INIBIOLP   05426
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE LA PLATA "PROF. DR. RODOLFO R. BRENNER"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Real-time study of the insertion of Alpha-hemolysin from E. coli into lipid bilayers and monolayers
Autor/es:
SABINA MATÉ; VANESA HERLAX; ROMINA VAZQUEZ; LAURA FANANI; M. ELENA VELA; LAURA BAKÁS
Reunión:
Congreso; Reunión anual de la Sociedad Argentina de Biofísica XLI; 2012
Resumen:
Escherichia coli α-hemolysin (HlyA), a member of the pore forming RTX toxin family, is an exotoxin that elicits a number of responses from mammalian target cell and also alters the membrane permeability of host cells, causing lysis and death. Three stages seem to be involved in the mechanism of action of HlyA, that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin in the membrane. In this concern we have previously found that irreversible insertion of the toxin into a membrane is favored by disordered liquid phases over gel or ordered liquid phases. However, we have also found that HlyA associates with detergent-resistant membranes (DRMs), enriched in SM and Cho. These apparently conflicting observations led us to examine the insertion of this toxin by an experimental system that allows the direct observation of this process. We explored at meso and nanoscale, in situ and in real-time, the influence of lipid phases on HlyA insertion into membranes. As first step in the study of HlyA membrane insertion we have examined the interaction of this toxin with lipid monolayers. HlyA was able to insert into monolayers of DOPC/16:0SM/Cho and DOPC/24:1SM/Cho. The time course of HlyA insertion was very similar in both mixtures. When surface topography of DOPC/16:0SM/Cho was visualized by Brewster Angle Microscopy (BAM), phase coexistence with a liquid ordered phase SM- Cho enriched, surrounded by a liquid expanded phase of DOPC was observed. The time course of insertion of HlyA into this lipid monolayer, at an initial lateral pressure of 20mN/m, did not cause domain coalescence, but the mean gray intensity diminished over time into both phases of the mixture. As further step we have followed HlyA insertion in lipid bilayers over time by Atomic force microscopy (AFM), providing nanometer lateral resolution and Ångström vertical resolution. We reported the first direct visualization of HlyA insertion preferentially into the boundary of lo-ld phases, followed by ld phases, of supported planar bilayers (SPB) composed of DOPC/16:0SM/Cho, using in situ and real time AFM. Yo lo sacaría porque dijiste arriba que vas a usa AFM. We can conclude that: - Defects and/or intrinsic instabilities in the bilayer may favor HlyA insertion. - HlyA does not recognize or bind to Cho that is sequestered into SM-Cho rich domains; however, HlyA insertion into de boundary of Lo-Ld domains leads to a change in the lipid organization at the interior of the Lo domains. - We corroborated a preferential insertion of the toxin into Ld phases.