Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins
constitute a major superfamily of pore-forming proteins that act as bacterial
virulence factors and effectors in immune defence. Upon binding to the membrane, they
convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using
real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic
structure fitting, we have mapped the structure and assembly pathways of a bacterial
CDC in unprecedented detail and accuracy, focussing on suilysin from
Streptococcus suis. We show that suilysin assembly is a
noncooperative process that is terminated before the protein inserts into the
membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped
assemblies are all seen to perforate the membrane, as also visible by the ejection of
its lipids. Membrane insertion requires a concerted conformational change of the
monomeric subunits, with a marked expansion in pore diameter due to large changes in
subunit structure and packing.
Many disease-causing bacteria secrete toxic proteins that drill holes into our cells
to kill them. Cholesterol-dependent cytolysins (CDCs) are a family of such toxins,
and are produced by bacteria that cause pneumonia, meningitis, and septicaemia.
The bacteria release CDC toxins as single protein molecules, which can bind to the
membrane that surrounds the host cell. After binding to the membrane, the toxin
molecules assemble in rings to form large pores in the host membrane. There are
several stages to this process, but our understanding of what happens at the
molecular level is incomplete.
Leung et al. studied suilysin, a CDC toxin produced by a bacterium that has a big
impact on the pig farming industry because it causes meningitis in piglets. The
bacterium can also cause serious diseases in humans through exposure to contaminated
pigs or pig meat.
Leung et al. used a technique called electron microscopy to obtain atomic-scale
snapshots of the toxin structures before and after the toxins were inserted into the
membrane. In addition, real-time movies of the process were gathered using another
technique called atomic force microscopy.
The experiments show that suilysin forms assemblies on the membrane that grow by one
molecule at a time, rather than by the merging of larger assemblies of molecules.
This results in a mixture of ring-shaped and arc-shaped toxin assemblies on the
membrane. The arcs of suilysin are incomplete ring assemblies, but they are still
able to make holes in the cell membrane. In order to insert into the membrane, the
toxin molecules in the arcs and rings undergo a dramatic change in shape.
Understanding how CDCs assemble in membranes will guide further work into the
development of new vaccines that can target these proteins to reduce the damage
caused by bacterial infections.