Biophysical and structural studies of bioactive peptides derived from the piscidin family in anionic membrane models

Abstract

Antimicrobial peptides (AMPs) represent a class of biomolecules essential to the innate immune system, playing a crucial role in defense against microbial infections and being found in diverse organisms. Taking into consideration the current context of increasing antimicrobial resistance, AMPs emerge as promising therapeutic candidates for the development of new pharmacological agents. In this regard, the mechanism of action of these peptides has been the subject of investigations, with predominant focus on peptide-membrane interactions, which frequently involve phospholipids as lipid bilayer models. Bacterial membranes present a phospholipid composition distinct from eukaryotic cells, characterized by a predominantly anionic nature. This property, combined with the positive net charge of most AMPs, results in preferential and selective electrostatic interactions with microbial membranes. Structurally, AMPs exhibit disordered conformation in aqueous medium, acquiring a defined amphipathic α-helical structure in the presence of lipid interfaces. The primary sequence and three-dimensional arrangement of amino acid side chains are determinants for their secondary structure and antimicrobial activity. Most AMPs interact directly with microbial membranes through various proposed mechanisms, being able to destabilize the phospholipid bilayer and consequent cell lysis. In this regard, detailed analysis of how AMPs interact and modify the phospholipid bilayer structure is of particular interest. Another relevant aspect is the incorporation of D-amino acids in antimicrobial sequences. Although the occurrence of enantiomeric amino acids was considered rare in peptides of animal origin, D-residues have been identified in natural AMPs, conferring greater resistance to proteolytic degradation compared to L-analogs. Studies demonstrate that peptides containing D-amino acids, such as the L- and D-Phenylseptin forms (FFFDTLKNLAGKVIGALT-NH2), present differentiated antimicrobial activities, with the D-epimer exhibiting greater efficacy against S. aureus and X. axonopodis. Additional examples include Bombinin H and the opioid peptides Dermorphins and Deltorphins, all isolated from amphibians. In this context, this thesis investigates the membrane interaction mechanisms of two antimicrobial peptides isolated from Epinephelus coioides: piscidin-4s (ecPis-4s) and piscidin-2s (ecPis-2s), including the epimeric form D-ecPis-2s. Through an integrated set of structural and biophysical techniques, it is proposed how specific molecular characteristics—including positive charge distribution, presence of aromatic residues, and secondary structure formation—modulate biological activity and peptide-membrane interaction patterns.