CONICET | Buscador de Institutos y Recursos Humanos

a-Hemolysin (HlyA) is a pore forming toxin secreted by pathogenic strains of Escherichia coli. The toxin is synthesized as a protoxin, pro-HlyA, which is matured in the cytosol to the active form by acylation at two internal lysines. This toxin with a wide target cell specificity is considered to be the prototype of a family of toxins called RTX (repeat in toxin), a series of protein toxins that share genetic and structural features. In this report, we studied the interaction of HlyA with lipid membranes using as experimental system sheep erythrocytes, one of the most studied target cell for this type of studies. A variety of pathogens, toxins and acylated proteins interact with microdomains enriched in cholesterol and sphingolipids (lipid rafts), therefore we studied the association of HlyA with these micro domains on sheep erythrocytes. Our experiments in functional properties of the toxin such as oligomerization and hemolitic activity studied by FRET and light scattering, respectively, using cholesterol- depleted erythrocytes indicate the possible interaction of the toxin with domain enriched in cholesterol and sphingolipids (lipid rafts). We study the effect of the toxin on the membrane fluidity using Laurdan Generalized Polarization (GP) images taken in a a two-photon Fluorescence Microscope. After incubation of the erythrocytes with sublytic concentration of the toxin, the GP images show a decrease in the GP values of the membrane, interpreted as a decrease of membrane fluidity when the toxin is bound, .and a decrease in the size of the erythrocytes .On the other side removal of cholesterol by CD show a decrease in the GP value indication of an increase in rigidity of the membrane. These results suggest that at sub-lytic concentration, the interaction of HlyA with the sheep erythrocytes membrane involve microdomains enriched in cholesterol and sphingolipids.