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.
-
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.
Metrics
-
- 1,640
- views
-
- 374
- downloads
-
- 6
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Single-molecule analysis of the entire perfringolysin O pore formation pathway
eLife 11:e74901.
https://doi.org/10.7554/eLife.74901
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here, we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry. Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å, which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II complexes.
-
- Biochemistry and Chemical Biology
- Stem Cells and Regenerative Medicine
Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.
