title: Boundary region between coexisting lipid phases as initial binding sites for Escherichia coli alpha-hemolysin: a real-time study.
abstract: α-Hemolysin (HlyA) is a protein toxin, a member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action of this toxin 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 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. Atomic-force-microscopy imaging reported that phase boundaries favor the initial binding of the toxin, whereas after a longer time period the HlyA becomes localized into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho. Our AFM images, however, showed that the HlyA interaction does not appear to match the general strategy described for other invasive proteins. We discuss these results in terms of the mechanism of action of HlyA.
authors.affiliation: Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), CCT-La Plata, CONICET, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 y 120, (1900) La Plata, Argentina. Electronic address: smate@med.unlp.edu.ar.
authors.forename: Sabina M
authors.initials: SM
authors.lastname: Maté
authors.affiliation: Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), CCT-La Plata, CONICET, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 y 120, (1900) La Plata, Argentina.
authors.forename: Romina F
authors.initials: RF
authors.lastname: Vázquez
authors.affiliation: Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), CCT-La Plata, CONICET, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 y 120, (1900) La Plata, Argentina.
authors.forename: Vanesa S
authors.initials: VS
authors.lastname: Herlax
authors.affiliation: Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Universidad Nacional de La Plata-CONICET, Sucursal 4 Casilla de Correo 16, 1900 La Plata, Argentina.
authors.forename: María A
authors.initials: MA
authors.lastname: Daza Millone
authors.affiliation: Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), UNC-CONICET, Dpto. de Química Biológica, Fac. Cs. Químicas, UNC, Haya de la Torre y Medina Allende, Ciudad Universtaria, Córdoba, Argentina.
authors.forename: María L
authors.initials: ML
authors.lastname: Fanani
authors.affiliation: Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), UNC-CONICET, Dpto. de Química Biológica, Fac. Cs. Químicas, UNC, Haya de la Torre y Medina Allende, Ciudad Universtaria, Córdoba, Argentina.
authors.forename: Bruno
authors.initials: B
authors.lastname: Maggio
authors.affiliation: Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Universidad Nacional de La Plata-CONICET, Sucursal 4 Casilla de Correo 16, 1900 La Plata, Argentina.
authors.forename: María E
authors.initials: ME
authors.lastname: Vela
authors.affiliation: Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), CCT-La Plata, CONICET, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 y 120, (1900) La Plata, Argentina; Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115, 1900 La Plata, Argentina.
authors.forename: Laura S
authors.initials: LS
authors.lastname: Bakás
mesh_terms: D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine
keywords: Liquid-disordered phase; Liquid-ordered phase; Monolayer; Pore-forming toxin; Supported lipid bilayer