Single‐molecule tracking of perfringolysin O assembly and membrane insertion uncoupling

Abstract

We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway and pore-formation of the archetypical cholesterol-dependent cytolysin nanopore, Perfringolysin O. We follow the stoichiometry and diffusion of Perfringolysin O complexes during assembly with 60 millisecond temporal resolution and 20 nanometre spatial precision. Our results suggest individual nascent complexes can insert into the lipid membrane where they continue active assembly. Overall, these data support a model of stepwise irreversible assem-1 .

Single-molecule analysis of the entire perfringolysin O pore formation pathway

“…Consistent with previous observations for PFO and other CDC/MACPF pore formers ( Leung et al, 2017 ; Leung et al, 2014 ; Senior et al, 2022 ), we find that PFO oligomerisation is irreversible. Notably, our data also shows that the PFO dimer is the first long-lived species on the membrane, such that dimerisation is the committed step for pore formation.…”

“…Membrane insertion leads to a drastic reduction of the diffusion rate of CDC complexes on the surface of flat membranes ( Senior et al, 2022 ; Leung et al, 2014 ). To determine whether the same effect could be observed on liposomes, we imaged PFO assembly and pore formation assay with high laser intensity to accurately localise the position (projected onto the x/y-plane) of growing PFO oligomers on liposomes over time with sub-pixel resolution.…”

“…Our experimental approach relying on immobilised liposomes is complementary to AFM ( Boyd et al, 2016 ; Leung et al, 2014 ; Mulvihill et al, 2015 ; Vögele et al, 2019 ) and single-molecule fluorescence microscopy ( Senior et al, 2022 ) studies on flat bilayers to image the dynamic process of CDC pore assembly in real time. The assay has sufficiently high throughput to study the effect of varying experimental conditions on pore formation (in this work, PFO concentrations and pH), whereby each experiment provides traces from hundreds to over a thousand liposomes providing statistical power to detect small changes in kinetic parameters.…”

“…We observed that AF647-PFO accumulation on the liposome continued unabated after opening of an arc pore, which we interpret as post-insertion oligomer growth. Using single-molecule tracking on droplet interface bilayers supported on an agarose layer, Wallace and colleagues also observed that PFO oligomers continue to grow by monomer addition after insertion into the bilayer ( Senior et al, 2022 ), whereby insertion was detected by the sudden drop in lateral diffusion of the assembling structure on the membrane. In contrast, AFM studies of suilysin pore formation on lipid bilayers supported on mica have shown that suilysin arcs do not continue to grow after insertion ( Leung et al, 2014 ).…”

“…To overcome the limitation of ensemble averaging, imaging methods have been developed to follow PFP assembly at the level of individual pores ( Benke et al, 2015 ; Ros et al, 2021 ; Sathyanarayana et al, 2018 ). Imaging modalities applied to CDC assembly on planar lipid bilayers include high speed atomic force microscopy, as shown for suilysin ( Leung et al, 2014 ) and listeriolysin O ( Ruan et al, 2016 ) and single-molecule fluorescence tracking, as shown for PFO assembly on a droplet interface bilayer ( Senior et al, 2022 ). These imaging studies support the insertion of incomplete arc-shaped membrane lesions, suggesting an alternative mechanism of pore formation that is distinct from the canonical prepore formation prior to insertion.…”