Biochem. J. (2002) 368, 91–100 (Printed in Great Britain) 91 Temporin L : antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles Andrea C. RINALDI* 1 , Maria Luisa MANGONI†, Anna RUFO‡, Carla LUZI‡, Donatella BARRA†, Hongxia ZHAO§, Paavo K. J. KINNUNEN§, Argante BOZZI‡, Antonio DI GIULIO‡ and Maurizio SIMMACO† *Cattedra di Chimica Biologica, Dipartimento di Scienze Mediche Internistiche, Universita  di Cagliari, I-09042 Monserrato (CA), Italy, †Dipartimento di Scienze Biochimiche ‘‘ A. Rossi Fanelli ’’, Azienda Ospedale S. Andrea, and CNR Centro di Biologia Molecolare, Universita  ‘‘ La Sapienza ’’, I-00185 Roma, Italy, ‡Dipartimento di Scienze e Tecnologie Biomediche, Universita  dell ’Aquila, Via Vetoio, Loc. Coppito, I-67100 L ’Aquila, Italy, and §Helsinki Biophysics & Biomembrane Group, Department of Medical Chemistry, Institute of Biomedicine, P.O. Box 63, Haartmaninkatu 8, FIN-00014, Finland The temporins are a family of small, linear antibiotic peptides with intriguing biological properties. We investigated the anti- bacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH  ), isolated from the skin of the European red frog Rana temporaria. The peptide displayed the highest activity of temporins studied to date, against both human erythrocytes and bacterial and fungal strains. At variance with other known temporins, which are mainly active against Gram-positive bacteria, temporin L was also active against Gram- negative strains such as Pseudomonas aeruginosa A.T.C.C. 15692 and Escherichia coli D21 at concentrations comparable with those that are microbiocidal to Gram-positive bacteria. In ad- dition, temporin L was cytotoxic to three different human tumour cell lines (Hut-78, K-562 and U-937), causing a necrosis-like cell death, although sensitivity to the peptide varied markedly with the specific cell line tested. A study of the interaction of temporin INTRODUCTION Antimicrobial peptides are small gene-encoded peptides which show a broad range of activity against Gram-negative and Gram-positive bacteria, fungi, mycobacteria and some enveloped viruses [1]. Originally identified in insects, they have subsequently been extracted from plants, crustaceans, ascidians and vertebrates [2–6], and it is now clear that these molecules constitute a key component of the innate immune system in multicellular organisms across the evolutionary scale. Amphibian skin has proved to be an especially rich source of such peptides, which form a remarkably heterogeneous ensemble with a broad spec- trum of antimicrobial activity and little sequence similarity [7]. The peptides are normally stored in the dermal glands of Anurans (frogs and toads), and are released into skin secretions in a holocrine fashion upon stress or injury, acting as the first line of defence against invading pathogens. Bombinins and magainins, isolated from skin secretions of Bombina species [8,9] and Xenopus laeis [10] respectively, are well-known examples of amphibian antimicrobial peptides. In most cases, several peptides of the same family, with overlapping sequence and structural features but distinct spectra of antimicrobial activity, are present sim- ultaneously on a single specimen, protecting the animal from a wider range of pathogens. Recently, the synthesis of antimicrobial peptides in the skin of Rana esculenta was demonstrated to be Abbreviations used : BMAP, bovine myeloid antimicrobial peptide ; CFU, colony-forming units ; FITC-D 4/20/70, fluorescein isothiocyanate dextrans of 4, 20 and 70 kDa average molecular mass respectively ; Gal-ONp, 2-nitrophenyl β-D-galactoside ; LB, Luria Bertani broth ; LC, lethal concentration ; LPS, lipopolysaccharide ; PC, egg yolk L-α-phosphatidylcholine ; PEG, poly(ethylene glycol) ; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. 1 To whom correspondence should be addressed (e-mail rinaldiunica.it). L with liposomes of different lipid compositions revealed that the peptide causes perturbation of bilayer integrity of both neutral and negatively charged membranes, as revealed by the release of a vesicle-encapsulated fluorescent marker, and that the action of the peptide is modulated to some extent by membrane lipid composition. In particular, the presence of negatively charged lipids in the model bilayer inhibits the lytic power of temporin L. We also show that the release of fluorescent markers caused by temporin L is size-dependent and that the peptide does not have a detergent-like effect on the membrane, suggesting that per- turbation of bilayer organization takes place on a local scale, i.e. through the formation of pore-like openings. Key words : cationic peptides, fluorescent markers, innate im- munity, liposomes, Rana temporaria. stimulated by micro-organisms, providing in io evidence for the induction of defence peptides in a vertebrate [11]. Spurred by the potential use of antimicrobial peptides in the treatment of infectious diseases that have become resistant to conventional antibiotics, an increasing public health problem, much research work has been conducted and is currently under way in order to understand the principles underlying the modes of interaction of these peptides with their target microbes. Although the exact mechanism(s) by which antimicrobial pep- tides exert their killing actions is not clearly understood, it is generally accepted that it involves interaction of the peptides with the cytoplasmic membrane of the target microbe, leading to membrane permeabilization and cell lysis and death [12,13]. However, it should be underlined that alternative andor co- existent antimicrobial mechanisms cannot be ruled out at this stage, and evidence is accumulating that some peptides might actually act by binding to intracellular targets, or by stimulating host defence mechanisms [14]. Focusing on membrane activity, a key problem is understanding the basis of the membrane-selective specificity of antimicrobial peptides, i.e. their ability to distinguish between prokaryotic versus eukaryotic cells, Gram-positive ver- sus Gram-negative bacteria, among the various bacterial strains, etc. Neither the impact of membrane composition nor the structural features of the peptides required for this specificity are as yet fully understood. The long-term goal in the field is to use  2002 Biochemical Society