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

Microvesicle release and micellar attack as the alternative mechanisms involved in the red-blood-cell-membrane solubilization induced by arginine-based surfactants

Two novel arginine-based surfactants, Bz-Arg-NHC10 and Bz-Arg-NHC12, were characterized with respect to surface properties and their interaction with human red-blood-cell (HRBC) membranes. The values for critical micellar concentration (CMC), the maximum surfactant adsorption at the air-liquid interface, and the area per molecule indicated better surface properties for Bz-Arg-NHC12. The observation of cylindrical worm-like aggregates of Bz-Arg-NHCnvia atomic-force microscopy supported the predictions based on the value of the surfactant-packing parameter (SPP). Erythrocyte-membrane solubilization was effected by surfactant aggregates since cell lysis became evident at only surfactant concentrations above the CMC. Changes in HRBC shape observed at different surfactant concentrations led to the conclusion that a slow mechanism based on the insertion of surfactant monomers into the HRBC membrane, followed by a shedding of microvesicles was responsible for the hemolysis produced by both surfactants at the lower concentrations tested. In contrast, the extraction of membrane lipids upon collisions between HRBCs and surfactant aggregates competes with and prevents microvesicle release at the higher concentrations assayed. © 2017 The Royal Society of Chemistry.Financial support of MINCyT (PICT 2013-00647), CONICET (PIP 0150) and UNLP (X11-682) are acknowledged. MEF and MH were awarded CONICET fellowship. HAA is UNLP Researcher. SRM and VH are members of CONICET Researcher Career. LB is member of CICPBA Researcher Career. EP is a member of the CONICET Support Professional Career Program. Dr Donald F. Haggerty, a retired academic career investigator and native English speaker, edited the final version of the manuscript.Peer reviewe

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

Histidine 19 Residue Is Essential for Cell Internalization of Antifungal Peptide SmAPα1-21 Derived from the α-Core of the Silybum marianum Defensin DefSm2-D in Fusarium graminearum

The synthetic peptide SmAPα1-21 (KLCEKPSKTWFGNCGNPRHCG) derived from DefSm2-D defensin α-core is active at micromolar concentrations against the phytopathogenic fungus Fusarium graminearum and has a multistep mechanism of action that includes alteration of the fungal cell wall and membrane permeabilization. Here, we continued the study of this peptide’s mode of action and explored the correlation between the biological activity and its primary structure. Transmission electron microscopy was used to study the ultrastructural effects of SmAPα1-21 in conidial cells. New peptides were designed by modifying the parent peptide SmAPα1-21 (SmAPH19R and SmAPH19A, where His19 was replaced by Arg or Ala, respectively) and synthesized by the Fmoc solid phase method. Antifungal activity was determined against F. graminearum. Membrane permeability and subcellular localization in conidia were studied by confocal laser scanning microscopy (CLSM). Reactive oxygen species (ROS) production was assessed by fluorescence spectroscopy and CLSM. SmAPα1-21 induced peroxisome biogenesis and oxidative stress through ROS production in F. graminearum and was internalized into the conidial cells’ cytoplasm. SmAPH19R and SmAPH19A were active against F. graminearum with minimal inhibitory concentrations (MICs) of 38 and 100 µM for SmAPH19R and SmAPH19A, respectively. The replacement of His19 by Ala produced a decrease in the net charge with a significant increase in the MIC, thus evidencing the importance of the positive charge in position 19 of the antifungal peptide. Like SmAPα1-21, SmAP2H19A and SmAP2H19R produced the permeabilization of the conidia membrane and induced oxidative stress through ROS production. However, SmAPH19R and SmAPH19A were localized in the conidia cell wall. The replacement of His19 by Ala turned all the processes slower. The extracellular localization of peptides SmAPH19R and SmAPH19A highlights the role of the His19 residue in the internalization