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

Abstract

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 full-length 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.

Keywords: Aggregation; Antibacterial activity; Anuran antimicrobial peptides; Membrane active peptides; Targeted DNA sequencing.