Structure, Antimicrobial Activities and Mode of Interaction with Membranes of Bovel Phylloseptins from the Painted-Belly Leaf Frog, Phyllomedusa sauvagii

Zahid Raja,Pierre Nicolas,Christophe Piesse,Sonia André,Bruno Oury,Denis Sereno,Thierry Foulon,Ali Ladram,Hubert Vaudry

doi:10.1371/journal.pone.0070782

Abstract

Transcriptomic and peptidomic analysis of skin secretions from the Painted-belly leaf frog Phyllomedusa sauvagii led to the identification of 5 novel phylloseptins (PLS-S2 to -S6) and also of phylloseptin-1 (PSN-1, here renamed PLS-S1), the only member of this family previously isolated in this frog. Synthesis and characterization of these phylloseptins revealed differences in their antimicrobial activities. PLS-S1, -S2, and -S4 (79–95% amino acid sequence identity; net charge = +2) were highly potent and cidal against Gram-positive bacteria, including multidrug resistant S. aureus strains, and killed the promastigote stage of Leishmania infantum, L. braziliensis and L. major. By contrast, PLS-S3 (95% amino acid identity with PLS-S2; net charge = +1) and -S5 (net charge = +2) were found to be almost inactive against bacteria and protozoa. PLS-S6 was not studied as this peptide was closely related to PLS-S1. Differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles combined with circular dichroism spectroscopy and membrane permeabilization assays on bacterial cells indicated that PLS-S1, -S2, and -S4 are structured in an amphipathic α-helix that disrupts the acyl chain packing of anionic lipid bilayers. As a result, regions of two coexisting phases could be formed, one phase rich in peptide and the other lipid-rich. After reaching a threshold peptide concentration, the disruption of lipid packing within the bilayer may lead to local cracks and disintegration of the microbial membrane. Differences in the net charge, α-helical folding propensity, and/or degree of amphipathicity between PLS-S1, -S2 and -S4, and between PLS-S3 and -S5 appear to be responsible for their marked differences in their antimicrobial activities. In addition to the detailed characterization of novel phylloseptins from P. sauvagii, our study provides additional data on the previously isolated PLS-S1 and on the mechanism of action of phylloseptins.

Highlights

The dermaseptin superfamily contains several families of host defense peptides that are synthetized in the skin of Hylidae frogs [1,2,3]
Results cDNA cloning of phylloseptins-S from P. sauvagii RT-PCR experiments were performed using Phyllomedusa sauvagii skin exudate mRNA as template and specific oligonucleotides designed to the conserved region (59- and 39-UTR) of previously cloned precursors of the dermaseptin superfamily
The remaining five cDNAs correspond to novel phylloseptins-S and were designated as PLS-S2, -S3, -S4, -S5 and -S6 (Table 1). All these PLS-S precursors had the characteristic signature of the preproforms of the dermaseptin superfamily, i.e. a signal peptide ending with a Cys residue, an acidic propiece with a typical prohormone processing signal Lys-Arg at the C-terminus, and a single downstream copy of the mature peptide progenitor sequence

Summary

Introduction

The dermaseptin superfamily contains several families of host defense peptides that are synthetized in the skin of Hylidae frogs [1,2,3]. The peptides are small, 10 to 50 amino acid residues long, cationic, and act in different ways, disrupting and permeabilizing the anionic bacterial cell membrane is the most frequent [4,5,6,7]. These peptide families include the dermaseptins (stricto sensu), the plasticins, the dermatoxins, the phylloxins, the phylloseptins, the raniseptins, the caerins, the caerin-related peptides, the hyposins, the fallaxidins, the frenatins, and the aureins. The strategy that the frogs have evolved over millions of years for the generation and design of an enormous array of small peptide antibiotics, each with high potency and specificity for particular microorganisms, may be adaptable for use in vitro

Methods

Results

Conclusion