Studies of the interaction between the antimicrobial peptide KHya1 and model membranes

Author(s)

Thais Azevedo Enoki

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Física (IF/SBI)
• Defense date: 2016-01-21

Examining board members

Maria Teresa Moura Lamy; Vera Bohomoletz Henriques; Amando Siuiti Ito; Elisabeth Andreoli de Oliveira; Joao Ruggiero Neto

• Advisor: Maria Teresa Moura Lamy; Karin do Amaral Riske

Abstract

Antimicrobial peptides are part of the innate defense immunity system of several plants and animals. In general, they exhibit strong activity against pathogen microorganisms, without affecting the host cells. The antimicrobial peptides selectivity against specific target pathogens is due to several factors, including the different lipid composition of prokaryotic and eukaryotic membranes: for instance, they can be distinguished by the presence or absence of negatively charged lipids at the cell surface, respectively. Therefore, the electrostatic interaction between cationic antimicrobial peptides and anionic membranes can play an important role in the selectivity and activity of these peptides. Here, we present a study of the antimicrobial peptide KHya1 with model membranes: liposomes prepared with controlled lipid composition that mimic the membrane outer leaflet of bacterial and eukaryotic cells. Peptide KHya1 (Ile - Phe - Gly - Ala - Ile - Leu - Phew - Leu - Ala - Leu - Gly - Ala - Leu - Lys - Ans - Leu - Ile - Lys - NH2) has a net charge of +4. KHya1 primary sequence originates from a modification of the sequence of Hylina1, a peptide isolated from the secretion of the skin of the frog Hipsiboas albopunctatus. Both peptides are known to exhibit effective action against bacteria and fungi. The interaction of peptide KHya1 with model membranes composed by neutral (DPPC, dipalmitoyl phosphatidyl choline), anionic (DPPG, dipalmitoyl phosphatidyl glycerol) and a mixture of both lipids (DPPC:DPPG, 1:1) was studied by the use of several different experimental techniques: differential scanning calorimetry (DSC), static and time-resolved fluorescence, using the peptide natural probe (Trp, Tryptophan) and an extrinsic bilayer probe (Laurdan), experiments of leakage of an entrapped fluorescent dye, dynamical light scattering (DLS), optical microscopy, electron spin resonance (ESR) and small-angle x-ray scattering (SAXS). The peptide KHya1 was found to interact differently with neutral and anionic membranes, located at different positions in the bilayer, and causing distinct membrane structural modifications. KHya1 preferentially interacts at the surface of neutral membranes, causing an average perturbation in the lipids. With DPPC, we also observed a larger partition of the peptide in aqueous solution, compared to the peptide aqueous partition in anionic lipid dispersions. In membranes composed of negatively charged lipids, the peptide KHya1 seems to be strongly anchored in a transmembrane position. SAXS results suggest that the peptide insertion causes membrane thinning, in both gel and fluid phases. For model systems composed by the mixture of neutral and anionic lipids, we observed that the peptide preferentially interacts with anionic lipids, and the changes the peptide causes in DPPC:DPPG vesicles are larger than those observed with pure anionic systems. This effect may be due to the larger peptide/PG molar ratio in DPPC:DPPG vesicles, and/or to lipid segregation caused by the peptide, and the consequent structural defects at the borders of neutral and anionic domains. Although KHya1 increases the permeability of neutral bilayers, optical microscopy and experiments of leakage of entrapped fluorescent dyes showed different mechanism of leakage for neutral and negatively charged bilayers. For high peptide concentrations, large pores are formed in anionic vesicles, leading to vesicle collapse. The new insights shown here about the different structural modifications caused by the antimicrobial peptide KHya1 in neutral and anionic vesicles can possibly explain the efficient action of this peptide against bacteria and its reduced effect in eukaryotic cells. (AU)

• FAPESP's process: 10/11671-5 - Interaction of antimicrobial peptides with model membranes.
• Grantee: Thais Azevedo Enoki Liarte
• Support Opportunities: Scholarships in Brazil - Doctorate