Volume 324, number 2. 159-161 FEBS 12571 8 1993 Federation of European Biochemical SometIes 00145793/93/$6.00 June 1993 Novel antimicrobial peptides from skin secretion of the European frog zyxwvutsrq Rana esculen ta Maurizio Simmaco, Giuseppina Mignogna, Donatella Barra and Francesco Bossa Dipartimento di Science Biochimiche A Rossi Fanelli, Centro di Biologia Molecolare de1 Consiglio Nazionale delle Ricerche. lJniversit2 La Sapienza. 00185 Roma, Italy Received 13 April 1993 Three antimicrobial peptides were isolated from skin secretion of the European frog. Rana esculentu. Two of them show similarity to brevinm-1 and brevinm-2. respectively. two antimrcrobial peptides recently isolated from a Japanese frog [Mortkawa. N., Hagiwara, K. and Nakajima. T. (1992) Biochem. Biophys. Res. Commun 189. 1841901. The third one, named esculentin, is 46 residues long and represents a different type of peptide. All these peptides have as a common motif an intramolecular dtsulfide bridge located at the COOH-terminal end. The peptides from R. esculentn show dtstmctive antibacterial activity against representative Gram-negative and Gram-posmve bacterial species. In parttcular, esculentm is the most active against Staphy lococcus aureus. and has a much lower hemolytic activity. Antrbactertal peptide; Hemolysis; Amphtbtan skin: Ranu esculenfo 1. INTRODUCTION As has been known for quite some time, skin extracts of frogs contain peptides with antimicrobial activity. The first observations were made in Bombina [ 1,2], al- though a thorough description of the structure and ac- tivity of the bombinins and their precursors has only recently been achieved [3,4]. The magainins, another class of potent antimicrobial peptides, were isolated from skin secretion of Xenopus luevis [5]. Dermaseptin is an antimicrobial peptide which was isolated from skin of Plzyllomedusa suuvagei [6]. All these molecules have since been the subject of intense multidisciplinary re- search in order to clarify their mechanism of action, biosynthesis, activity towards different microorganisms and potential therapeutical applications [7.8]. In the course of a research project aimed at the isola- tion and pharmacological characterization of bradyki- nin-like peptides in methanolic extracts from the skin of the European green frog, Ranu esculenta, we have iso- lated a family of hydrophobic peptides which possessed cytolytic activity [9]. Preliminary structural analysis of these peptides aroused suspicion about their numerosity as being at least in part originated from artifacts due to the relatively crude extraction procedure. We therefore decided to investigate the possibility of obtaining a pep- tide fraction using a more physiological procedure and Correspondence address. F. Bossa, Dipartimento di Sctenze Biochtm- iche, Universita La Sapienza. Piazzale Aldo Moro 5, 00185 Roma, Italy. Fax: (39) (6) 444 0062. to undertake a detailed study of both its structural and lytic properties. Here we present our results on the structure and the antimicrobial and hemolytic activities of three different peptides isolated from skin secretion of Runu esculentu. While this paper was in preparation, the sequence of two related peptides was presented which were isolated from skin extracts of the Japanese frog Runu brevipodu porsu [lo]. ‘. MATERIALS AND METHODS The pepttde-containing secretion, induced by mild electrtcal shock. was collected from the surface of the skin of I specimen of R esculentu by washing the dorsal region of the frog with 120 ml of 0.9% NaCl. The solution was then lyophilized and redtssolved in 12 ml btdistilled water. l-ml ahquots were filtered and fractionated by HPCL on a Beckman model 332 system using a reverse-phase column (Aquapore RP-300. 7 mm x 250 mm. Brownlee Labs, Applied Biosystems) eluted with a 35 min-gradient of lo-70% acetomtrtle/isopropanol (4:l) m 0.2% (by vol ) trifluoroacetic acid, at a flow rate of 2.0 ml/mitt. Elutton was momtored on a Beckman 165 spectrophotometer at 220 nm. The effluent from three such HPLC separattons. developed under identtcal experimental condtttons. was collected in 2-ml fractions in the same set of tubes and lyophilized. A 1% ahquot of the material from each tube was then used for assay of btological activity. 2.2. Antrbacterlal USS~I‘S The antibacterial activtty was tested using an inhibttion zone assay on agarose plates seeded wtth Escherichm coli D21. Bacrllus megutrrrurt~ Bml 1 or Stuph~lococcus aweus Cowan I zyxwvutsrqponmlkjih [l 11. The peptide fractions (3 ~1) were placed in small wells of thin agarose plates con- taming rich medium and about 1 zyxwvutsrqponmlkjihgfedcbaZYXWV x 10” bacterial cells. The plates were incubated overnight at 30°C. The zones of inhibition were measured, and the lethal concentration (LC, the lowest concentration that inhib- Published by Elsevrer Science Puhhshers B V 159