus, this peptide was identified as the skipjack tuna GAPDH-related antimicrobial peptide (SJGAP). SJGAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 1.2–17.0 μg/mL), Gram-negative bacteria, such as Aeromonas hydrophila, Escherichia coli D31, and Vibrio parahaemolyticus (MECs, 3.1–12.0 μg/mL), and against Candida albicans (MEC, 16.0 μg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide is heat-stable but salt-sensitive. According to the secondary structural prediction and the homology modeling, this peptide consists of three secondary structural motifs, including one α-helix and two parallel β-strands, and forms an amphipathic structure. This peptide showed neither membrane permeabilization ability nor killing ability, but did display a small degree of leakage ability. These results suggest that SJGAP acts through a bacteriostatic process rather than bactericidal one. SJGAP is another GAPDH-related antimicrobial peptide isolated from skipjack tuna and likely plays an important role for GAPDH in the innate immune defense of tuna fish.     

Gene cloning and characterization of novel antinociceptive peptide from the brain of the frog, Odorrana grahami

Amphibian opiate peptides including dermorphins and deltorpins have been recently found only in the skin of South American frogs belonging to the subfamily Phyllomedusinae (Phyllomedusa, Agalychnis and Pachymedusa species). No opiate peptides have ever been identified from other amphibians or organs except skin. Here we report the purification and characterization of a novel antinociceptive peptide named odorranaopin from the homogenates of the frog brains, Odorrana grahami, which is also the first antinociceptive peptide found in Ranidae amphibian. Odorranaopin comprises 17 amino acid residues with the sequence of DYTIRTRLHQESSRKVL (Mr 2102 Da). The cDNA encoding odorranaopin was cloned from the frog brain cDNA library, and it was confirmed to be a specific gene. The odorranaopin precursor deduced is composed of 61 amino acid residues including the predicted signal peptide, acidic spacer peptide and mature odorranaopin positioned at the C-terminus. Odorranaopin could inhibit nociceptive responses induced by formalin and acetic acid. It also inhibited the contractile responses of ileum smooth muscle induced by bradykinin, implying that the antinociceptive activity of odorranaopin possibly results from its blockade on bradykinin or bradykinin receptor functions. Odorranaopin is the first antinociceptive peptide found in Ranidae amphibian.     

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.     

A family of antimicrobial and immunomodulatory peptides related to the frenatins from skin secretions of the Orinoco lime frog Sphaenorhynchus lacteus (Hylidae)

Peptidomic analysis of norepinephrine-stimulated skin secretions of the Orinoco lime tree frog Sphaenorhynchus lacteus (Hylidae, Hylinae) revealed the presence of three structurally related host-defense peptides with limited sequence similarity to frenatin 2 from Litoria infrafrenata (Hylidae, Pelodryadinae) and frenatin 2D from Discoglossus sardus (Alytidae). Frenatin 2.1S (GLVGTLLGHIGKAILG.NH₂) and frenatin 2.2S (GLVGTLLGHIGKAILS.NH₂) are C-terminally α-amidated but frenatin 2.3S (GLVGTLLGHIGKAILG) is not. Frenatin 2.1S and 2.2S show potent bactericidal activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MIC ≤16 μM) but are less active against a range of Gram-negative bacteria. Frenatin 2.1S (LC₅₀ = 80 ± 6 μM) and 2.2S (LC₅₀ = 75 ± 5 μM) are cytotoxic against non-small cell lung adenocarcinoma A549 cells but are less hemolytic against human erythrocytes (LC₅₀ = 167 ± 8 μM for frenatin 2.1S and 169 ± 7 μM for 2.2S). Weak antimicrobial and cytotoxic potencies of frenatin 2.3S demonstrate the importance of C-terminal α-amidation for activity. Frenatin 2.1S and 2.2S significantly (P < 0.05) increased production of proinflammatory cytokines IL-1β and IL-23 by lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and frenatin 2.1S also enhanced production of TNF-α. Effects on IL-6 production were not significant. Frenatin 2.2S significantly downregulated production of the anti-inflammatory cytokine IL-10 by LPS-stimulated cells. The data support speculation that frenatins act on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms. They may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.     

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.     

Two novel families of antimicrobial peptides from skin secretions of the Chinese torrent frog, Amolops jingdongensis

The characterization of new natural antimicrobial peptides (AMPs) can help to solve the serious problem of bacterial resistance to currently used antibiotics. In the current study, we analyzed two families of AMPs from the Chinese torrent frog Amolops jingdongensis with a range of bioactivities. The first family of peptides, named jindongenin-1a, is 24 amino acids in length; a BLAST search of jindongenin-1a revealed no sequence similarity with other AMPs. The second family consists of two peptides containing 29 amino acid residues each. These peptides have high sequence similarity with the AMPs of palustrin-2 and are therefore designated palustrin-2AJ1 and palustrin-2AJ2. The cDNA sequences encoding these AMPs have been cloned and the deduced protein sequence of each AMP has been determined by protein sequencing. Sequence and structural analysis showed that each precursor is composed of a putative signal peptide, an N-terminal spacer, a processing site and a disulfide-bridged heptapeptide segment at the C-terminus. We synthesized jindongenin-1a and palustrin-AJ1 to test their antimicrobial, hemolytic, antioxidative and cytotoxic activities. These two peptides showed broad-spectrum antimicrobial activity to standard and clinically isolated strains of bacteria. In addition, they exhibited weak hemolytic activity to human and rabbit erythrocytes under our experimental conditions. Moreover, these peptides also displayed cytotoxic activity against the K562 and HT29 mammalian cell lines and low anti-oxidant activity. These findings provide helpful insight that will be useful in the design of anti-infective peptide agents.