Abstract
Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.
Highlights
Pore formation is a strategy for host infection and the instigation of disease employed by many pathogenic organisms, and especially by bacteria [1,2]
This paper describes a study designed to test whether this is the case by imaging pores of the cholesterol-dependent cytolysin (CDC) pneumolysin in situ in membranes, without stain or model bias derived from iterative two-dimensional image alignment [22]
Purified pneumolysin was added to cholesterol-containing liposomes and incubated at 378C for 5 s or 1 min prior to the collection of cryo-electron tomograms
Summary
Pore formation is a strategy for host infection and the instigation of disease employed by many pathogenic organisms, and especially by bacteria [1,2]. One of the largest and most intensely studied families of pore-forming proteins are the cholesterol-dependent cytolysins (CDCs) first identified in the Gram-positive bacterial genera Streptococcus, Clostridium, Listeria and Bacillus [3,4]. Prominent examples of CDCs include listeriolysin from Listeria monocytogenes and perfringolysin from Clostridium perfringens, as well as pneumolysin from Streptococcus pneumoniae. The CDCs are highly immunogenic and are vaccine candidates for their producing organisms [8], while listeriolysin in particular is being employed in vaccines against other diseases, including tuberculosis and cancer, primarily because of its capacity to confer intracellular growth [9,10,11].
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