Efeito da carga dos lipídios na interação do BP100 em modelos de membrana / Charge effect of lipids in interaction of BP100 and model membranes

Mariana Canale Manzini

Antimicrobial peptides (AMPs) have been a promising alternative therapy to antibiotics. These molecules, consisting of less than 80 amino acids, with different structures and amino acids composition, have amphipathic character which gives them the ability to act on lipid bilayers. They act, by different mechanisms, over bacteria to mammal cells. The AMPs are found in all kinds of living creatures, bacteria, plants, insects and mammals. Two important antimicrobial peptides have been studied because of their high efficiency: the Cecropin A, a non-hemolytic peptide which undergoes degradation by proteases, and Melittin, which has high antimicrobial potential but with hemolytic activity. In order to obtain peptides with better therapeutic action, hybrids peptides of Cecropin and Mellitin were synthesized. One of them, BP100, which has the sequence KKLFKKILKYL-NH2, is a target of several studies because it acts on bacteria´s membrane causing it´s death, but do not cause hemolysis and has a high selectivity for negatively charged membranes, characteristic of bacterial membranes. Here we study the properties of BP100 and its interaction with large unilamellar vesicles, LUVs, prepared with egg phosphatidylcholine (PC) and phosphatidylglycerol (PG) by extrusion. Fluorescence techniques, circular dichroism, CD, electrophoretic mobility, dynamic light scattering and optical microscopy with giant vesicles (GUVs) were used in order to obtain informations about its action mechanisms. Our results showed that BP100 is able to cause leakage of vesicles prepared with different mixtures of PC:PG, even at high ionic strength. The increase in negative charge of the vesicles significantly raises the activity of the peptide whereas the presence of cholesterol decreases its activity. The secondary structure of BP100 was studied by CD technique in water and a random coil structure was observed. In the presence of PC vesicles, BP100 did not change its structure. However, it aquires a typical alpha-helix structure in the presence of negative vesicles of PC:PG. Analysis of hydrodynamic diameter of LUVs in the presence of BP100 showed that BP100 didn\ t cause significant change in PC vesicles diameter but,with anionic vesicles at low ionic strength, large aggregates are formed. BP100 also changed the electrophoretic mobility of both LUVs containing only PC and mixtures of PC:PG, confirming the binding of BP100 at all membranes. The data set obtained is suggestive that BP100 acts binding to the vesicle membrane forming pores through which the internal contents overflows. The negative surface of the vesicle induces BP100 to acquire an α -helix conformation which favors the formation of a pore structure. BP100 also forms aggregates at the membrane surface of giant lipid vesicles (GUVs) and reduce its diameter. Aggregation of vesicles, formation of buds on the vesicle´s surface and finally the rupture and leakage of internal contents was also observed in GUVs of PC and PC:PG

Efeito da carga dos lipídios na interação do BP100 em modelos de membrana / Charge effect of lipids in interaction of BP100 and model membranes

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