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

Abstract

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...

Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

“…The subunit structure of perfringolysin O was studied by electron microscopy [9] and image processing which showed an oligomeric structure containing an average of 50 subunits, each of which spans the ring and consists of a periodic repeat of 2.4 nm on the outer rim of the ring. A similar analysis was reported for streptolysin O [10] in which the authors proposed an oligomeric structure consisting of two concentric rings of subunits.…”

Are amyloid diseases caused by protein aggregates that mimic bacterial pore-forming toxins?

“…In the a-hemolysin barrel, each monomer contributes an amphipathic hairpin to a 14-stranded heptameric b-barrel (Menestrina et al 2001). PFTs form pores ranging in diameter from 1 to 2 nm for staphylococcal a-hemolysin (Fussle et al 1981) and Vibrio cholerae cytolysin (Zitzer et al 1995), and from 15 to 45 nm for streptolysin O (Sekiya et al 1993) and perfringolysin O (Olofsson et al 1993). For some PFTs, some degree of heterogeneity (pore size and oligomeric state) has been reported (Sharpe & London, 1999 ;Tadjibaeva et al 2000).…”

Electron microscopic evaluation of a two-step theory of pore formation by streptolysin O

“…Both ring-shaped and arc-shaped structures can be observed on erythrocyte membranes that have been treated with 9,14).…”