Stellato, Marco Ignazio (2016) Physico-chemical characterization of model bio-membranes and their interaction with potential therapeutic peptides. [Tesi di dottorato]

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In the present Ph.D. thesis an extensive structural and functional study on model bio-membranes is presented. The first part of my research has been focused to understand how the lipid composition of the bio-membranes affects their biophysical properties and modulates their interactions with peptides. One of the biological processes involving lipid composition is the interaction between antimicrobial peptides (AMPs) and biological membranes. In fact, the selective interaction of AMPs with prokaryotic cells arises from the difference in the chemical composition between prokaryotic and eukaryotic membranes. Different mechanisms of membrane destruction have been proposed, depending on physico-chemical properties of AMPs and of the target bio-membranes. Among the large number of AMPs present in nature, Myxinidin, from hagfish (Myxine glutinosa L.), is a promising antimicrobial candidate due to its antibacterial activity against different pathogenic Gram negative and Gram positive bacteria. This thesis reports a comparative study of the interaction between Myxinidin and its mutant WMR with two model bio-membranes at different composition and complexity. In particular, in order to understand the role of lipid composition in the peptide-membrane interaction, two different models of bio-membranes have been studied mimicking P. aeruginosa and E. coli cell cytoplasmic membranes. The final goal was to elucidate the effect of amino acid residues substitutions of the peptides and the role of lipid composition on the antibacterial activity of Myxinidin and WMR against these two model bio-membranes.The collected data have allowed to recognize the AMPs specificity for a particular lipid composition and to propose a mechanism of membrane destabilization. In order to study the role of lipid composition in biological processes, another important model bacterial bio-membrane has been studied. In particular the work has been focused on a particular model of bio-membrane representative of Bradyrhizobium BTAi1 Gram negative bacterium, containing an unusual lipopolysaccharide (LPS) in which the lipid A is covalently linked to a hopanoid moiety The aim of this study was to understand the effect of this unique lipid A in modulating the stability and rigidity of the outer membrane of Bradyrhizobium BTAi1 strain. To obtain a wide physico-chemical characterization of the analyzed systems, a combined experimental strategy has been adopted, including spectroscopic and calorimetric techniques such as Circular Dichroism (CD) to study the secondary structure of peptides and its changes in lipid environment; Fluorescence to estimate the microenvironment of the peptides in the vesicles; Dynamic Light Scattering (DLS) to estimate the size and distribution of the liposomes in the absence and in the presence of peptides; (NMR) to obtain information about the conformation of the peptides in membrane environment; Electron Paramagnetic Resonance (EPR) to investigate the dynamics of the lipid hydrophobic tails in the bilayer; Differential Scanning Calorimetry (DSC) to understand the thermotropic behavior of liposomes and the effect of peptides on their phase transition; Isothermal Titration Calorimetry (ITC) to study the energetic of the interaction process between peptides and liposomes.

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