Single-molecule analysis of the entire perfringolysin O pore formation pathway
- University of New South Wales, Australia
- Monash University, Australia
- University of Melbourne, Australia
- St Vincents Institute of Medical Research, Australia
Abstract
The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.
Data availability
The image analysis software is available at https://github.com/lilbutsa/JIM-Immobilized-Microscopy-Suite. Microscopy image stacks for Figure 1 and Figures 3-8; files containing single-molecule tracks extracted from all image stacks for Figure 2 (single-molecule binding); and a representative subset of image stacks recorded for Figure 2 are available on Dryad (doi: https://doi.org/10.5061/dryad.8w9ghx3q4). The complete set of image stacks collected for Figure 2 is too large (>10 TB) to be included in this repository such that these data are stored on the UNSW data archive (data management plan number D0240569) and can be obtained for research (including commercial) by submitting a request to research.soms@unsw.edu.au.
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Single-molecule analysis of the entire perfringolysin O pore formation pathwayDryad Digital Repository, doi:10.5061/dryad.8w9ghx3q4.
Article and author information
Author details
Charles Bayly-Jones
Funding
National Health and Medical Research Council (APP1182212)
- Till Böcking
Australian Research Council (FT150100049)
- Michelle A Dunstone
National Health and Medical Research Council (APP1194263)
- Michael W Parker
Australian Research Council (DP160101874)
- Michael W Parker
Australian Research Council (DP200102871)
- Craig J Morton
- Michael W Parker
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Janice L Robertson, Washington University in St Louis, United States
Version history
- Preprint posted: October 19, 2021 (view preprint)
- Received: October 21, 2021
- Accepted: August 16, 2022
- Accepted Manuscript published: August 24, 2022 (version 1)
- Version of Record published: September 8, 2022 (version 2)
- Version of Record updated: December 7, 2022 (version 3)
Copyright
© 2022, McGuinness et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Single-molecule analysis of the entire perfringolysin O pore formation pathway
eLife 11:e74901.
https://doi.org/10.7554/eLife.74901
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