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.     

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).     

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

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

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.     

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.     

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.     

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC₅₀ values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC₅₀ value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC₅₀ = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.     

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).     

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.     

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.     

Antimicrobial peptide defenses of the Tarahumara frog,Rana tarahumarae

Populations of the Tarahumara frog Rana tarahumarae have decreased markedly in recent years in the northern part of their range. Infection by the chytrid fungus Batrachochytrium dendrobatidis has been implicated in these declines. To determine whether antimicrobial peptides in the skin provide protection against this pathogen, norepinephrine-stimulated skin secretions were tested for their ability to inhibit growth of B. dendrobatidis in vitro. After concentration, crude mixtures of skin peptides inhibited the growth of the chytrid in a concentration-dependent manner. Proteomic analysis led to the identification and characterization of three peptides belonging to the brevinin-1 family of antimicrobial peptides and three belonging to the ranatuerin-2 family. The two most abundant peptides, ranatuerin-2TRa (GIMDSIKGAAKEIAGHLLDNLKCKITGC) and brevinin-1TRa (FLPVIAGIAANVLPKLFCKLTKRC), were active against B. dendrobatidis (MIC of 50 microM for ranatuerin-2TRa and 12.5 microM for brevinin-1TRa against zoospores). These data clearly show that antimicrobial peptides in the skin secretions of the Tarahumara frog are active against B. dendrobatidis and should provide some protection against infection. Therefore, the observed susceptibility of these frogs to this pathogen in the wild may be due to the effects of additional environmental factors that impair this innate defense mechanism, leading to the observed population declines.     

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.     

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.     

Leishmanicidal activity of synthetic antimicrobial peptides in an infection model with human dendritic cells

Different species of Leishmania are responsible for cutaneous, mucocutaneous or visceral leishmaniasis infections in millions of people around the world [14]. The adverse reactions caused by antileishmanial drugs, emergence of resistance and lack of a vaccine have motivated the search for new therapeutic options to control this disease. Different sources of antimicrobial molecules are under study as antileishmanial agents, including peptides with antimicrobial and/or immunomodulatory activity, which have been considered to be potentially active against diverse species of Leishmania [7] and [39]. This study evaluated the cytotoxicity on dendritic cells, hemolytic activity, leishmanicidal properties on Leishmania panamensis and Leishmania major promastigotes and effectiveness on parasite intracellular forms (dendritic cells infected with L. panamensis and L. major promastigotes), when each parasite in culture was exposed to different concentrations of a group of synthetic peptides with previously reported antimicrobial properties, which were synthesized based on their naturally occurring reported sequences. Dermaseptin, Pr-2 and Pr-3 showed inhibitory activity on the growth of L. panamensis promastigotes, while Andropin and Cecropin A (with a selectivity index of 4 and 40, respectively) showed specific activity against intracellular forms of this species. The activities of Andropin and Cecropin A were exclusively against the intracellular forms of the parasite, therefore indicating the relevance of these two peptides as potential antileishmanial agents. In the case of L. major promastigotes, Melittin and Dermaseptin showed inhibitory activity, the latter also showed a selectivity index of 8 against intracellular forms. These findings suggest Andropin, Cecropin A and Dermaseptin as potential therapeutic tools to treat New and Old World cutaneous leishmaniasis.     

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.     

Novel motifs in amino acid permease genes from Leishmania

Eight amino acid permease genes from the protozoan parasite Leishmania donovani (AAPLDs) were cloned, sequenced, and shown to be expressed in promastigotes. Seven of these belong to the amino acid transporter-1 and one to the amino acid polyamino-choline superfamilies. Using these sequences as well as known and characterized amino acid permease genes from all kingdoms, a training set was established and used to search for motifs, using the MEME motif discovery tool. This study revealed two motifs that are specific to the genus Leishmania, four to the family trypanosomatidae, and a single motif that is common between trypanosomatidae and mammalian systems A1 and N. Interestingly, most of these motifs are clustered in two regions of 50-60 amino acids. Blast search analyses indicated a close relationship between the L. donovani and Trypanosoma brucei amino acid permeases. The results of this work describe the cloning of the first amino acid permease genes in parasitic protozoa and contribute to the understanding of amino acid permease evolution in these organisms. Furthermore, the identification of genus-specific motifs in these proteins might be useful to better understand parasite physiology within its hosts.     

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.     

Peptides with antimicrobial and anti-inflammatory activities that have therapeutic potential for treatment of acne vulgaris

The pathogenesis of acne vulgaris is multifactorial involving infection of the pilosebaceous unit with Propionibacterium acnes and a cytokine-mediated inflammatory response. Five frog skin-derived antimicrobial peptides ([D4k]ascaphin-8, [G4K]XT-7, [T5k]temporin-DRa, brevinin-2GU, and B2RP-ERa), chosen for their low hemolytic activity against human erythrocytes, were assessed for their effects on the growth of clinical isolates of P. acnes and on the release of pro-inflammatory and anti-inflammatory cytokines from peripheral blood mononuclear (PBM) cells. All peptides inhibited the growth of P. acnes with the highest potency exhibited by [D4k]ascaphin-8 (minimum inhibitory concentration, MIC=3-12.5 μM). Release of TNF-α from concanavalin A (ConA)-stimulated PBM cells was significantly reduced by [D4k]ascaphin-8, [G4K]XT-7, brevinin-2GU, and B2RP-ERa (1 and 20 μg/ml) and by [T5k]temporin-DRa (20 μg/ml). Release of IFN-γ from unstimulated PBM cells was significantly reduced by [D4k]ascaphin-8 and brevinin-2GU (1 and 20 μg/ml). No peptide showed significant effects on Il-17 release. Release of the anti-inflammatory cytokines TGF-β, IL-4, and IL-10 from both unstimulated and ConA-treated PBM cells was significantly increased by [T5k]temporin-DRa and B2RP-ERa (1 and 20μg/ml). The potent activities of [D4k]ascaphin-8 and [T5k]temporin-DRa in inhibiting the growth of P. acnes and the release of pro-inflammatory cytokines, and in stimulating the release of anti-inflammatory cytokines suggest a possible therapeutic role in the treatment of acne vulgaris.     

The role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines

Amphibian species have experienced population declines and extinctions worldwide that are unprecedented in recent history. Many of these recent declines have been linked to a pathogenic skin fungus, Batrachochytrium dendrobatidis, or to iridoviruses of the genus Ranavirus. One of the first lines of defense against pathogens that enter by way of the skin are antimicrobial peptides synthesized and stored in dermal granular glands and secreted into the mucus following alarm or injury. Here, I review what is known about the capacity of amphibian antimicrobial peptides from diverse amphibians to inhibit B. dendrobatidis or ranavirus infections. When multiple species were compared for the effectiveness of their in vitro antimicrobial peptides defenses against B. dendrobatidis, non-declining species of rainforest amphibians had more effective antimicrobial peptides than species in the same habitat that had recently experienced population declines. Further, there was a significant correlation between the effectiveness of the antimicrobial peptides and resistance of the species to experimental infection. These studies support the hypothesis that antimicrobial peptides are an important component of innate defenses against B. dendrobatidis. Some amphibian antimicrobial peptides inhibit ranavirus infections and infection of human T lymphocytes by the human immunodeficiency virus (HIV). An effective antimicrobial peptide defense against skin pathogens appears to depend on a diverse array of genes expressing antimicrobial peptides. The production of antimicrobial peptides may be regulated by signals from the pathogens. However, this defense must also accommodate potentially beneficial microbes on the skin that compete or inhibit growth of the pathogens. How this delicate balancing act is accomplished is an important area of future research.     

Leishmania (Kinetoplastida): species typing with isoenzyme and PCR-RFLP from cutaneous leishmaniasis patients in Ethiopia

Cutaneous leishmaniasis (CL) is an increasing public health problem in Ethiopia. There is a concern that it is spreading with increased incidence. In this study, we used isoenzyme electrophoresis and internal transcribed spacer one (ITS1) PCR-RFLP techniques to identify Leishmania species from CL patients in Ethiopia. We obtained isolates from 55 localized cutaneous leishmaniasis (LCL), 3 diffused cutaneous leishmaniasis (DCL) and 36 biopsy samples from 34 LCL and 2 DCL cases from All Africa Leprosy and Tuberculosis Rehabilitation and Training Center (ALERT) and clinically diagnosed CL cases from Ochollo village. Both isoenzyme and ITS1 PCR-RFLP techniques showed that Leishmania aethiopica (L. aethiopica) was the aetiologic agent in all cases. Our study also showed that ITS1 PCR-RFLP could identify Leishmania species from biopsy samples and suggests the method could be used for epidemiological surveillance of leishmaniasis in Ethiopia and for species-specific diagnosis.