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

Abstractα-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

Novel evidence for the specific interaction between cholesterol and α-haemolysin of Escherichia coli

Several toxins that interact with animal cells present some kind of interaction with cholesterol (Cho) or sphingomyelin. In the present work we demonstrate that alpha hemolysin of E. coli (HlyA) interacts directly with Chocholesterol, resulting in one of the first reported toxins secreted by Gram negative bacteria and the first reported member of the RTX toxin family that participates in the interaction with this sterol. We have recently reported that HlyA became associated with detergent-resistant membranes enriched in sphingomyelin and Chocholesterol; moreover, after Cho depletion, toxin oligomerization and hence hemolytic activity diminishes. Considering these results we studied the insertion process by monolayer technique, finding that HlyA insertion into membranes is favouredfavored in sphingomyelin and Chocholesterol-containing membranes. Taking into account this result, the direct interaction with either of the lipids was studied by lipid dot blot, lysis inhibition and surface plasmon resonance assays. Results demonstrated that there isit exists a direct interaction between Chocholesterol and HlyA that seems to favoursfavors a conformational state of the protein that allows the correct insertion into the membrane and further oligomerization to pore formation.Fil: Vazquez, Romina Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Bioquímicas de La Plata; ArgentinaFil: Maté, Sabina María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Bioquímicas de La Plata; ArgentinaFil: Bakás, Laura S.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Bioquímicas de La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: Fernández, Marisa Mariel. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Malchiodi, Emilio Luis. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Herlax, Vanesa Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Bioquímicas de La Plata; Argentin