Vanesa Herlax

Relevance of Fatty Acid Covalently Bound to Escherichia coli α-Hemolysin and Membrane Microdomains in the Oligomerization Process

α-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of Escherichia coli that causes lysis of several mammalian cells, including erythrocytes of different species. HlyA is synthesized as a protoxin, pro-HlyA, which is activated by acylation at two internal lysines Lys-563 and Lys-689. It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes, but these experiments have yielded conflicting results about the structure of the pore. In this study, HlyA cysteine replacement mutant proteins of amino acids have been labeled with Alexa-488 and Alexa-546. Fluorescence resonance energy transfer measurements, employing labeled toxin bound to sheep ghost erythrocytes, have demonstrated that HlyA oligomerizes on erythrocyte membranes. As the cytotoxic activity is absolutely dependent on acylation, we have studied the role of acylation in the oligomerization, demonstrating that fatty acids are essential in this process. On the other hand, fluorescence resonance energy transfer and the hemolytic activity decrease when the erythrocyte ghosts are cholesterol-depleted, hence indicating the role of membrane microdomains in the clustering of HlyA. Simultaneously, HlyA was found in detergent-resistant membranes. Pro-HlyA has also been found in detergent-resistant membranes, thus demonstrating that the importance of acyl chains in toxin oligomerization is the promotion of protein-protein interaction. These results change the concept of the main role assigned to acyl chain in the targeting of proteins to membrane microdomains.

N-Nervonoylsphingomyelin (C24:1) Prevents Lateral Heterogeneity in Cholesterol-Containing Membranes

This study was conducted to explore how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture SM/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), focusing on the importance of the hydrophobic part of the SM molecule for domain formation. Atomic force microscopy (AFM) measurements showed that the presence of a double bond in the 24:1 SM molecule in mixtures with cholesterol (CHO) or in pure bilayers led to a decrease in the molecular packing. Confocal microscopy and AFM showed, at the meso- and nanoscales respectively, that unlike 16:0 and 24:0 SM, 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM force spectroscopy measurements, demonstrating that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep, and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights into how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specifically, the data suggest that unsaturated SM may help to keep membrane lipids in a homogeneous mixture rather than in separate domains.

Alpha Hemolysin Induces an Increase of Erythrocytes Calcium: A FLIM 2-Photon Phasor Analysis Approach

α-hemolysin (HlyA) from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin), a group of proteins that share genetic and structural features. HlyA is an important virulence factor in E. coli extraintestinal infections, such as meningitis, septicemia and urinary infections. High concentrations of the toxin cause the lysis of several cells such as erythrocytes, granulocytes, monocytes, endothelial and renal epithelial cells of different species. At low concentrations it induces the production of cytokines and apoptosis. Since many of the subcytolytic effects in other cells have been reported to be triggered by the increase of intracellular calcium, we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA. Calcium concentration was monitored using the calcium indicator Green 1, 2-photon excitation, and fluorescence lifetime imaging microscopy (FLIM). Data were analyzed using the phasor representation. In this report, we present evidence that, at sublytic concentrations, HlyA induces an increase of calcium concentration in rabbit erythrocytes in the first 10 s. Results are discussed in relation to the difficulties of measuring calcium concentrations in erythrocytes where hemoglobin is present, the contribution of the background and the heterogeneity of the response observed in individual cells.

Induction of eryptosis by low concentrations of E. coli alpha-hemolysin.

Uropathogenic strains of Escherichia coli deliver the toxin alpha-hemolysin (HlyA) to optimize the host environment for the spread of infection. It was reported that at high concentrations, the toxin forms pores in eukaryotic membranes, leading to cell lysis, while lower concentrations have appeared to interfere with host-cell-signaling pathways causing cell death by apoptosis. Nevertheless, what is not clear is how often HlyA reaches levels that are high enough to lyse host target cells during the course of an infection. In the present investigation, we demonstrate that a low toxin concentration induces the suicidal death of erythrocytes (eryptosis), the major cell type present in blood. Eryptosis is triggered both by an increment in intracellular calcium and by ceramide. Since we have previously demonstrated that a low concentration of HlyA induces an increase in intraerythrocyte calcium, in the present experiments we have shown that this ion activates calpains, which hydrolyze skeleton proteins such as spectrin, ankyrin, protein 4.1 and the electrophoretic Band-3 species, thus resulting in morphologic changes in the erythrocytes. We furthermore observed that a low toxin concentration induced the activation of endogenous sphingomyelinases that in turn increased the amount of ceramide in erythrocyte membranes. Both spectrin proteolysis and ceramide formation may cause the exposure of phosphatidylserine on the membrane so as to trigger a macrophage engulfment of the erythrocyte. By this means eryptosis may be an advantageous mechanism for removing defective erythrocytes before hemolysis.

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

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.

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

Several toxins that act on animal cells present different, but specific, interactions with cholesterol or sphingomyelin. In the present study we demonstrate that HlyA (α-haemolysin) of Escherichia coli interacts directly with cholesterol. We have recently reported that HlyA became associated with detergent-resistant membranes enriched in cholesterol and sphingomyelin; moreover, toxin oligomerization, and hence haemolytic activity, diminishes in cholesterol-depleted erythrocytes. Considering these results, we studied the insertion process, an essential step in the lytic mechanism, by the monolayer technique, finding that HlyA insertion is favoured in cholesterol- and sphingomyelin-containing membranes. On the basis of this result, we studied the direct interaction with either of the lipids by lipid dot blotting, lysis inhibition and SPR (surface plasmon resonance) assays. The results of the present study demonstrated that an interaction between cholesterol and HlyA exists that seems to favour a conformational state of the protein that allows its correct insertion into the membrane and its further oligomerization to form pores.

Interaction of acylated and unacylated forms of E. coli alpha-hemolysin with lipid monolayers: a PM-IRRAS study

Uropathogenic strains of Escherichia coli produce virulence factors, such as the protein toxin alpha-hemolysin (HlyA), that enable the bacteria to colonize the host and establish an infection. HlyA is synthetized as a protoxin (ProHlyA) that is transformed into the active form in the bacterial cytosol by the covalent linkage of two fatty-acyl moieties to the polypeptide chain before the secretion of HlyA into the extracellular medium. The aim of this work was to investigate the effect of the fatty acylation of HlyA on protein conformation and protein-membrane interactions. Polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) experiments were performed at the air-water interface, and lipid monolayers mimicking the outer leaflet of red-blood-cell membranes were used as model systems for the study of protein-membrane interaction. According to surface-pressure measurements, incorporation of the acylated protein into the lipid films was faster than that of the nonacylated form. PM-IRRAS measurements revealed that the adsorption of the proteins to the lipid monolayers induced disorder in the lipid acyl chains and also changed the elastic properties of the films independently of protein acylation. No significant difference was observed between HlyA and ProHlyA in the interaction with the model lipid monolayers; but when these proteins became adsorbed on a bare air-water interface, they adopted different secondary structures. The assumption of the correct protein conformation at a hydrophobic-hydrophilic interface could constitute a critical condition for biologic activity.

Dynamic regulation of extracellular ATP in Escherichia coli

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

The unfavorable lipid environment reduced caveolin-1 expression in apical membranes from human preeclamptic placentas.

Abstract Syncytialization process is associated with a reduction in the number of caveolas, and a decreased of caveolin-1 (Cav-1). Differentiation of syncytiotrophoblast affects the membranes phospholipid composition. Thus, disturbances in these processes are related to pathological conditions such as preeclampsia. Objective To analyse the lipid composition of the apical (MVM) and the basal (BM) membranes of syncytiotrophoblast and its relationship with Cav-1 expression in normal and preeclamptic placentas. Molecular expression of Cav-1 was determined in MVM and BM from normal and preeclamptic placentas and in detergent-resistant membranes (DRMs). Phospholipids were analyzed by thin layer chromatography. Cholesterol was also determined by enzymatic assay. Membrane fluidity was evaluated by electron paramagnetic resonance. Sphingomyelin (SM) molecular species were analyzed and quantified by gas-liquid chromatography and mass spectrometry. Cav-1 was significantly reduced in MVM from preeclamptic placentas. Regarding Cav-1 localization, it was barely detectable in syncytiotrophoblast but it was present in the endothelium. Western blots also showed a significantly decrease of Cav-1 in the apical DRMs from preeclamptic placentas. Lipid analysis showed an increase SM in MVM from preeclamptic placentas without changes in cholesterol. Preeclamptic MVM fluidity decreased significantly and we found an increase in C18:1 fatty acids of SM. We concluded that preeclamptic-MVMs are more rigid than normal ones, possible due to an increment on SM. Moreover, the increase of long and unsaturated SM molecular specie found in these vesicles may disrupt the ability of SM to assemble into lipid rafts in the luminal leaflet of the bilayer, creating an unfavorable environment for Cav-1.

Study of rabbit erythrocytes membrane solubilization by sucrose monomyristate using laurdan and phasor analysis

The study of surfactant and bio membranes interaction is particularly complex due to the diversity in lipid composition and the presence of proteins in natural membranes. Even more difficult is the study of this interaction in vivo since cellular damage may complicate the interpretation of the results, therefore for most of the studies in this field either artificial or model systems are used. One of the model system most used to study biomembranes are erythrocytes due to their relatively simple structure (they lack nuclei and organelles having only the plasma membrane), their convenient experimental manipulation and availability. In this context, we used rabbit erythrocytes as a model membrane and Laurdan (6-lauroyl-2-dimethylaminonaphthalene) as the fluorescent probe to study changes promoted in the membrane by the interaction with the sucrose monoester of myristic acid, β-d-fructofuranosyl-6-O-myristoyl-α-d-glucopyranoside (MMS). Surfactant and erythrocytes interaction was studied by measuring hemoglobin release and the changes in water content in the membrane sensed by Laurdan. Using two-photon excitation, three types of measurements were performed: Generalized Polarization (analyzed as average GP values), Fluorescence Lifetime Imaging, FLIM (analyzed using phasor plots) and Spectral imaging (analyzed using spectral phasor). Our data indicate that at sublytical concentration of surfactant (20μM MMS), there is a decrease of about 35% in erythrocytes size, without changes in Laurdan lifetime or emission spectra. We also demonstrate that as hemolysis progress, Laurdan lifetime increased due to the decrease in hemoglobin (strong quencher of Laurdan emission) content inside the erythrocytes. Under these conditions, Laurdan spectral phasor analyses can extract the information on the water content in the membrane in the presence of hemoglobin. Our results indicate an increase in membrane fluidity in presence of MMS.