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

CANCELARICH, N. L.; BASSO, N. G.; MARANI, M. M:

Teresina, Piauí

Congreso; III Simpósio Latino-Americano de Biotecnología do Nordeste; 2015

SLAB

The search for new therapeutic options to combat resistance to conventional antibiotics by certain pathogens is of great interest because of threat to public health worldwide. Antimicrobial peptides (AMPs) have proven effective against a broad spectrum of pathogens and several have been approved for clinical use. These are obtained from various natural sources being the skin of amphibians one of the most abundant.. Amphibians belonging to different families, genera and species store different sets of AMPs with varying chain lengths, net charge, hydrophobicity and spectrum of action. The Patagonian region has a broad biodiversity with more than 60 species of unexplored amphibians, so it is a promising source of new peptides.The objective of this work was the identification and characterization of skin AMPs Patagonian frog Pleurodema thaul by mRNA isolation, cDNA cloning and sequencing. Adult specimen of P. thaul was collected in Llao Llao Municipal Park wetland meadow, San Carlos de Bariloche, Río Negro, Argentina. Total RNA was extracted using Trizol reagent and Agarose gel and spectrophotometer analysis techniques were used for quality and quantity analysis of the RNA. Reactions of 3′RACE were performed for cDNA synthesis. PCR products were purified using ADN PuriPrep-GP Kit. Purified fragments were used in cloning and transfection of E. coli DH5α competent cells. Following the selection and growth of bacterial colonies, the resulting plasmids were purified and subjected to sequencing. The amino acid sequence and the 3D structure of each of the peptides is deduced and synthesized in solid phase. Preliminar test of antimicrobial activity and citotoxicity were performed to select best candidate. Finally, tests of circular dichroism, antimicrobial activity, cytotoxicity, AFM and Surface Plasmon Resonance assays were performed.Five new peptides, with similar 3D structures to other PAMs, were identified. Preliminar antimicrobial and citotoxicity test demonstrates that Thaulin-1 was the best candidate. Secondary structure analysis, by circular dichroism, exhibited an α-helix content. Thaulin-1 inhibited E. coli, K. pneumoniae and S. aureus growth and AFM studies demonstrates morphological alterations in E. coli membrane structure. In addition, our citotoxicity studies demonstrated that Thaulin-1 present acceptable levels of tolerance at MIC concentrations on eukaryotic cells. These findings support that Thaulin-1 is a suitable candidate for antimicrobial application and highlights the potential of the Patagonian´s unexplored biodiversity as a source for new molecule discovery.