INIBIOLP   05426
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE LA PLATA "PROF. DR. RODOLFO R. BRENNER"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Lipid raft are involved in alpha-Hemolysin of E.coli oligomerization
Autor/es:
SABINA MATÉ; VANESA HERLAX; LAURA BAKÁS
Lugar:
Acapulco, Mexico
Reunión:
Conferencia; 2nd Latin American Protein Society Meeting; 2007
Institución organizadora:
LAPS
Resumen:
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. 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.