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

Skin secretions of many tested anuran species have been shown to contain a variety of antimicrobial peptides (AMPs) acting directly towards pathogens, including multidrug resistant isolates, and also showing immunomodulatory properties [1]. We have selectively amplified transcripts likely to encode for AMPs, thus providing their sequences. After RNA extraction from frog skin tissue samples, cDNA synthesis followed by PCR amplification was performed. For this purpose, forward degenerate primers were designed based on highly conserved signal peptide regions [2], together with a reverse primer designed on the poly-A tail of mRNA. Signal peptide regions were derived from sequences deposited in DADP database [3] and transcriptome data in SRA database [4]. Resulting amplicons were size-selected and processed by ion semiconductor sequencing, obtaining several thousand sequencing reads. Those were then assembled into contigs representing nearly full-length AMP-encoding transcripts. Analysis of the assembled sequencing output allowed to identify more than a hundred full-length mature peptides from 5 different specimens belonging to 5 different frog species, mostly from Ranidae species. Based on appropriate biophysical properties (e.g. charge, hydrophobicity, amphipathicity) six of the most promising candidates were chosen for chemical synthesis and extensive characterization. All peptides were tested against a panel of Gram-negative and Gram-positive bacteria, as well on tumour cell lines. One peptide, identified in Rana arvalis, proved to be active against both reference ATCC strains and epithelial cancer cell lines, while less toxic for circulating MEC-1 cells in a range of concentrations several fold higher then minimal inhibitory concentration (MIC) and IC50 values. Furthermore, the peptide was found to disrupt the bacterial membrane even at sub-MIC concentrations, as observed by flow cytometry and visualised by atomic force microscopy (AFM). [1] A. Nijnik and R. Hancock, Emerg. Health Threats J. 2, (2009). [2] V. Tessera, F. Guida, D. Juretić, and A. Tossi, FEBS J. 279, 724 (2012). [3] M. Novković, J. Simunić, V. Bojović, A. Tossi, and D. Juretić, Bioinformatics 28, 1406 (2012). [4] Y. Kodama, M. Shumway, and R. Leinonen, Nucleic Acids Res. 40, D54 (2012).