Alteration by cereolysin of the structure of cholesterol-containing membranes

Cowell, J L; Kim, Kwang-Shin; Bernheimer, Alan W.

doi:10.1016/0005-2736(78)90419-4

Cited by 50 publications

(17 citation statements)

References 29 publications

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“…We have previously reported [11,14] that water-soluble monomeric pneumolysin incorporates into erythrocyte membranes to form large oligomeric ring and arc structures, as reported for many other of the membrane-damaging thiol-activated toxins [4][5][6][7][8]. The precise mechanism of oligomer formation is currently unclear and consequently a detailed knowledge of the subunit structure of the oligomer may provide further clues.…”

Section: Discussionmentioning

confidence: 93%

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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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Section: Discussionmentioning

confidence: 93%

“…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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“…All of these primarily water-soluble toxins share the following properties. They consist of single polypeptide chains in the Mr range 50,000 to 80,000; they are reversibly inactivated by atmospheric oxygen and require the presence of a reducing agent for maximal expression of biological activity; they invoke membrane damage after binding to surface-exposed membrane cholesterol; and membrane damage has in several cases been shown to be accompanied by the appearance of circular, semicircular, and compound arc structures visible by electron microscopy (4,12,13,15,22,25,26). These structures are widely believed to represent toxin-cholesterol complexes (1,4,15).…”

mentioning

confidence: 99%

Mechanism of membrane damage by streptolysin-O

Bhakdi¹,

Tranum‐Jensen²,

Sziegoleit³

1985

Infect Immun

373

221

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Streptolysin-O (SLO) is a thiol-activated, membrane-damaging protein toxin of Mr 69,000 that is produced by most strains of I-hemolytic group A streptococci. Native, primarily water-soluble toxin molecules bind to cholesterol-containing target membranes to assemble into supramolecular curved rod structures (25 to 100 nm long by ca. 7.5 nm wide), forming rings and arcs that penetrate into the apolar domain of the bilayer. Electron microscopic analyses of toxin polymers in their native and reconstituted membrane-bound form indicate that the convex surface of the rod structures is a hydrophobic, lipid-binding domain, whereas the concave surfaces appear to be hydrophilic. The embedment of the rings and arcs generates large transmembrane slits or pores of up to 30-nm diameter that can be directly visualized by negative staining and freeze-fracture electron microscopy. SLO oligomers were isolated in extensively delipidated form in detergent solution, and cholesterol was found not to detectably contribute to the observed rod structures. The rods are stable structures that resist prolonged exposure to trypsin and chymotrypsin. They can be reincorporated into cholesterol-free phosphatidylcholine liposomes to generate lesions identical to those observed on erythrocytes lysed by native SLO. Thus, although cholesterol plays a key role in the initial binding of SLO to the membrane, it does not directly participate in the formation of the membrane-penetrating toxin channels. Membrane damage by SLO is basically analogous to that mediated by previously studied channel formers, namely, the C5b-9 complement complex and staphylococcal ot-toxin. Lancefield group A P-hemolytic streptococci are the ma-* Corresponding author.

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“…These proteins bind to cholesterol-containing target membranes and irreversibly damage bilayers of erythrocytes and nucleated mammalian cells. Several of these toxins have been shown to generate circular and semicircular "lesions" that can be visualized by electron microscopy (4,13,14,16,25,28,29).…”

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confidence: 99%

Use of a monoclonal antibody to determine the mode of transmembrane pore formation by streptolysin O

Hugo¹,

Reichwein²,

Arvand³

et al. 1986

Infect Immun

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Murine monoclonal antibodies were generated against streptolysin 0. One out of 10 tested immunoglobulin clones exhibited strong neutralizing activity; in solution, the presence of approximately two to four antibody molecules per toxin monomer effected 50% neutralization of hemolytic toxin activity. An enzyme-linked immunosorbent assay performed with target cell membranes that were treated with streptolysin 0 in the presence and absence of neutralizing antibodies showed that the antibodies did not block primary binding of the toxin to the cells. When membranes were solubilized in deoxycholate detergent and centrifuged in sucrose density gradients, those lysed with streptolysin 0 contained detergent-resistant, high-molecular-weight oligomers identical to the pore lesions, whereas those given toxin and neutralizing antibody contained the toxin exclusively in low-molecular-weight, nonoligomerized form. The process of pore formation by streptolysin 0 must thus involve two distinct steps, i.e., the primary binding of toxin molecules to the membrane followed by oligomerization of bound toxin monomers by lateral aggregation in the lipid bilayer to form the transmembrane pores.

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Alteration by cereolysin of the structure of cholesterol-containing membranes

Cited by 50 publications

References 29 publications

Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

Mechanism of membrane damage by streptolysin-O

Use of a monoclonal antibody to determine the mode of transmembrane pore formation by streptolysin O

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