Temporin-Ra

The skin of amphibians is widely exploited as rich sources of membrane active peptides that differ in chain size, polypeptide net charge, secondary structure, target selectivity and toxicity. In this study, two small antimicrobial peptides, temporin-Ra and temporin-Rb, originally isolated from the skin of the European marsh frog (Rana ridibunda), described as active against pathogen bacteria and presenting low toxicity to eukaryotic cells were synthesized and had their physicochemical properties and mechanism of action investigated. The temporin peptides were examined in aqueous solution and in the presence of membrane models (lipid monolayers, micelles, lipid bilayers and vesicles). A combined approach of bioinformatics analyses, biological activity assays, surface pressure measurements, synchrotron radiation circular dichroism spectroscopy, and oriented circular dichroism spectroscopy were employed. Both peptides were able to adsorb at a lipid-air interface with a negative surface charge density, and efficiently disturb the lipid surface packing. A disorder-to-helix transition was observed on the secondary structure of both peptides when either in a non-polar environment or interacting with model membranes containing a negative net charge density. The binding of both temporin-Ra and temporin-Rb to membrane models is modulated by the presence of negatively charged lipids in the membrane. The amphipathic helix induced in temporin-Ra is oriented parallel to the membrane surface in negatively charged or in zwitterionic lipid bilayers, with no tendency for realignment after binding. Temporin-Rb, instead, assumes a β-sheet conformation when deposited into oriented stacked lipid bilayers. Due to their short size and simple composition, both peptides are quite attractive for the development of new classes of peptide-based anti-infective drugs.

In this study, two novel antimicrobial peptides from the skin secretions of the marsh frog, Rana ridibunda, named temporin-Ra and temporin-Rb, were identified and purified using RP-HPLC. Temporin-Ra and temporin-Rb are composed of 14 and 12 amino acids, respectively. Our results show that these peptides have inhibitory effects on both gram-negative and gram-positive bacteria, especially antibiotic resistant strains prevalent in hospitals, such as Staphylococcus aureus and Streptococcus agalactiae. The sequences and molecular weights of these peptides were determined using tandem MS. The molecular masses were found to be 1242.5 Da for temporin-Rb and 1585.1 Da for temporin-Ra. Human red blood cells tolerated well exposure to temporin-Ra and temporin-Rb, which, at a concentration of 60 µg/ml, induced 1.3% and 1.1% hemolysis, respectively. MIC values of these peptides are suitable for potent antimicrobial peptides. The low hemolytic effect and wide-spectrum antimicrobial activity suggest a possible therapeutic application of these novel peptides.