Structural analysis of the protein/lipid complexes associated with pore formation by the bacterial toxin pneumolysin

\Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membr anes of cells by forming ring-shaped oligomers. Magic angle spinning and wi deline static (31)p NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydra ted model membranes containing cholesterol and phosphatidylcholine and dice tyl phosphate (10:10:1 molar ratio), NMR spectra show that the interaction of pneumolysin with cholesterol-containing Liposomes results in the formati on of a nonbilayer phospholipid phase and vesicle aggregation. The amount o f the nonbilayer phase increases with increasing protein concentration. Fre eze-fracture electron microscopy indicates the coexistence of aggregated ve sicles and free ring-shaped structures in the presence of pneumolysin. On t he basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid m embranes is a complex combination of pore formation within the bilayer, ext raction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of sma ll vesicles.