Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

Abstract

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].

Pneumolysin generates multiple conductance pores in the membrane of nucleated cells

“…This seems unlikely. Many studies have shown the presence of arc and ring structures in membrane exposed to CDCs [8][9][10], but 8 provided no evidence that complete oligomers of CDCs (rings) of greatly differing sizes enter the membrane. The arcs are believed by some to be membrane-inserted but incomplete oligomers that can function as channels [10][11][12].…”

“…Membrane currents were recorded with an Axopatch 200 amplifier (Axon Instruments, Foster City, CA, USA) connected to a PC for data acquisition and subsequent analysis. Patch-pipettes were made from thick walled, filamented borosilicate glass capillaries, which were coated with Sylgard and fire-polished to give resistances of [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] MΩ. The holding potential was set to 0mV and a 1.6 s pulse to +40 followed by a 1.6 s pulse to -40 mV was applied every second throughout the recordings.…”

Pathogenesis of microbial keratitis

“…Other than capsule, the most studied virulence factor of pneumococcus is pneumolysin, a toxin belonging to the family of bacterial cholesterol-dependent cytolysin. It causes direct cell damage by binding to the cholesterol in the host cell membrane followed by polymerization into 30-50 mers, thereby forming pores in the host cell membranes [124,197]. In addition to forming pores, it initiates immune-derived damage by activating complement system and induces inflammation [145].…”

Pathogenesis of pneumococcal infection

“…Oligomeric pneumolysin exists as large arc and ring structures measuring 30±40 nm in diameter in cell membranes [47]. The problem posed by this is the apparent absence of hydrophobic areas on both the surface of pneumolysin and at the domain interfaces making membrane insertion impossible.…”