Boundary region between coexisting lipid phases as initial binding sites for escherichia coli alpha-hemolysin: a real-time study

SABINA MATÉ; ROMINA VAZQUEZ; VANESA HERLAX; MARÍA A. DAZA MILLONE; MARIA L. FANANI; BRUNO MAGGIO; MARIA E. VELA; LAURA S. BAKAS

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2014 vol. 1838 p. 1832 - 1841

0005-2736

Escherichia coli α-hemolysin (HlyA) is a member of the pore-forming Repeat-in Toxin (RTX) family (α-hemolysin (HlyA) is a protein toxin, member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action seems to involve three stages that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane. Since the influence of phase segregation on HlyA binding and insertion in lipid membranes is not clearly understood, we explored at the meso- and nanoscale?both in situ and in real-time?the interaction of HlyA with with lipid monolayers and bilayers. Our results demonstrate that HlyA could insert into monolayers of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/16:0SM/Cho) and DOPC/24:1SM/Cho. The time course for HlyA insertion was similar in both lipidic mixtures. HlyA insertion into DOPC/16:0SM/Cho monolayers, visualized by Brewster-angle microscopy (BAM), suggest an integration of the toxin into both the liquid-ordered and liquid-expanded phases. By atomic-force?microscopy imaging we reported the first direct visualization of HlyA insertion preferentially into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho, although lipid-packing defects arising at the interfaces between coexisting lipid phases function as initial binding sites for the toxin. Finally?and most significantly?we have visualized here the insertion of an acylated and detergent-resistant membrane-associated protein preferentially into Ld phases shedding light on a controversial point related with the interaction of these type of proteins with model membranes.