Structural basis for tuning activity and membrane specificity of bacterial cytolysins

Nita R Shah,Courtney M Boyd,Doryen Bubeck,Edward S Parsons,Bart W Hoogenboom,Tomas B Voisin

doi:10.1038/s41467-020-19482-6

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.

Highlights

Cholesterol-dependent cytolysins (CDCs) are pore-forming proteins that serve as major virulence factors for pathogenic bacteria
A long extended domain 2 (D2) flexibly links domain 4 (D4) to the membrane attack complex perforin (MACPF)/CDC domain, which is responsible for pore formation[11]
ILY from Streptococcus intermedius targets human cells by binding the GPIanchored complement regulator CD599 through an extended βhairpin of D417

Summary

Introduction

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. Pore-forming proteins play a crucial role in immune defence, killing Gram-negative bacteria, cancer cells, and phagocytosed microbes[2] Understanding how these proteins discriminate between self-cells and target membranes will provide insight into fundamental virulence strategies, as well as facilitate the application of engineered CDCs that lyse new cellular targets. Other CDCs, such as intermedilysin (ILY) secreted by Streptococcus intermedius, achieve species-specificity for their hosts by hijacking the cell surface receptor CD59 to initiate membrane binding[9] For this subgroup of CDCs, the interaction with CD59 is sufficient for attachment and the role of cholesterol is restricted to pore formation[10]. These rearrangements are facilitated by stabilizing interactions between a number of amino acids at the MACPF/CDC domain interface[16], but structural details of intermediate conformations remain unresolved

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