Structural basis for tuning activity and membrane specificity of bacterial cytolysins

Abstract

Cholesterol-dependent cytolysins (CDCs) are pore-forming proteins that serve as major virulence factors for pathogenic bacteria. They target eukaryotic cells using different mechanisms, but all require the presence of cholesterol to pierce lipid bilayers. How CDCs use cholesterol to selectively lyse cells is essential for understanding virulence strategies of several pathogenic bacteria, and for repurposing CDCs to kill new cellular targets. Here we address that question by trapping an early state of pore formation for the CDC intermedilysin, bound to the human immune receptor CD59 in a nanodisc model membrane. Our cryo electron microscopy map reveals structural transitions required for oligomerization, which include the lateral movement of a key amphipathic helix. We demonstrate that the charge of this helix is crucial for tuning lytic activity of CDCs. Furthermore, we discover modifications that overcome the requirement of cholesterol for membrane rupture, which may facilitate engineering the target-cell specificity of pore-forming proteins.

Single molecule tracking the uncoupling of assembly and membrane insertion in Perfringolysin O

“…3) with rapid oligomerization in early stages followed by a period of slower extension of the oligomer. These kinetics are reminiscent of the two-step mechanism reported for streptolysin-O, where membrane-bound dimerisation is followed by sequential oligomerization [50], and for the initial conformation change reported for PFO [51] A B and other CDCs [16,52]. A two-step process is also consistent with the observations of Böcking and co-workers [35] reported alongside this manuscript.…”

“…Overall, we observe bi-component assembly kinetics (3) with rapid oligomerization in early stages followed by a period of slower extension of the oligomer. These kinetics are reminiscent of the two-step mechanism reported for streptolysin-O, where membrane-bound dimerisation is followed by sequential oligomerization ([49]), and for the initial conformation change reported for PFO ([50]) and other CDCs ([21, 51]).…”

“…Only 1 in 340 collisions lead to successful oligomerization, surprisingly many compared to reversible assembly of other oligomeric proteins ( [37]). It's possible that this difference may be accounted for by the cooperative nature of α-HL assembly, whereas the scale of CDC oligomers may instead require some kind of switching mechanism to retain monomers once they join ( [51]). This supports an irreversible model of CDC assembly as also evidenced by cryoEM and AFM ( [45,20]).…”

Diversity of β‐hemolysins produced by the human opportunistic streptococci

“…20–30 nm in diameter) on the target cells. Recently, atomic force microscopy and cryo‐electron microscopy, employing high resolution, have revealed more detailed structures of the generated pores and pore‐formation processes of CDCs 51–57 …”

Challenges and approaches to studying pore-forming proteins

“…Consequently, studies of intermediate stages of pore formation are rare or require biochemical modifications to the PFP in order to 'trap' and visualise intermediates [18,[58][59][60][61][62][63][64]. Moreover, these modifications also raise questions about the validity of the manipulated state, specifically how closely it reflects a true state in nature, if at all.…”