Membrane-active antimicrobial peptide identified in Rana arvalis by targeted DNA sequencing

Antimicrobial peptides (AMPs) are naturally produced, gene encoded molecules with a direct antimicrobial activity against pathogens, often also showing other immune-related properties. Anuran skin secretions are rich in bioactive peptides, including AMPs, and we have reported a novel targeted sequencing approach to identify novel AMPs simultaneously in different frog species, from small quantities of skin tissue. Over a hundred fulllength peptides were identified from specimens belonging to five different Ranidae frog species, out of which 29 were novel sequences. Six of these were selected for synthesis and testing against a panel of Gram-negative and Gram-positive bacteria. One peptide, identified in Rana arvalis, proved to be a potent and broad-spectrum antimicrobial, active against ATCC bacterial strains and a multi-drug resistant clinical isolate. CD spectroscopy suggests it has a helical conformation, while surface plasmon resonance (SPR) that it may self-aggregate/oligomerize at the membrane surface. It was found to disrupt the bacterial membrane at sub-MIC, MIC and above- MIC concentrations, as observed by flow cytometry and/or visualized by atomic force microscopy (AFM). Only a limited toxicity was observed towards peripheral blood mononuclear cells (PBMC) with a more pronounced effect observed against the MEC-1 cell line.