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

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

San Javier, Tucumán

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica (SAB)- Simposio BIOTOX; 2012

Sociedad Argentina de Biofísica

Escherichia coli α-hemolysin (HlyA) is a member of the pore forming RTX toxin family. Three stages seem to be involved in their mechanism of action that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin in the membrane. 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. 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. HlyA insertion into DOPC/16:0SM/Cho at an initial lateral pressure of 20mN/m and visualized by BAM, did not cause domain coalescence, but the mean gray intensity decreased over time in both phases of the mixture. No phase coexistence at surface pressure higher than 4mN/m was observed by BAM for DOPC/ 24:1SM/Cho. 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. We can conclude that HlyA does not insert into SMCho 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.