Structural Analysis of the Protein/Lipid Complexes Associated with Pore Formation by the Bacterial Toxin Pneumolysin

Abstract

Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static 31 P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the 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 membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.Pneumolysin is a major virulence factor from the human pathogen Steptococcus pneumoniae and is a member of a family of cholesterol-binding toxins (CBTs), 1 which also includes perfringolysin from Clostridium perfringens, listeriolysin from Listeria monocytogenes, and streptolysin from Streptococcus pyogenes. Streptolysin is well known for its use in cell biology for the permeabilization of cells. As well as pore formation leading to the breakdown of membrane barriers into cells and between tissues, these toxins act in a complex manner involving the activation of intracellular signaling pathways and the induction of inflammation, among other responses (see Morgan et al.(1) for review). Understanding the mechanisms by which CBTs act has been greatly assisted by the determination of a crystal structure for soluble, monomeric perfringolysin (2) and the use of electron cryo-microscopy (cryo-EM) to obtain threedimensional reconstructions of the oligomeric and pore-forming states of CBTs using pneumolysin (3). These direct structural insights have been augmented by results obtained by Tweten and co-workers (4) using fluorescence spectroscopy (5), which demonstrate that a pendant domain of the CBTs interacts with phospholipid molecules during pore formation via two ␣-helix bundles refolded into two ␤-hairpins.

ESAT-6 fromMycobacterium tuberculosisDissociates from Its Putative Chaperone CFP-10 under Acidic Conditions and Exhibits Membrane-Lysing Activity

“…The two long helices in the ESAT-6 hairpin structure formed by residues Phe8 to Trp43 and Glu49 to Ala79 are indeed twice the length required to insert into a lipid bilayer. A number of studies showed the importance of cholesterol in protein-lipid interactions in bacterial infection processes (2,13,17). Although our results suggest that both cholesterol and PC are needed for membrane interaction of ESAT-6, under different experimental conditions, the presence of PC alone has shown some effect (18,29).…”

Perforin activity at membranes leads to invaginations and vesicle formation

“…4E, Movies S9 and S10). The ability of pneumolysin to cause blebbing has been documented before (43). We also used rotenone (inhibiting mitochondrial complex I) or azide (inhibiting complex IV) (see SI Materials and Methods) to show that ATP depletion does not prevent PFNinduced vesiculation (Movies S11, S12, S13, and S14).…”

Identification and Characterization of an Amphipathic Leucine Zipper-like Motif in Escherichia coli Toxin Hemolysin E

“…Since it is a pore-forming toxin, membraneinteraction and assembly therein are the key steps, associated with its mechanism of action. Recently membrane interaction of several protein and peptide toxins like pneumolysin (37), Vibrio cholerae cytolysin (38), equinatoxin (39), E. coli ␣-hemolysin (40, 41) B. thuringiensis ␦-toxin (31, 42) and pardaxin (21) have been studied in detail in order to understand their mechanism of action. Although, pore-formation has been demonstrated by electrophysiological experiments (4, 7) and visualized by electron microscopy (10) Both the Wild Type and Mutant Peptides Derived from the Leucine Zipper-like Motif Bind to the Phospholipid Membrane-In order to detect the ability of the peptides to bind to phospholipid vesicles, they were labeled by fluorescent probe NBD.…”