Directly Observing the Lipid-Dependent Self-Assembly and Pore-Forming Mechanism of the Cytolytic Toxin Listeriolysin O.

Listeriolysin O (LLO) is the major virulence factor of Listeria monocytogenes and a member of the cholesterol-dependent cytolysin (CDC) family. Gram-positive pathogenic bacteria produce water-soluble CDC monomers that bind cholesterol-dependent to the lipid membrane of the attacked cell or of the phagosome, oligomerize into prepores, and insert into the membrane to form transmembrane pores. However, the mechanisms guiding LLO toward pore formation are poorly understood. Using electron microscopy and time-lapse atomic force microscopy, we show that wild-type LLO binds to membranes, depending on the presence of cholesterol and other lipids. LLO oligomerizes into arc- or slit-shaped assemblies, which merge into complete rings. All three oligomeric assemblies can form transmembrane pores, and their efficiency to form pores depends on the cholesterol and the phospholipid composition of the membrane. Furthermore, the dynamic fusion of arcs, slits, and rings into larger rings and their formation of transmembrane pores does not involve a height difference between prepore and pore. Our results reveal new insights into the pore-forming mechanism and introduce a dynamic model of pore formation by LLO and other CDC pore-forming toxins.