Short and unique membrane-active antimicrobial peptides from frog skin

Gene-encoded antimicrobial peptides (AMPs) are key components of the innate immune system in all species of life. Interestingly, several kinds of plants and animals can synthesize a considerable number of similar cationic AMPs. In Amphibia, temporins represent the largest family and are among the shortest (10 to 14 residues) AMPs, with up to ten isoforms within the same specimen (Simmaco, M., Mignogna, G., Canofeni, S., Miele, R., Mangoni, M. L., and Barra, D. (1996) Eur. J. Biochem. 242, 788-792). The biological significance of the presence of such isoforms in a single living organism is not clear. We addressed this question using temporins A, B, and L isolated from Rana temporaria skin secretion. We found that temporins A and B are weakly active towards Gram-negative bacteria, but a marked synergism occurs when each is mixed with temporin L. To understand the underlying mechanisms involved in these activities, we investigated: (i) the effect of the peptides’ interaction on both the viability and membrane permeability of intact bacteria and spheroplasts; (ii) their interaction and binding affinity to the major component of the outer membrane of Gram-negative bacteria, the anionic lipopolysaccharides (LPS), by means of various biophysical methods including spectroscopic measurements and calorimetric studies (iii) the effect of LPS on the oligomeric state of temporins, alone or combining one with each other; (iii) their structure in solution and when bound to LPS. Our results shed light into a new mechanism used by native AMPs to overcome bacterial resistance imposed by the LPS protective layer. Such studies should also contribute to the development of a new peptide-based anti-infective drugs, urgently needed because of the increasing microbial resistance to the available antibiotics.