Lipid rafts are involved in alpha-Hemolysin of E. coli oligomerization

SABINA MATÉ; VANESA HERLAX.; LAURA BAKÁS

Acapulco, Mexico.

Congreso; 2nd Latin American Protein Society Meeting en conjunto con el 1st Congreso de la Rama de Fisicoquímica, Estructura y Diseño de Proteínas de la Sociedad Mexicana de Bioquímica A.C.; 2007

Latin American Protein Society and Sociedad Mexicana de Bioquímica A.C.

Lipid rafts are involved in a-Hemolysin of E.coli oligomerization Sabina Mate a, Vanesa Herlaxa and Laura Bakásb aInstituto de Investigaciones Bioquímicas La Plata, INIBIOLP, Argentina b Instituto de Investigaciones Bioquímicas La Plata, INIBIOLP, Argentina; Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas,1900 La Plata Argentina 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 erythrocyte membranes and that FRET efficiency decreases when ghost erythrocytes were cholesterol depleted with b-methylcyclodextrin. Simultaneously, we determined that HlyA physically associates with lipid rafts. Ghost erythrocytes were incubated with HlyA and detergent resistant membranes (DRMs) were obtained by incubation with Triton X-100 and sucrose density gradient ultracentrifugation. Immunoblot analysis and lipid characterization revealed that a substantial proportion of cell-associated toxin was associated with DRMs. Instead, sheep erythrocytes treated with b-methylcyclodextrin showed a significant 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.