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

Abstract

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.

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

“…Occasionally, arcs and slits combined into ring‐shaped structures, which were not yet perfectly circular and only partially associated with the formation of membrane pores. Several pore‐forming toxins (PFTs) have shown the ability to bind to lipid membranes as oligomers forming arcs or slits that can dynamically rearrange on the membrane surface to form larger pores (Leung et al , 2014; Sonnen et al , 2014; Mulvihill et al , 2015; Podobnik et al , 2015). In agreement with this observation, GSDMD arcs and slits were found that presumably fused into perfect ring‐shaped GSDMD Nterm oligomers of variable diameters, with an average value of 21 nm (Fig 6F–H).…”

“…Each experimental condition characterized by AFM was reproduced at least three times. Liposomes made from E. coli polar lipids incubated in buffer solution but in the absence of GSDMD showed no arc‐, slit‐ or ring‐like structures when imaged by AFM (Mulvihill et al , 2015). …”

The membrane attack complex, perforin and cholesterol-dependent cytolysin superfamily of pore-forming proteins

“…It has been previously applied to perfringolysin O (Czajkowsky et al, 2004) to reveal the height change during the prepore-to-pore transition of a CDC. More recently, several AFM studies have focused on other CDCs (Leung et al, 2014;Podobnik et al, 2015;Mulvihill et al, 2015); these all show heterogeneous distributions of arc-and ring-shaped assemblies.…”

“…By visualising membrane removal from the lumen of suilysin and listeriolysin O assemblies, AFM studies have established that both arc-and ring-shaped assemblies can perforate the adjacent membrane (Leung et al, 2014;Podobnik et al, 2015;Mulvihill et al, 2015). This implies that pores with an incomplete β-barrel create an unsealed edge of the lipid bilayer (Fig.…”

“…Furthermore, upon subsequent injections of suilysin monomers in solution, new pore assemblies appear, but no growth was observed for any previously formed pore assemblies (Leung et al, 2014). However, when arc-shaped pore assemblies of listeriolysin O are incubated at 37°C for up to several hours, they continue to move and merge in some cases, resulting in larger lesions of the membrane (Podobnik et al, 2015;Mulvihill et al, 2015). Hence, the prepore-to-pore transition inhibits further assembly, but in some cases, separate pore assemblies can join together to form large holes in the membrane.…”