Elsevier

Peptides

Volume 24, Issue 7, July 2003, Pages 955-961
Peptides

Characterization of novel antimicrobial peptides from the skins of frogs of the Rana esculenta complex

https://doi.org/10.1016/S0196-9781(03)00193-1Get rights and content

Abstract

Rana esculenta is a hybridogenetic hybrid between Rana ridibunda and Rana lessonae and so is best considered as a complex of interbreeding species rather than a discrete single species. In this study, antimicrobial peptides were isolated from a pooled extract of the skins of specimens of the R. esculenta complex collected in the wild. In addition to several peptides belonging to the brevinin and esculentin families that have been previously isolated from skin secretions of a single specimen of R. esculenta, three newly described members of the brevinin-2 family (brevinin-2Ei, brevinin-2Ej, and brevinin-2Ek) and one member of the temporin family (temporin-1Ec) were purified and characterized. In addition, three structurally related peptides with no sequence similarity with antimicrobial peptides isolated from other species of ranid frogs, that potently and selectively inhibit the growth of the Gram-positive bacterium Escherichia coli (minimal inhibitory concentration (MIC<5 μM)), were identified. These peptides show limited amino acid sequence similarity to the homologous exon gene products that encode the N-terminal flanking peptides of preprocaerulein, preproxenopsin, and preprolevitide and so have been termed caerulein precursor-related fragments (CPRF-Ea, CPRF-Eb, and CPRF-Ec). The data suggest that there may be considerable polymorphism among specimens from different populations of the R. esculenta complex. It is proposed that the distribution and amino acid sequences of skin antimicrobial peptides may be useful markers for taxonomic classification of particular sub-populations and for an understanding of phylogenetic interrelationships.

Introduction

The common edible frog Rana esculenta Linnaeus 1758 is widely distributed throughout Europe from France to the Volga river basin [8]. Its taxonomic status is not completely certain but it is generally regarded as having a hybrid origin arising from the marsh frog, Rana ridibunda Pallas 1771 and the pool frog, Rana lessonae Camerano 1882 [21]. R. ridibunda is a relatively large frog (size range for adult specimens 74–94 mm) that is distinguishable from the smaller R. lessonae (42–71 mm), not only on the basis of body size, but by numerous morphological and biochemical features. Studies in the field indicate that the original hybridizations that produce R. esculenta are between female R. ridibunda and male R. lessonae. Thereafter, R. esculenta lineages are maintained largely by matings of R. esculenta females with R. lessonae males [6]. The ranges of the three species often overlap so that collection of specimens of the intermediate sized (54–89 mm) R. esculenta in the wild will almost inevitably contain individuals belonging R. ridibunda and R. lessonae species as well as the various hybrids. Hence, the term “R. esculenta complex” is used to describe the diverse interbreeding populations of the three species. Phylogenetic analysis of the nucleotide sequences of ribosomal DNAs indicates that the R. esculenta complex is an outgroup of other Holarctic and Neotropical Rana families [10].

A previous study by Simmaco et al. [18] has described the isolation of multiple antimicrobial peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families from the electrically-stimulated skin secretions of a single specimen of R. esculenta. Similarly, analysis of an extract of gastric tissue from pooled specimens of R. esculenta collected in the wild led to the isolation of four antimicrobial peptides of the brevinin-2 family, two of which had been previously described by Simmaco et al. [18] and two with novel amino acid sequences [24]. In the present study, a pooled extract of the skin of approximately 100 specimens belonging to the R. esculenta complex collected in the field was analyzed for the presence of antimicrobial peptides. In addition to several components that have already been characterized, seven previously undescribed peptides were isolated and their primary structures and antimicrobial properties determined.

Section snippets

Animals

Adult specimens of frogs belonging to the R. esculenta complex were collected in different regions of Albania by a commercial enterprise and shipped to the University of Rouen. Approximately 100 animals of both sexes were anaesthetized by immersion in crushed ice and sacrificed by decapitation and pithing. Skin was immediately removed, frozen in liquid nitrogen, and freeze-dried.

Tissue extraction

The dried tissue (27 g) was extracted by homogenization in ethanol/0.7 M HCl (3:1 v/v; 500 ml) at 0 °C using a Waring

Purification of the peptides

The skin extract, after partial purification on Sep-Pak C-18 cartridges, was chromatographed on a Vydac C-18 semipreparative reverse-phase HPLC column and the elution profile is shown in Fig. 1. Aliquots of the fractions were tested for their ability to inhibit growth of the Gram-negative bacterium, E. coli and the Gram-positive bacterium, S. aureus. Antimicrobial activity was associated with fractions with retention times between 54 and 78 min. Analysis of these fractions by electrospray mass

Discussion

The present study extends the work of Simmaco et al. [17], [18], [20] and has led to the isolation of seven previously undescribed antimicrobial peptides from the skins of frogs of the R. esculenta complex. The amino acid sequence of three peptides indicates that they are members of the brevinin-2 family, first identified in the skin of the Asian frog Rana brevipoda porsa [13]. Their primary structures are compared in Fig. 4 with those of brevinin-2 peptides previously isolated from the skin

Acknowledgements

This work was supported by a grant from the National Science Foundation (EPS-9720643). The authors thank Donald Babin and Eva Lovas, Creighton University Medical School for amino acid analyses and mass spectrometry measurements.

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