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

  1. Conall McGuinness
  2. James C Walsh  Is a corresponding author
  3. Charles Bayly-Jones
  4. Michelle A Dunstone
  5. Michelle P Christie
  6. Craig J Morton
  7. Michael W Parker
  8. Till Böcking  Is a corresponding author
  1. University of New South Wales, Australia
  2. Monash University, Australia
  3. University of Melbourne, Australia
  4. 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.

The following data sets were generated

Article and author information

Author details

  1. Conall McGuinness

    EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
  2. James C Walsh

    EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia
    For correspondence
    james.walsh@unsw.edu.au
    Competing interests
    The authors declare that no competing interests exist.
  3. Charles Bayly-Jones

    Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7573-7715
  4. Michelle A Dunstone

    Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  5. Michelle P Christie

    Department of Biochemistry and Pharmacology, University of Melbourne, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  6. Craig J Morton

    Department of Biochemistry and Pharmacology, University of Melbourne, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5452-5193
  7. Michael W Parker

    Australian Cancer Research Foundation Rational Drug Discovery Centre, St Vincents Institute of Medical Research, Fitzroy, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3101-1138
  8. Till Böcking

    EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia
    For correspondence
    till.boecking@unsw.edu.au
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1165-3122

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

  1. Janice L Robertson, Washington University in St Louis, United States

Version history

  1. Preprint posted: October 19, 2021 (view preprint)
  2. Received: October 21, 2021
  3. Accepted: August 16, 2022
  4. Accepted Manuscript published: August 24, 2022 (version 1)
  5. Version of Record published: September 8, 2022 (version 2)
  6. 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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  1. Conall McGuinness
  2. James C Walsh
  3. Charles Bayly-Jones
  4. Michelle A Dunstone
  5. Michelle P Christie
  6. Craig J Morton
  7. Michael W Parker
  8. Till Böcking
(2022)
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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