Ocellatin-PT Antimicrobial Peptides: Structural Characterization, Membrane Interactions and Anti-Leishmania Activity

SOUSA CARLA; OLIVEIRA MAYARA; ALVES GEORGINA; MARANI MARIELA M.; PLACIDO ALEXANDRA; DELERUE-MATOS MARÍA C.; GAMEIRO PAULA; KUCKELHAUS SELMA A.S.; TOMAS ANA M.; LEITE JOSÉ R. S. A.; EATON PETER

Porto

Encuentro; XV EPI 15° Encontro Peptidico Ibérico; 2016

Faculdade de Ciencias-Universidade do Porto

Antimicrobial peptides (AMPs) are ubiquitous molecules as a part ofinnate immunity. Ocellatins are a family of AMPs isolated from the skinsecretions of the genus Leptodactyluswith antibacterial activity. Recently, we isolated eight new ocellatin peptidesfrom the skin secretion of Leptodactyluspustulatus and determined their amino acid sequences by de novo sequencing and cDNA cloning. AllOcellatin-PT peptides exhibited low antimicrobial activity but caused membraneperturbation in E. coli. Moreover, therewas no haemolytic activity and cytotoxicity to murine fibroblasts at thedetermined minimal inhibitory concentrations (MICs). (Marani et al. 2015)In this work we continued the study of these Ocellatin-PT peptides,testing their activity against promastigotes and axenic amastigote forms of Leishmania infantum, describing theeffect of these peptides on parasite membranes using atomic force microscopy (AFM)and scanning electron microscopy (SEM), and testing their cytotoxicity against bonemarrow-derived macrophages. Moreover, we characterized by circular dichroism(CD) the secondary structure of Ocellatin-PT peptides and we further exploredthe different interaction affinity of these peptides for bacteria, parasite andmammalian cell membranes, using surface plasmon resonance (SPR). All peptides,except OcellatinPT2, showed moderate activity against promastigote forms of L. infantum, but only Ocellatin-PT4 andOcellatinPT6 showed activity against the axenic amastigote stage. AFM and SEMimages confirmed the perturbation of the membrane of L. infantum promastigotes caused by Ocellatin-PT1 and Ocellatin-PT8.Selectivity for parasite versus human cell was also determined, with all Ocellatin-PTpeptides showing low toxicity against bone-marrow macrophages. In summary,binding affinities calculated by SPR show a higher affinity of both Ocellatin-PT1and Ocellatin-PT8 for bacteria and Leishmanialipid membranes compared to mammalian lipid membranes.