Real-time study of the insertion of Alphahemolysin from E. coli into lipid bilayers and monolayers

SABINA MATÉ; VANESA HERLAX; ROMINA VAZQUEZ; MARIA L. FANANI; ANTONIETA DAZA MILLONE; MARÍA ELENA VELA ; LAURA BAKÁS

Tucuman

Congreso; XLI Reunion Annual Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

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 decreased over time in both phases of the mixture. DOPC/ 24:1SM/Cho visualized by BAM did not show phase coexistence at surface pressure higher than 4mN/m. HlyA insertion did not induce phase separation and the mean gray value of the unique phase decreased over the time assayed. 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 in the boundary of lo-ld phases, followed by ld phases, of supported planar bilayers (SPB) composed of DOPC/16:0SM/Cho. Upon the addition of HlyA to SPB of DOPC/24:1SM/Cho in situ AFM imaging revealed that HlyA was homogeneously inserted in the unique phase formed by this ternary lipid mixture. We can conclude that: - Defects and/or intrinsic instabilities in the bilayer may favor HlyA insertion. - HlyA does not insert 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.