Structural Basis of Pore Formation by the Bacterial Toxin Pneumolysin

Abstract

The bacterial toxin pneumolysin is released as a soluble monomer that kills target cells by assembling into large oligomeric rings and forming pores in cholesterol-containing membranes. Using cryo-EM and image processing, we have determined the structures of membrane-surface bound (prepore) and inserted-pore oligomer forms, providing a direct observation of the conformational transition into the pore form of a cholesterol-dependent cytolysin. In the pore structure, the domains of the monomer separate and double over into an arch, forming a wall sealing the bilayer around the pore. This transformation is accomplished by substantial refolding of two of the four protein domains along with deformation of the membrane. Extension of protein density into the bilayer supports earlier predictions that the protein inserts beta hairpins into the membrane. With an oligomer size of up to 44 subunits in the pore, this assembly creates a transmembrane channel 260 A in diameter lined by 176 beta strands.

Irreversible loss of membrane-binding activity of Listeria-derived cytolysins in non-acidic conditions: a distinct difference from allied cytolysins produced by other Gram-positive bacteria

“…The mechanism of pore formation by proteins of the CDC family involves a series of complex events including recognition and binding to the target membrane, the formation of pre-pore complexes on the membrane (oligomerization), and insertion of a transmembrane bbarrel (Heuck et al, 2003;Ramachandran et al, 2004;Tilley et al, 2005). Recently, it was reported that rapid denaturation of LLO was triggered at neutral pH by the premature unfolding of domain 3 transmembrane b-hairpins from the normal transmembrane b-barrel form (Schuerch et al, 2005).…”

Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation

“…This correlates reasonably with the 'growing pore' model 46 where the toxin binds and immediately inserts the membrane to form small pores, which successively grow to full-size pores. Whether LLO also forms complete prepore rings, as in the case of pneumolysin 7 and PFO 47 remains to be shown experimentally, for example, by locking LLO in a prepore form like PFO 47 . However, at sublytic toxin concentrations (o500 ng ml À 1 ), we see no structures on the ghosts in our EM analysis that could be correlated with Ca 2 þ influx.…”

“…CDC monomers are secreted by the pathogenic bacteria in a water-soluble form that binds to a receptor on the target cell, where they oligomerize into a ring of up to 50 monomers with a diameter of around 300 Å 4,5 . Pore formation occurs through a subsequent conformational change, in which two helix bundles in each monomer convert into a pair of amphiphilic transmembrane b-hairpins that insert into the membrane 3,6,7 .…”

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].…”