Biophysical characterization of the insertion of two potent antimicrobial peptides-Pin2 and its variant Pin2[GVG] in biological model membranes

• Published in: Biochimica et biophysica acta. Biomembranes 2020-02, Vol.1862 (2), p.183105-183105, Article 183105
• Main Authors: Bertrand, Brandt, Munusamy, Sathishkumar, Espinosa-Romero, José-Francisco, Corzo, Gerardo, Arenas Sosa, Iván, Galván-Hernández, Arturo, Ortega-Blake, Iván, Hernández-Adame, Pablo Luis, Ruiz-García, Jaime, Velasco-Bolom, José-Luis, Garduño-Juárez, Ramón, Munoz-Garay, Carlos
• Format: Article
• Language: English
• Subjects: Animals; Antimicrobial Cationic Peptides - chemistry; Antimicrobial Cationic Peptides - pharmacology; Antimicrobial peptides; Bacteria; Biophysical characterization; Cell Membrane - chemistry; Cell Membrane - drug effects; Erythrocyte Membrane - drug effects; Fungi; Lipid membrane models; Lipid-protein interaction; Membrane Fluidity; Membrane Potentials; Pin2; Pin2[GVG]; Sterols - chemistry; Unilamellar Liposomes - chemistry; Viscosity
• ISSN: 0005-2736; 1879-2642
• DOI: 10.1016/j.bbamem.2019.183105

The aim of this study was to investigate the factors that govern the activity and selectivity of two potent antimicrobial peptides (AMPs) using lipid membrane models of bacterial, erythrocyte and fungal cells. These models were used in calcein liposome leakage experiments to explore peptide efficiency. The AMPs (Pin2 and its variant Pin2[GVG]) showed highest affinity towards the bacterial models in the nanomolar range, followed by the erythrocyte and fungal systems. The presence of sterols modulated the variant's selectivity, while the wild type was unaffected. Liposome leakage experiments with Fluorescein Isothiocyanate-dextran (FITC)-dextran conjugates indicated that pore size depended on peptide concentration. Dynamic Light Scattering revealed peptide aggregation in aqueous solution, and that aggregate size was related to activity. The interacting peptides did not alter liposome size, suggesting pore forming activity rather than detergent activity. Atomic Force Microscopy showed differential membrane absorption, being greater in the bacterial model compared to the mammalian model, and pore-like defects were observed. Electrophysiological assays with the Tip-Dip Patch Clamp method provided evidence of changes in the electrical resistance of the membrane. Membrane potential experiments showed that liposomes were also depolarized in the presence of the peptides. Both peptides increased the Laurdan Generalized Polarization of the bacterial model indicating increased viscosity, on the contrary, no effect was observed with the erythrocyte and the fungal models. Peptide membrane insertion and pore formation was corroborated with Langmuir Pressure-Area isotherms and Brewster Angle Microscopy. Finally, molecular dynamics simulations were used to get an insight into the molecular mechanism of action. [Display omitted] •Pin2 presented higher affinity than Pin2 [GVG] in all liposome models.•Sterols govern Pin2 [GVG] activity and selectivity.•Peptide activity was related to degree of peptide aggregation.•Pore size was dependent of peptide concentration.•Peptides formed toroidal pores.