INIBIOLP   05426
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE LA PLATA "PROF. DR. RODOLFO R. BRENNER"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Study of the hemolytic process of E.coli Ą- hemolysin
Autor/es:
VANESA HERLAX; SABINA MATÉ; LAURA BAKÁS
Lugar:
Buenos aires, Argentina
Reunión:
Workshop; 1st South-American Workshop on Advanced Fluorescence Microscopy Techniques; 2007
Institución organizadora:
UBA y Laboratory of Fluorescence Dynamics
Resumen:
a- hemolysin (HlyA) is a protein toxin (110 kDa) with a wide target cell specificity. It belongs to the so-called RTX (repeats in toxin) family, a series of protein toxins that contain a number of glycine and aspartate-rich nonapeptide tandem repeats near their C-terminal end (1). Synthesis, maturation, and secretion of E. coli HlyA are determined by the hlyCABD operon (2). The gene A product is a 110-kDa polypeptide corresponding to protoxin (ProHlyA), which is matured in cytosol to the active form (HlyA) by HlyC directed acylation at K 564 and K 690. Like other members of the family, HlyA is extracellularly secreted as a soluble protein, although an alternative secretion mechanism by outer membrane vesicles was recently identified (3) (Herlax, V., unpublished results). In the extracellular medium, HlyA must associate with calcium in order to bind to membranes in the lytically active form (4, 5). Once HlyA is activated, the toxin appears to have a two stage interaction with membranes: reversible adsorption, sensitive to electrostatic forces, and irreversible insertion (6). Moreover, ProHlyA, although non-acylated, also interacts with membranes. This is not surprising because the amino acid sequence of the polypeptide shows amphipathic helices in the amino acid 250-400 region. We found that the presence of two acyl chains in HlyA confers this protein with the property of irreversible binding to membranes, which is essential for the lytic process to take place (7).   On the other hand, we demonstrated that in soluble toxin, the fatty acids covalently bound promote the exposure of intrinsically disordered regions (8). In the context that these regions may participate in protein-protein interaction and that the toxin promotes the formation of proteo-lipidic pores at lytic concentration, we studied the formation of the oligomer on red blood cell membranes by Fluorescent Resonance Energy Transfer (FRET). For that purpose, we used different cystein mutants derivatized with fluorescent probes ALEXA-488 or ALEXA-546. Several controls were done to confirm that FRET efficiency measured corresponds to the formation of an oligomer and not to random distribution of the toxin in membranes. The results show that HlyA oligomerizases and that acyl chains are involved in this process, being acylation of K690 an essential requisite. In recent years, it has been recognized that a variety of pathogens and toxins interact with microdomains in the plasma membrane enriched in cholesterol and sphingolipids known as lipid rafts. 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 many acylated proteins interact with these microdomains our objective was to study the putative association of HlyA with these microdomains on sheep erythrocytes. 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. Immunoblot analysis and lipid characterization revealed that a substantial proportion of cell-associated toxin was associated with lipid rafts.