Does a-hemolysin of E. coli form an oligomer structure when it is bound to membranes?

SABINA M. MATÉ; VANESA S. HERLAX; LAURA BAKÁS

Rosario

Congreso; XXXV Reunion Anual de la Sociedad Argentina de Biofísica; 2006

SAB

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. Postraslational modification of the protein with fatty acids is required for all known cytotoxic activities which occur at two internal lysine residues (K564 y K690).   This toxin promotes the formation of proteo-lipidic pores at lytic concentration rather than forming purely proteinaceous ionic channels.  We studied the interaction of this toxin with phospholipid membrane using artificial planar lipid membranes composed of asolectin. Addition of nanomolar concentrations of toxin resulted in an increase of bilayer conductance at a step concentration dependent fashion, suggesting that several toxin molecules could be involved in the conductive unit. Histograms of single channel conductance show a dependence with HlyA concentration indicating that the pore is not a unique rigid structure. To obtain conclusive information on the formation of the oligomer we used different cystein mutants of the toxin. These mutants were derivatized with fluorescent probes ALEXA-488 or ALEXA-546 (fluorescein and rhodamine derivatives) in order to study the formation of an oligomer on red blood cell membranes by Fluorescent Resonance Energy Transfer (FRET). It seems that the acyl chains may be involved in the oligomerization process, and acylation of K690 is an essential requisite. The oligomerization is temperature dependent as seen by the low efficiency of FRET when the experiment was done at 4ºC. On the other hand the FRET efficiency decreases when ghost erythrocytes where cholesterol depleted, indicating the role of ordered membrane microdomains for HlyA concentration on the membrane and thus for oligomerization.