Repair of tissue wounds is a fundamental process to re-establish tissue integrity and regular function. Importantly, infection is a major factor that hinders wound healing. Multicellular organisms have evolved an arsenal of host-defence molecules, including antimicrobial peptides (AMPs), aimed at controlling microbial proliferation and at modulating the host's immune response to a variety of biological or physical insults. In this brief review we provide the evidence for a role of AMPs as endogenous mediators of wound healing and their promising therapeutic potential for treatment of non-life threatening skin and other epithelial injuries.

A cDNA,library from the skin of Kuna temporaria has been screened using a cDNA fragment probe that encodes the signal peptide of the precursor of esculentin from the skin secretion of Rana esculentu. With this approach, the cDNAs encoding the precursors of three peptides were isolated. Subsequently, the peptides predicted from the sequence of the cloned cDNAs as well as several structurally related peptides could be isolated from the skin secretion of R. temporuria. These peptides, which were named temporins, have a length of 10-13 residues and show some sequence similarity to hemolytic peptides Natural and synthetic temporins have antibacterial activity against gram-positive bacteria, but they are not hemolytic. Temporins are the smallest antibacterial peptides hitherto found in nature.

The temporins are a family of small, linear antibiotic peptides with intriguing biological properties. We investigated the antibacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH2), 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 addition, 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 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 perturbation of bilayer organization takes place on a local scale, i.e. through the formation of pore-like openings.

Temporins, antimicrobial peptides of 10±13 residues, were isolated from secretions of Rana temporaria [Simmaco, M., Mignogna, G., Canofeni, S., Miele, R., Mangoni, M.L. & Barra, D. (1996) Eur. J. Biochem. 242, 788±792]. These molecules are specific to this amphibian species, which is also able to secrete on its skin other antimicrobial peptides similar to those found in different Rana species. The effect of temporins A, B and D (13 residues, net charge +2), and H (10 residues, net charge +1 and +2, respectively) against both artificial membranes of differing lipid composition and bacteria has been investigated in order to gain insight into their mechanisms of action. The results indicate that: the lytic activity of temporins is not greatly affected by the membrane composition; temporins A and B allow the leakage of large-size molecules from the bacterial cells; temporin H renders both the outer and inner membrane of bacteria permeable to hydrophobic substances of low molecular mass; and temporin D, although devoid of antibacterial activity, has a cytotoxic effect on erythrocytes. The results allow important conclusions to be drawn about the minimal structural requirements for lytic efficiency and specificity of temporins.

Temporins are short and homologous antimicrobial peptides (AMPs) isolated from the frog skin of Rana genus. To date, very little is known about the biological significance of the presence of closely related AMPs in single living organisms. Here we addressed this question using temporins A, B, and L isolated from Rana temporaria. We found that temporins A and B are only weakly active toward Gram-negative bacteria. However, a marked synergism occurs when each is mixed with temporin L. To shed light on the underlying mechanisms involved in these activities, we used various experimental strategies to investigate: (i) the effect of the peptides' interaction on both the viability and membrane permeability of intact bacteria and spheroplasts; (ii) their interaction with lipopolysaccharides (LPS) and the effect of LPS on the oligomeric state of temporins, alone or combining one with another; (iii) their structure in solution and when bound to LPS, by using circular dichroism and ATR-FTIR spectroscopies. Our data reveal that temporin L synergizes with A and B by preventing their oligomerization in LPS. This should promote their translocation across the outer membrane into the cytoplasmic membrane. To the best of our knowledge, this is the first study that explains how a combination of native AMPs from the same species can overcome bacterial resistance imposed by the LPS leaflet.

Antimicrobial peptides (AMPs) kill bacteria mainly through the perturbation of their membranes and are promising compounds to fight drug resistance. Models of the mechanism of AMPs-induced membrane perturbation were developed based on experiments in liposomes, but their relevance for bacterial killing is debated. We determined the association of an analogue of the AMP PMAP-23 to Escherichia coli cells, under the same experimental conditions used to measure bactericidal activity. Killing took place only when bound peptides completely saturated bacterial membranes (10 6 −10 7 bound peptides per cell), indicating that the "carpet" model for the perturbation of artificial bilayers is representative of what happens in real bacteria. This finding supports the view that, at least for this peptide, a microbicidal mechanism is possible in vivo only at micromolar total peptide concentrations. We also showed that, notwithstanding their simplicity, liposomes represent a reliable model to characterize AMPs partition in bacterial membranes.

Due to the rapid emergence of resistant microbes to the currently available antibiotics, cationic antimicrobial peptides have attracted considerable interest as a possible new generation of anti-infective compounds. However, low cost development for therapeutic or industrial purposes requires, among other properties, that the peptides will be small and with simple structure. Therefore, considerable research has been devoted to optimizing peptide length combined with a simple design. This review focuses on the similarities and differences in the mode of action and target cell specificity of two families of small peptides: the naturally occurring temporins from the skin of amphibia and the engineered ultrashort lipopeptides. We will also discuss the finding that acylation of cationic peptides results in molecules with a more potent spectrum of activity and a higher resistance to proteolytic degradation. Conjugation of fatty acids to linear native peptide sequences is a powerful strategy to engineer novel successful anti-infective drugs.

Antimicrobial peptides are produced by all organisms in response to microbial invasion and are considered as promising candidates for future antibiotics. There is a wealth of evidence that many of them interact and increase the permeability of bacterial membranes as part of their killing mechanism. However, it is not clear whether this is the lethal step. To address this issue, we studied the interaction of the antimicrobial peptide temporin L with Escherichia coli by using fluorescence, confocal and electron microscopy. The peptide previously isolated from skin secretions of the frog Rana temporaria has the sequence FVQWFSKFLGRIL-NH2. With regard to fluorescence microscopy, we applied, for the first time, a triple-staining method based on the fluorochromes 5-cyano-2,3-ditolyl tetrazolium chloride, 4',6-diamidino-2-phenylindole and FITC. This technique enabled us to identify, in the same sample, both living and total cells, as well as bacteria with altered membrane permeability. These results reveal that temporin L increases the permeability of the bacterial inner membrane in a dose-dependent manner without destroying the cell's integrity. At low peptide concentrations, the inner membrane becomes permeable to small molecules but does not allow the killing of bacteria. However, at high peptide concentrations, larger molecules, but not DNA, leak out, which results in cell death. Very interestingly, in contrast with many antimicrobial peptides, temporin L does not lyse E. coli cells but rather forms ghost-like bacteria, as observed by scanning and transmission electron microscopy. Besides shedding light on the mode of action of temporin L and possibly that of other antimicrobial peptides, the present study demonstrates the advantage of using the triple-fluorescence approach combined with microscopical techniques to explore the mechanism of membrane-active peptides in general.