Novel antimicrobial peptides from the skin secretion of the Patagonian specie Pleurodema thaul (Anura Leptodactylidae: Leiuperinae)

CANCELARICH NATALIA L.; RODRIGUES DE ARAUJO ALYNE; PLACIDO ALEXANDRA; SOUSA CARLA; PEREZ LUIS O.; BASSO NÉSTOR G.; LEITE JOSÉ R. S. A.; MARANI MARIELA M.

Parnaíba

Simposio; III Simposio latino-americano de Biotecnología do Nordeste y I Encontro luso brasileiro de Biotecnología; 2015

Universidad Federal do Piauí (UFPI) Núcleo de Pesquisa en Biodiversidade e Biotecnología (BIOTEC)

The search for new therapeutic options tocombat resistance to conventional antibiotics by certain pathogens, is of greatinterest because of threat to public health worldwide. Antimicrobial peptides(AMPs) have proven effective against a broad spectrum of pathogens and severalhave been approved for clinical use. These are obtained from various naturalsources being the skin of amphibians one of the most abundant.. Amphibiansbelonging to different families, genera and species store different sets of AMPswith varying chain lengths, net charge, hydrophobicity and spectrum of action.The Patagonian region has a broad biodiversity with more than XX species ofunexplored amphibians, so it is a promising source of new peptides.The objective of this work was theidentification and characterization of skin AMPs Patagonian frog Pleurodema thaul by mRNA isolation, cDNAcloning and sequencing. Adult specimen of P. thaul was collected in Llao LlaoMunicipal Park wetland meadow, San Carlos de Bariloche, Río Negro, Argentina.Total RNA was extracted using Trizol reagent and Agarose gel andspectrophotometer analysis techniques were used for quality and quantityanalysis of the RNA. Reactions of 3′RACE were performed for cDNA synthesis. PCRproducts were purified using ADN PuriPrep-GP Kit. Purified fragments were usedin cloning and transfection of E. coliDH5α competent cells. Following the selection and growth of bacterial colonies,the resulting plasmids were purified and subjected to sequencing. The aminoacid sequence and the 3D structure of each of the peptides is deduced andsynthesized in solid phase. Preliminar test of antimicrobial activity andcitotoxicity were performed to select best candidate. Finally, tests ofcircular dichroism, antimicrobial activity, cytotoxicity, AFM and SurfacePlasmon Resonance assays were performed.Five new peptides, with similar 3Dstructures to other PAMs, were identified. Preliminar antimicrobial andcitotoxicity test demonstrates that Thaulin-1 was the best candidate. Secondarystructure analysis, by circular dichroism, exhibited an α-helix content. Thaulin-1inhibited E. coli, K. pneumoniae andS. aureus growth and AFM studiesdemonstrates morphological alterations in E.coli membrane structure. In addition, our citotoxicitystudies demonstrated that Thaulin-1 present acceptable levels of tolerance atMIC concentrations on eukaryotic cells. These findings support that Thaulin-1 is asuitable candidate for antimicrobial application and highlights the potentialof the Patagonian´s unexplored biodiversity as a source for new moleculediscovery.