Antimicrobial peptides from the skin secretions of the South-East Asian frog Hylarana erythraea (Ranidae)

doi:10.1016/j.peptides.2009.12.013

Peptides

Volume 31, Issue 4, April 2010, Pages 548-554

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Abstract

Peptidomic analysis of norepinephrine-stimulated skin secretions of the South-East Asian frog Hylarana erythraea (formerly Rana erythraea partim) has led to the identification of multiple peptides with antimicrobial activity. Structural characterization of the peptides demonstrated that they belong to the brevinin-1 (3), brevinin-2 (2), esculentin-2 (4), and temporin (1) families. The values in parentheses indicate the number of paralogs. In addition, a peptide (GVIKSVLKGVAKTVALG ML.NH₂) was isolated that shows some structural similarity to the brevinin-2-related peptides (B2RP) previously isolated from North American frogs of the genus Lithobates. A synthetic replicate of the species B2RP showed broad-spectrum growth inhibitory activity against reference strains of Escherichia coli (MIC = 12.5 μM), Staphylococcus aureus (MIC = 12.5 μM) and Candida albicans (MIC = 50 μM) and was active against multidrug-resistant clinical isolates of Acetinobacter baumannii (MIC in the range 6–12.5 μM). The hemolytic activity of the peptide was relatively low (LC₅₀ = 280 μM). Phylogenetic analysis based upon the amino acid sequences of 47 brevinin-2 peptides from 17 Asian species belonging to the family Ranidae provides support for the placement of H. erythraea in the genus Hylarana.

Introduction

Skin secretions from a variety of species of Anura (frogs and toads) contain peptides with broad-spectrum antibacterial and antifungal activities and the ability to permeabilize mammalian cells (reviewed in [9]). They constitute a component of the system of innate immunity that defends the animal against invasion by pathogenic microorganisms [19]. Such peptides have excited interest because of their potential for development into therapeutically valuable anti-infective agents [19], [22] and for their value as phylogenetic markers for gaining insight into the evolutionary history of the different frog families [10], [11]. The antimicrobial peptides themselves may be grouped together in families on the basis of limited similarities in amino acid sequence [6]. Skin secretions from a single species frequently contain several members of a particular peptide family with varying degrees of potency and selectivity towards microorganisms that are presumed to have arisen from multiple duplications of an ancestral gene [38].

Skin secretions from frogs belonging to the family Ranidae (“true frogs”) have proved to be a particularly rich source of antimicrobial peptides and more than 200 such peptides from approximately 60 species have been described (reviewed in [10], [11]). The Green Paddy frog Hylarana erythraea (Schlegel, 1837) (formerly classified as Rana erythraea) is a small (snout-vent length up to 45 mm for males and 75 mm for females), mainly nocturnal frog that is readily identified by a pair of cream-colored bands along the side of the body. It is widely distributed in South-East Asia (Cambodia, Laos, Vietnam, Java, Borneo, West and East Malaysia, Singapore, and Thailand) and has been introduced to the Philippines [17], [25]. Populations in Northern India that were formerly assigned to R. erythraea are now included within Hylarana tytleri [26]. The animal's natural habitats are tropical and subtropical moist lowland forests and freshwater marshes but the species has adapted well to human activity and is frequently found in agricultural areas, rural gardens, and irrigation ditches and ponds. The species is not currently considered to be threatened or endangered but declines due to loss of habitat and water pollution by agrochemicals have taken place [15].

This study describes the purification and structural characterization of 11 peptides with cytolytic activity against microorganisms from norepinephrine-stimulated skin secretions of H. erythraea. Nomenclature adopted for antimicrobial peptides from frogs of the Ranidae family follows recent guidelines [6]. Peptides from previously described families are given the suffix ER and isoforms are denoted by lower case letters e.g. brevinin-1ERa.

Section snippets

Collection of skin secretions

All experiments with live animals were approved by the Animal Research Ethics Committee of UAE University and were carried out by authorized investigators. Five adult and sub-adult specimens of H. erythraea (weights 6–12 g; sexes unknown) were collected in central Vietnam and imported into the U.S. by a U.S. Fish and Wildlife Service licensed importer. Each animal was injected via the dorsal lymph sac with norepinephrine hydrochloride (40 nmol/g body weight) and placed in a solution (100 ml) of

Purification of the peptides from H. erythraea

The elution profile on a preparative Vydac C-18 column of the norepinephrine-stimulated skin secretion from H. erythraea after partial purification on Sep-Pak cartridges, is shown in Fig. 1. Ten peaks (designated 1–10 in Fig. 1) were associated with differential antimicrobial activity. Under the condition of assay, material from peak 1 inhibited growth of E. coli only, peaks 8–10 inhibited growth of S. aureus only, and peaks 2–7 inhibited growth of both E. coli and S. aureus. Subsequent

Discussion

This study has demonstrated that antimicrobial peptides present in norepinephrine-stimulated skin secretions of H. erythraea belong to the well-characterized brevinin-1, brevinin-2, esculentin-2, and temporin families (Fig. 3). Brevinin-1 and brevinin-2 peptides were first identified in the skin of R. brevipoda porsa (reclassified as Pelophylax porosus) [24] but subsequent work has shown that members of the brevinin-1 family are widely distributed in both Eurasian and N. American species

Acknowledgments

This work was supported by an Interdisciplinary Grant (01-03-2-12/08) and a Faculty Support Grant from U.A.E. University. The authors thank Tibor Pal and Agnes Sonnevend, Department of Medical Microbiology, UAE University for supply of bacterial strains and help with the microbiological assays.

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Conlon's proposed nomenclature [13] may effectively avoid this confusion. Use of the primary sequences of antimicrobial peptides from amphibians as phylogenetic or taxonomic markers is well established [2,23,24]. The brevinin-1, brevinin-2, and esculentin-2 families antimicrobial peptides have been especially valuable as phylogenetic biomarkers for studying the relationships among Ranidae species [2,19,21,23,26].
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.

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Antimicrobial peptides from the skin secretions of the South-East Asian frog Hylarana erythraea (Ranidae)

Abstract

Introduction

Section snippets

Collection of skin secretions

Purification of the peptides from H. erythraea

Discussion

Acknowledgments

Comp Biochem Physiol

Mol Phylogenet Evol

Peptides

Regul Pept

Biochim Biophys Acta

Biochim Biophys Acta

Toxicon

Toxicon

Regul Pept

Lancet Infect Dis

Peptides

Lancet Infect Dis

Biochem Biophys Res Commun

Peptides

Biophys J

J. Biol. Chem.

Peptides

Biochem Syst Ecol

Biochimie

Peptides