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.
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
Further reading
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
