INTERACTION OF NOVEL INTRAGENIC ANTIMICROBIAL PEPTIDES WITH BIOMIMETIC BACTERIAL MEMBRANES

Intragenic antimicrobial peptides (IAPs) are internal sequences of proteins provided by a software that searches for short amino acid sequences within the human proteome with certain physical-chemical characteristics so that they have a potential antimicrobial activity. Among these, Hs02 is a 16-amino acid cationic IAP that exhibited significant antimicrobial activity and affinity to negatively charged model membranes and 13.2 is an analogue of Hs02 that lacks the three last residues of the C-terminus. In this work, the interaction of Hs02 and 13.2 with biomimetic membranes composed of both synthetic lipids and B. subtilis bacterial lipid extract were investigated with different biophysical approaches. To obtain the lipid extract of B. subtilis bacteria (PY79 wild type), an extraction protocol based on a two-phase ternary solvent mixture (chloroform, methanol and water) was employed, in which the lipid extract was recovered from the nonpolar phase. Optical microscopy was used to determine the line tension of giant unilamellar vesicles (GUVs) of B. subtilis lipid extract. This parameter gives the energy cost for maintaining a pore opened across the membrane, and therefore can be associated with membrane stability. The results showed a decreased value of around 25 pN, which indicates that pores in this membrane closes relatively slowly. Mixtures of a zwitterionic and anionic were used to mimic bacterial and tumoral membranes, and to investigate the changes in liposome size and zeta potential in the presence of both peptides. In parallel, the interaction between the peptides and liposomes was investigated by Isothermal Titration Calorimetry (ITC). Results show that the binding of the peptides to anionic vesicles leads to charge neutralization, concomitant with liposome aggregation and that the interaction is exothermic for 13.2, whereas an additional endothermic component is present for the analogue 13.2. ITC was also used to determine the temperature in which heat of partitioning of peptides into the membranes is zero. Optical microscopy of GUVs was used to compare the interaction of both peptides with GUVs composed of synthetic and bacterial lipid extract by determining the minimal peptide concentration that causes bursting of the majority of GUVs.