Association of E. coli alpha-Hemolysin with lipid rafts"

SABINA M. MATÉ; VANESA HERLAX.; LAURA BAKÁS

Universidad Nacional de Quilmes.

Taller; Taller de la Sociedad Argentina de Biofísica “Biofísica de macromoléculas: aspectos estructurales e implicancias biológicas y biotecnológicas”.; 2007

Sociedad Argentina de Biofísica.

Association of E.coli a-Hemolysin with lipid rafts 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 is an extracellular protein toxin (107 kDa) secreted by Escherichia coli that acts at the level of plasma membranes of target eukaryotic cells. Posttranslational modification of the protein with fatty acids which occur at two internal lysine residues (K564 y K690) is required for all known cytotoxic activities. In recent years, it has been recognized that a variety of pathogens and toxins interact with microdomains in the plasma membrane known as lipid rafts. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Certain bacterial toxins utilize rafts as a site for high affinity binding and oligomerization on the surface of host cells. Taking also into account that acylated proteins interact with these microdomains our objective was to study the putative association of HlyA with these microdomains on sheep erythrocytes. In a previous work we demonstrated that HlyA forms an oligomer on erythrocytes membranes. For this purpose cystein mutants of the toxin were derivatized with fluorescent probes ALEXA-488 or ALEXA-546 (fluorescein and rhodamine derivatives respectively) in order to evaluate the formation of the oligomer by Fluorescent Resonance Energy Transfer (FRET). Using the same technique we observed that FRET efficiency decreases when ghost erythrocytes were cholesterol depleted with b-methylcyclodextrin. Simultaneously the hemolytic activity of the toxin was measured comparing control erythrocytes with ones which were cholesterol depleted using egg SUV. A diminished in the hemolytic rate was observed for the cholesterol depleted erythrocytes. Both results suggest the implication of lipid rafts in the action mechanism of the toxin. Finally we determined whether HlyA physically associates with lipid rafts. Ghost erythrocytes were incubated with HlyA. Detergent insoluble 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 lipid rafts.