CONICET | Buscador de Institutos y Recursos Humanos

Lipid rafts of erythrocytes are involved in the action mechanism of E. coli a-Hemolysin Sabina Mate a, Vanesa Herlaxa and Laura Bakásb a INIBIOLP, Fac. Cs. Médicas, UNLP, La Plata b Fac. Cs Exactas, UNLP, La Plata e-mail: sabinamate@atlas.med.unlp.edu.ar a-Hemolysin (HlyA) is an extracellular protein toxin (107 kDa) secreted by Escherichia coli that acts at the level of plasma membranes of target eukaryotic cells. Considering that certain bacterial toxins utilize lipid rafts as a site for high affinity binding and oligomerization on the surface of host cells our objective was to study the role of these microdomains in the action mechanism of HlyA. Using Fluorescent Resonance Energy Transfer (FRET) technique we demonstrated that HlyA forms an oligomer on erythrocytes membranes and that FRET efficiency decreases when ghost erythrocytes were cholesterol depleted with b-methylcyclodextrin. The cholesterol depletion was quantified by HPTLC and [4-14C]cholesterol incorporation. Simultaneously, we determined whether HlyA physically associates with lipid rafts. Ghost erythrocytes were incubated with HlyA. Detergent resistant membranes (DRMs) were obtained by incubation with Triton X-100 and sucrose density gradient ultracentrifugation. Inmunoblot analysis and lipid characterization revealed that a substantial proportion of cell-associated toxin was associated with DRMs. Instead, sheep erythrocytes treated with b-methylcyclodextrin show a mark decrease in the HlyA association with DRMs. Finally, the hemolytic activity of the toxin diminished when erythrocytes were cholesterol depleted using egg Small Unilamellar Vesicles. These results suggest the implication of lipid rafts in the oligomerization of the toxin on the sheep erythrocyte membranes.