Ultrastructural analysis of the membrane insertion of domain 3 of streptolysin O

Streptolysin O (SLO) is a membrane-damaging toxic protein produced by group A streptococci. We performed an ultrastructural analysis of pore formation and the mechanism of hemolysis by SLO, using a mutant form of SLO [SLO(C/A)-SS] and native SLO. SLO(C/A)-SS was unable to penetrate the erythrocyte membrane as a consequence of immobilization that was due to a disulfide bond between domains. The SLO(C/A)-SS molecules that bound to membranes formed numerous single-layered ring-shaped structures that did not result in pores on the membranes. These structures were similar to the structures formed by native SLO at 0 degrees C. After treatment with dithiothreitol, SLO(C/A)-SS that had bound to membranes formed double-layered rings with pores on the membranes, as does native SLO at room temperature. Our morphological evidence demonstrates that an increase in temperature is necessary for the occurrence of conformational changes and for the formation of double-layered rings after the insertion of domain 3 into the host cell membrane. On the basis of a model of the oligomeric structure of SLO, we propose some new details of the mechanism of hemolysis by SLO.     

Effect of streptolysin O on erythrocyte membranes, liposomes, and lipid dispersions. A protein-cholesterol interaction

The effect of the bacterial cytolytic toxin, streptolysin O (SLO), on rabbit erythrocyte membranes, liposomes, and lipid dispersions was examined. SLO produced no gross alterations in the major erythrocyte membrane proteins or lipids. However, when erythrocytes were treated with SLO and examined by electron microscopy, rings and "C"-shaped structures were observed in the cell membrane. The rings had an electron-dense center, 24 nm in diameter, and the overall diameter of the structure was 38 nm. Ring formation also occurred when erythrocyte membranes were fixed with glutaraldehyde and OsO4 before the addition of toxin. In contrast, rings were not seen when erythrocytes were treated with toxin at 0 degrees C, indicating that adsorption of SLO to the membrane is not sufficient for ring formation since toxin is known to bind to erythrocytes at that temperature. The ring structures were present on lecithin-cholesterol-dicetylphosphate liposomes after SLO treatment, but there was no release of the trapped, internal markers, K2CrO4 or glucose. The crucial role of cholesterol in the formation of rings and C's was demonstrated by the fact that these structures were present in toxin-treated cholesterol dispersions, but not in lecithin-dicetylphosphate dispersions nor in the SLO preparations alone. The importance of cholesterol was also shown by the finding that no rings were present in membranes or cholesterol dispersions which had been treated with digitonin before SLO was added. Although rings do not appear to be "holes" in the membrane, a model is proposed which suggests that cholesterol molecules are sequestered during ring and C-structure formation, and that this process plays a role in SLO-induced hemolysis.     

A ring-shaped structure with a crown formed by streptolysin O on the erythrocyte membrane.

Streptolysin O (SLO) is a membrane-damaging toxin produced by most strains of group A beta-hemolytic streptococci. We performed ultrastructural analysis of SLO-derived lesions on erythrocyte membranes by examining electron micrographs of negatively stained preparations. SLO formed numerous arc- and ring-shaped structures with or without holes on membranes. Rings formed on intact cell membranes had an inner diameter of ca. 24 nm and had distinct borders of ca. 4.9 nm in width, but the diameter of rings varied from 24 to 30 nm on membranes of erythrocyte ghosts. Image analysis of electron micrographs demonstrated that each ring was composed of an inner and an outer layer. Each layer contained an array of 22 to 24 SLO molecules. On the top of the ring, we found a characteristic crown that projected from the cell membrane. The crown was separated by an electron-dense layer from the basal part of the ring that was embedded in the lipid bilayer of the erythrocyte membrane. Heights of the three parts, namely, the crown (head), the space (neck), and the basal portion (base), were ca. 3.2, 1.6, and 5.0 nm, respectively, and we postulated that these parts are the constituents of a single SLO molecule. The volumes of SLO molecules in the inner and outer layers were calculated to be 77 and 88 nm3. On the basis of a model of the structure of SLO, we propose some new details of the mechanisms of hemolysis by SLO toxin.     

Mechanism of haemolysis by Vibrio vulnificus haemolysin

The haemolytic action of Vibrio vulnificus haemolysin (VVH) was compared to that of streptolysin O (SLO). Both were cholesterol-binding haemolysins, but differed in the release of haemoglobin (Hb). In the first step of haemolysis, the haemolysins were temperature-independently bound to the cholesterol site on the target erythrocyte membrane. This was followed by the rapid release of K+, which is an intra-erythrocyte marker. Hb was then released, in different ways. In the case of VVH, Hb was released slowly after a relatively long lag, whereas with SLO, Hb was released as rapidly as K+. Haemolysis by VVH was inhibited by the addition of 30 mM-dextran 4 (mean Mr 4000), which is considered to be an effective colloid-osmotic protectant. The results therefore indicated that haemolysis by VVH (like that by Escherichia coli alpha-haemolysin and Staphylococcus aureus alpha-toxin) was caused by a colloid-osmotic mechanism. Both K+ and Hb release caused by VVH proceeded temperature-dependently, and the membrane fluidity of liposomes prepared with lipids extracted from sheep red blood cell membranes increased above 20 degrees C. These results suggest that the temperature-dependence of the haemolysis by VVH is due to the requirement for an increase in the membrane fluidity during the formation of a transmembrane pore.     

Specificity of streptolysin O in cytolysin-mediated translocation

Cytolysin-mediated translocation (CMT) is a recently described process in the Gram-positive pathogen Streptococcus pyogenes that translocates an effector protein of streptococcal origin into the cytoplasm of a host cell. At least two proteins participate in CMT, the pore-forming molecule streptolysin O (SLO) and an effector protein with the characteristics of a signal transduction protein, the Streptococcus pyogenes NAD-glycohydrolase (SPN). In order to begin to elucidate the molecular details of the translocation process, we examined whether perfringolysin O (PFO), a pore-forming protein related to SLO, could substitute for SLO in the translocation of SPN. When expressed by S. pyogenes, PFO, like SLO, had the ability to form functional pores in keratinocyte membranes. However, unlike SLO, PFO was not competent for translocation of SPN across the host cell membrane. Thus, pore formation by itself was not sufficient to promote CMT, suggesting that an additional feature of SLO was required. This conclusion was supported by the construction of a series of mutations in SLO that uncoupled pore formation and competence for CMT. These mutations defined a domain in SLO that was dispensable for pore formation, but was essential for CMT. However, introduction of this domain into PFO did not render PFO competent for CMT, implying that an additional domain of SLO is also critical for translocation. Taken together, these data indicate that SLO plays an active role in the translocation process that extends beyond that of a passive pore.     

Mechanism of human erythrocyte hemolysis induced by short-chain phosphatidylcholines and lysophosphatidylcholine

The incorporation and accumulation of a certain amount of short-chain phosphatidylcholine or lysophosphatidylcholine into lipid bilayers of erythrocyte membranes is the first step causing membrane perturbation in the process of hemolysis. Accumulation of dilauroylglycerophosphocholine into membranes makes human erythrocytes "permeable cells"; Ions such as Na+ or K+ can permeate through the membrane, though large molecules such as hemoglobin can not. The "pore" formation was partially reproduced in liposomes prepared from lipids extracted from human erythrocyte membranes; C12:0PC induced the release of glucose from liposomes but did not significantly induce the release of dextran. It was suggested that the phase boundary between dilauroylglycerophosphocholine and the host membrane bilayer or dilauroylglycerophosphocholine rich domain itself behaves as "pores." Erythrocytes could expand to 1.5 times the original cell volume without any appreciable hemolysis when incubated with C12:0PC at 37 degrees C. The capacity of the erythrocytes to expand was temperature dependent. The capacity may play an important role in the resistance of the cells against lysis. The "permeable cell" stage could be hardly observed when erythrocytes were treated with didecanoylglycerophosphocholine and lysophosphatidylcholine. Perturbation induced by accumulation of didecanoylglycerophosphocholine or lysophosphatidylcholine may cause non specific destruction of membranes rather than formation of a kind of "pore."     

Changes in erythrocyte morphology induced by imipramine and chlorpromazine

Chlorpromazine (CPZ), a phenothiazine derivative, is a potent antipsychotic agent and imipramine (IP) is a widely used tricyclic antidepressant. The interaction between these molecules and erythrocyte membranes is of particular interest considering the role of these cells in the transport and release of these drugs at the central nervous system. In the present paper, we intend to study the effects of IP on erythrocyte membranes and to compare these effects with those of CPZ. Erythrocytes from adult Sprague-Dawley rats were incubated separately with different concentrations of IP or CPZ for lh at room temperature, fixed and stained by Giemsa. Changes in erythrocyte morphology were quantified by an image analysis system. The interaction of both drugs, CPZ and IP, with the erythrocyte membrane causes similar changes in cell shape. Increasing concentrations of both drugs induces the formation of stomatocytes, spherostomatocytes and spherocytes, because of an irreversible loss of area and volume, probably due to endovesiculation. Our results also show that the CPZ is more potent than IP.     

A novel cholesterol‐insensitive mode of membrane binding promotes cytolysin‐mediated translocation by Streptolysin O

Cytolysin‐mediated translocation (CMT), performed by Streptococcus pyogenes, utilizes the cholesterol‐dependent cytolysin Streptolysin O (SLO) to translocate the NAD⁺‐glycohydrolase (SPN) into the host cell during infection. SLO is required for CMT and can accomplish this activity without pore formation, but the details of SLO's interaction with the membrane preceding SPN translocation are unknown. Analysis of binding domain mutants of SLO and binding domain swaps between SLO and homologous cholesterol‐dependent cytolysins revealed that membrane binding by SLO is necessary but not sufficient for CMT, demonstrating a specific requirement for SLO in this process. Despite being the only known receptor for SLO, this membrane interaction does not require cholesterol. Depletion of cholesterol from host membranes and mutation of SLO's cholesterol recognition motif abolished pore formation but did not inhibit membrane binding or CMT. Surprisingly, SLO requires the coexpression and membrane localization of SPN to achieve cholesterol‐insensitive membrane binding; in the absence of SPN, SLO's binding is characteristically cholesterol‐dependent. SPN's membrane localization also requires SLO, suggesting a co‐dependent, cholesterol‐insensitive mechanism of membrane binding occurs, resulting in SPN translocation.     

Inhibition of human breast cancer Matrigel invasion by Streptolysin O activation of the EGF receptor ErbB1

Streptolysin O (SLO) is a protein cytotoxin derived from Group A beta-hemolytic streptococci that associates with membranes and permeabilizes cells. Oxidation inactivates SLO, eliminating the characteristic hemolytic and cytotoxic activities. However, oxidized SLO produces beneficial therapeutic effects in vivo on scleroderma, scar formation and wound healing. Here we report that oxidized SLO also significantly inhibited invasion by human metastatic breast cancer MDA-MB-231 cells through Matrigel in an in vitro model of metastatic disease. This dose-dependent response corresponded to selective SLO activation of epidermal growth factor receptor (EGFR) ErbB1. SLO and EGF were equally selective in activation of EGFR, but EGF elicited larger relative increases in phosphorylation at various sites, especially pronounced for Tyr845. Addition of SLO did not affect either ERK1/2 or Akt kinases and altered the expression of only 10 of 84 metastasis-related genes in MDA-MB-231 cells. Neither SLO nor EGF promoted growth of several human breast cancer cell lines. Knockdown of EGFR by siRNA ablated the inhibitory effect of SLO on cancer cell invasion, showing SLO selectively activated ErbB1 kinase to reduce invasion without increasing cell growth. The results suggest SLO might have promise as a new therapy to inhibit metastasis.     

The cell resealing technique for manipulating,visualizing, and elucidating molecular functions in living cells

BackgroundCell-based assays are essential for analyzing molecular functions and spatiotemporal information. The cell resealing technique, in which pore-forming toxins are used to permeabilize cell membranes, enables the delivery of various membrane-impermeable molecules inside cells.Scope of reviewWe review the basics of the resealed cell system, including optimized protocols, assessment of cellular damage, and recovery following permeabilization of the membrane. Additionally, we introduce the streptolysin O (SLO)-type and listeriolysin O (LLO)-type resealing techniques. In SLO, the formation of larger pores (~30 nm) enables the passage of a wider range of molecules. Then, we discuss the advantages and applications of the semi-intact cell system, in which ongoing permeabilization is selected to maintain and analyze a specific cellular environment.Major conclusionsAs confirmed by the effective use of quantitative image analysis, the SLO-type resealing system is successful for establishing and phenotyping diabetic model cells by introducing cytosol from diabetic mice. The LLO-type resealing technique enables the delivery of mid-sized molecules with high efficiency and low damage. As each technique has specific advantages, understanding the characteristics of LLO and SLO is necessary for choosing the appropriate technique.General significanceSLO-type resealing is optimal for creating disease model cells and drug screening, especially lifestyle-related diseases. LLO-type resealing is expected to be suitable for screening mid-sized biological drugs. Semi-intact cells can contribute to elucidating various cellular phenomena that have remained intractable due to their complexity.     

Lipid organization in erythrocyte membrane microvesicles.

The aminophospholipids of microvesicles released from human erythrocytes on storage or prepared from erythrocyte ghosts by shearing under pressure are susceptible to the action of 2,4,6-trinitrobenzenesulphonic acid. The aminophospholipids of the former vesicles are also susceptible to attack by phospholipase A2. Under the same conditions, the aminophospholipids of erythrocytes undergo little reaction. This suggests that the phospholipids in microvesicle membranes are more randomly distributed than those in erythrocyte membranes. Measurements have also been made of the ability of filipin to react with the cholesterol of sealed and unsealed erythrocyte ghosts and of microvesicles prepared from them. From the initial rates of reaction, it was concluded that there is no preferential transfer of cholesterol molecules from one side of the bilayer to the other during the formation of the microvesicles.     

Proteolytic self-digestion of bovine erythrocyte membranes

"Self-digestion" of bovine erythrocyte membrane proteins was studied in isolated membrane preparations during prolonged incubation at 37 C. Protease activities associated with the membrane result in progressive degradation of all main erythrocyte membrane proteins, in particular spectrin and Band 3, and formation of lower molecular weight products which have been tentatively assigned to parent molecules. Membrane protein "self-digestion" occurs in a broad pH range (2-11), is inhibited by increased ionic strength and by inhibitors of metalloproteases, cysteine and serine proteases, and activated by low concentrations of SDS. "Self-digestion" also takes place in NaOH-stripped erythrocyte membranes. The activity of a protease involved in the "self-digestion", of apparent molecular weight of about 35,000, was renatured after SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins.     

Phosphorescent analysis of lipid peroxidation products in liposomes]

It was found that lipid peroxidation products incorporated into liposomes prepared from oxidized preparations of bovine heart phosphatidylcholine and the total lipid fraction of human erythrocyte membranes are able to phosphoresce at room temperature was studied. The temperature dependences of kinetic and spectral parameters of phosphorescence were measured. It is shown that mechanism of phosphorescence quenching of lipid chromophores has a dynamic nature. It is proposed to use endogenic molecules of the lipid peroxidation products capable of phosphorescence as intrinsic phosphorescence probes for studying the slow molecular dynamics of lipids in artificial and biological membranes in a millisecond range.     

Assembly mechanism of the oligomeric streptolysin O pore: the early membrane lesion is lined by a free edge of the lipid membrane and is extended gradually during oligomerization.

Streptolysin O (SLO) is a bacterial exotoxin that binds to cell membranes containing cholesterol and then oligomerizes to form large pores. Along with rings, arc-shaped oligomers form on membranes. It has been suggested that each arc represents an incompletely assembled oligomer and constitutes a functional pore, faced on the opposite side by a free edge of the lipid membrane. We sought functional evidence in support of this idea by using an oligomerization-deficient, non-lytic mutant of SLO. This protein, which was created by chemical modification of a single mutant cysteine (T250C) with N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid, formed hybrid oligomers with active SLO on membranes. However, incorporation of the modified T250C mutant inhibited subsequent oligomerization, so that the hybrid oligomers were reduced in size. These appeared as typical arc lesions in the electron microscope. They formed pores that permitted passage of NaCl and calcein but restricted permeation of large dextran molecules. The data indicate that the SLO pore is formed gradually during oligomerization, implying that pores lined by protein on one side and an edge of free lipid on the other may be created in the plasma membrane. Intentional manipulation of the pore size may extend the utility of SLO as a tool in cell biological experiments.     

Structural changes in erythrocyte membranes during cooling studied by a spin probe method

Peculiarities of structural changes in erythrocyte membranes during freezing (from -20 degrees to -50 degrees) were studied by electron paramagnetic resonance method using spin-labelled derivative of stearic acid-5-doxylstearate. It was established that membranes underwent a number of structural reconstructions due to the temperature decrease and water freezing-out. Differences were found in temperature dependences that characterize lipid ordering during probe insertion into membranes of native erythrocytes, white ghosts, and liposomes from total lipids of erythrocyte membranes. The data obtained indicate the impairment in the structure of lipid components and lipid-protein interactions in erythrocyte membranes during cooling.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

The Effect of PS Content on the Ability of Natural Membranes to Fuse with Positively Charged Liposomes and Lipoplexes

Supramolecular aggregates containing cationic lipids have been widely used as transfection mediators due to their ability to interact with negatively charged DNA molecules and biological membranes. First steps of the process leading to transfection are partly electrostatic, partly hydrophobic interactions of liposomes/lipoplexes with cell and/or endosomal membrane. Negatively charged compounds of biological membranes, namely glycolipids, glycoproteins and phosphatidylserine (PS), are responsible for such events as adsorption, hemifusion, fusion, poration and destabilization of natural membranes upon contact with cationic liposomes/lipoplexes. The present communication describes the dependence of interaction of cationic liposomes with natural and artificial membranes on the negative charge of the target membrane, charges which in most cases were generated by charging the PS content or its exposure. The model for the target membranes were liposomes of variable content of PS or PG (phosphatidylglycerol) and erythrocyte membranes in which the PS and other anionic compound content/exposure was modified in several ways. Membranes of increased anionic phospholipid content displayed increased fusion with DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) liposomes, while erythrocyte membranes partly depleted of glycocalix, its sialic acid, in particular, showed a decreased fusion ability. The role of the anionic component is also supported by the fact that erythrocyte membrane inside-out vesicles fused easily with cationic liposomes. The data obtained on erythrocyte ghosts of normal and disrupted asymmetry, in particular, those obtained in the presence of Ca²⁺, indicate the role of lipid flip-flop movement catalyzed by scramblase. The ATP-depletion of erythrocytes also induced an increased sensitivity to hemoglobin leakage upon interactions with DOTAP liposomes. Calcein leakage from anionic liposomes incubated with DOTAP liposomes was also dependent on surface charge of the target membranes. In all experiments with the asymmetric membranes the fusion level markedly increased with an increase of temperature, which supports the role of membrane lipid mobility. The decrease in positive charge by binding of plasmid DNA and the increase in ionic strength decreased the ability of DOTAP liposomes/lipoplexes to fuse with erythrocyte ghosts. Lower pH promotes fusion between erythrocyte ghosts and DOTAP liposomes and lipoplexes. The obtained results indicate that electrostatic interactions together with increased mobility of membrane lipids and susceptibility to form structures of negative curvature play a major role in the fusion of DOTAP liposomes with natural and artificial membranes.     

Hyperglycaemia alters the physico-chemical properties of proteins in erythrocyte membranes of diabetic patients.

1. The dynamic properties of erythrocyte membranes in diabetic children and of control erythrocyte membranes subjected to in vitro glycation have been investigated by means of fluorescence quenching of membrane tryptophan residues and ESR spectroscopy. 2. The apparent distance separating the membrane protein tryptophan and the bound 1-anilino-8-naphthalenesulphonate (ANS) molecules was decreased in erythrocyte membranes from children with diabetes. This resulted in a significant increase of the maximum energy transfer efficiency in diabetic membranes. 3. The relevant alterations occurred in the above parameters due to the in vitro nonenzymatic glycosylation of control membranes. 4. These changes were accompanied by the decreased hw/hs parameter of MSL and the increased relative rotational correlation time (tau c) of ISL in diabetic membranes and in the membranes subjected to in vitro glycation. 5. The results suggest that the conformational changes in membrane proteins may occur at both the intrinsic and exposed thiol groups. 6. Both the in vivo and the in vitro data indicate that nonenzymatic glycosylation of membrane proteins may be the major factor attributable to the alterations in the dynamic properties of erythrocyte membrane in diabetic state.     

Topo-optical reactions and polarization optical analysis of human lymphocytes

The latent birefringence of lymphocyte membranes of various species may readily be studied and analysed by various topo-optical reactions. The membranes of glutaraldehyde-fixed and PBS-washed lymphocytes show continuous birefringence with thiazine- and quinoline dyes. According to polarization optical analysis thiazine dye-stained cells are radially positive, whereas quinoline dye-stained cells are radially negative spherites, i.e. thiazine dye molecules are in a perpendicular, quinoline dye molecules in a parallel orientation relative to the membrane surface. These findings suggest that in lymphocyte membranes glycoproteins are primarily responsible for the topo-optical reactions. The actual conformational state of the glycoprotein components is a decisive factor not only in dye binding but also in the orientation of dye molecules. Heparin treatment directs attention to an important interaction between heparin and membrane glycoproteins. With the aid of the critical electrolyte concentration (CEC) technique we were able to demonstrate an ultrastructural differences between human erythrocyte and human lymphocyte membranes. After this procedure the birefringence of erythrocyte membranes was lost, whereas that of lymphocyte membranes did not change. There were no differences between the topo-optical reactions of T and B lymphocytes.     

Simvastatin. A new therapeutic approach for Smith-Lemli-Opitz syndrome

The Smith-Lemli-Opitz syndrome (SLOS) is caused by deficient Delta(7)-dehydrocholesterol reductase, which catalyzes the final step of the cholesterol biosynthetic pathway, resulting in low cholesterol and high concentrations of its direct precursors 7-dehydrocholesterol (7DHC) and 8DHC. We hypothesized that i) 7DHC and 8DHC accumulation contributes to the poor outcome of SLOS patients and ii) blood exchange transfusions with hydroxymethylglutaryl (HMG)-CoA reductase inhibition would improve the precursor-to-cholesterol ratio and may improve the clinical outcome of SLO patients. First, an in vitro study was performed to study sterol exchange between plasma and erythrocyte membranes. Second, several exchange transfusions were carried out in vivo in two SLOS patients. Third, simvastatin was given for 23 and 14 months to two patients. The in vitro results illustrated rapid sterol exchange between plasma and erythrocyte membranes. The effect of exchange transfusion was impressive and prompt but the effect on plasma sterol levels lasted only for 3 days. In contrast, simvastatin treatment for several months demonstrated a lasting improvement of the precursor-to-cholesterol ratio in plasma, erythrocyte membranes, and cerebrospinal fluid (CSF). Plasma precursor concentrations decreased to 28 and 33% of the initial level, respectively, whereas the cholesterol concentration normalized by a more than twofold increase. During the follow-up period all morphometric parameters improved. The therapy was well tolerated and no unwanted clinical side effects occurred. This is the first study in which the blood cholesterol level in SLOS patients is normalized with a simultaneous significant decrease in precursor levels. There was a lasting biochemical improvement with encouraging clinical improvement. Statin therapy is a promising novel approach in SLOS that deserves further studies in larger series of patients.