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

Duncan, James L.; Schlegel, Robert

doi:10.1083/jcb.67.1.160

Cited by 134 publications

(79 citation statements)

References 26 publications

(30 reference statements)

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“…The resulting structures have been reported for many of the thiol-activated toxins [4][5][6][7][8]. However the detailed topology of these oligomers is still unclear.…”

Section: Introductionmentioning

confidence: 99%

Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

et al. 1995

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We present a detailed analysis of the oligomeric subunit organisation of pneumolysin by the use of negative stain electron microscopy and image processing to produce a projection density map. Analysis of the rotational symmetry has revealed a large and variable subunit number, between 40-50. The projected subunit density by rotational averaging shows at least two distinct subunit domains at different radial positions. Side views of the rings reveal further details concerning the dimensions of the oligomer in the membrane. On the basis of these observations and our previous knowledge of the monomer domain structure we propose that the 4-domain subunits are packed in a square planar arrangement to form the pneumolysin oligomer.Key words." Pneumolysin; Pore-forming toxin; Electron microscopy; Image processing copy. This model consists of a single ring of subunits which contains ~30 subunits. The incorporation of pneumolysin oligomers into liposomes rather than sheep erythrocytes and the use of image processing have enabled a more refined analysis of the pneumolysin oligomeric structures. Here we present (i) a symmetry-averaged projected density map of a pneumolysin oligomer and (ii) side views of the oligomers. On the basis of these images we propose a compact 4-domain subunit model for the oligomer. Materials and methods Pneumolysin expression and purificationWildtype pneumolysin protein was expressed in Escherichia coli JMI01 and purified as described previously [12]. Sample purity was checked by SDS PAGE analysis and haemolytic activity assayed as described previously [12].

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“…The resulting structures have been reported for many of the thiol-activated toxins [4][5][6][7][8]. However the detailed topology of these oligomers is still unclear.…”

Section: Introductionmentioning

confidence: 99%

Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

et al. 1995

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“…It has been shown by electron microscopy that SLO forms arc-or ring-shaped structures (arcs or rings) on erythrocyte membranes as do other thiol-activated toxins after binding to cholesterol molecules (7,9,12,13).…”

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confidence: 99%

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

et al. 1993

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Streptolysin 0 (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.Streptolysin 0 (SLO) is a membrane-damaging protein toxin. It is known as a thiol-activated or oxygen-sensitive toxin and is produced in the extracellular medium by most strains of group A beta-hemolytic streptococci (1-3, 6, 17, 21).It has been shown by electron microscopy that SLO forms arc-or ring-shaped structures (arcs or rings) on erythrocyte membranes as do other thiol-activated toxins after binding to cholesterol molecules (7,9,12,13).Two hemolytic forms of SLO molecules have been reported: an acidic type with a molecular weight of 69,000 t 3,000 and a pI of 6.0 to 6.4 and a neutral and degraded type of 57,000 ± 5,000 in molecular weight with a pI of 7.0 to 7.5 (4).The calculated volume of the ring indicates that one ring is composed of between 20 and 100 monomers of the toxin molecule (4, 6). Results of hemolytic titrations indicate that 70 to 125 monomers of SLO are required for formation of a single functional lesion on an erythrocyte (4). However, the mechanism of hemolysis after formation of these rings is not known. There are also no definitive reports on the ultrastructure of the rings.In this report, we describe the presence of a crown on the top of an SLO ring and the arrangement of the discrete SLO molecules in the ring. We also propose a model for the ring that involves discrete molecules of SLO. MATERIALS AND METHODSPreparation of SLO. The toxin was kindly provided by Noboru Nakagawa (The Kitasato Institute, Tokyo, Japan), and it was prepared by a modified version of the methods of Suzuki et al. (18) and Duncan and Schlegel (9) total of 40 liters of Todd-Hewitt broth (Difco Laboratories, Det...

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“…Electron-microscopy studies have shown that 0-toxin and other thiol-activated cytolysins form ring-shaped and arc-shaped structures on erythrocyte ghosts and cholesterol dispersions (Mitsui et al, 1979;Duncan and Schlegel, 1975). The formation of these structures by wildtype and mutant recombinant toxins was investigated (Fig.…”

mentioning

confidence: 99%

Contribution of Individual Tryptophan Residues to the Structure and Activity of θ‐Toxin (Perfringolysin O), A Cholesterol‐Binding Cytolysin

Sekino-Suzuki

Nakamura

Mitsui³

et al. 1996

European Journal of Biochemistry

107

101

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&Toxin (perfringolysin 0), secreted by Clostridium perfringens, shares with other known thiol-activated toxins a conserved undecapeptide, ECTGLAWEWWR, located in the C-terminal region of the protein and containing the unique cysteine of the molecule. Single and double amino acid substitutions were created in the 8-toxin molecule to investigate the role of individual tryptophan residues in the lytic activity of &toxin. Wild-type and mutant &toxins were overproduced in Escherichia coli by means of a T7 RNA polymerase/promoter system and purified. The relative hemolytic activities of four mutant toxins, each with a Trp to Phe substitution outside the common Cys-containing region, were more than 60% that of wild-type 8-toxin. In contrast, mutant toxins with Phe replacements within the Cys-containing region (at Trp436, Trp438 or Trp439) showed significantly reduced hemolytic and erythrocyte-membranebinding activities. The largest reduction in binding affinity, more than 100-fold, was observed for Trp438 mutant toxins. However, the mutants retain binding specificity for cholesterol and the ability to form arcshaped and ring-shaped structures on membranes. These results indicate that the low hemolytic activities of these mutant toxins can be ascribed, at least in part, to reduced binding activities. With respect to protease susceptibility and far-ultraviolet circular-dichroism spectra, only the W436-F mutant toxin, showed any considerable difference from wild-type toxin in secondary or higher-order structures, indicating that Trp436 is essential for maintenance of toxin structure.

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

Cited by 134 publications

References 26 publications

Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

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

Contribution of Individual Tryptophan Residues to the Structure and Activity of θ‐Toxin (Perfringolysin O), A Cholesterol‐Binding Cytolysin

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