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

The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by 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 fluor...

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Bibliographic Details
Published in:eLife 2022-08, Vol.11
Main Authors: McGuinness, Conall, Walsh, James C, Bayly-Jones, Charles, Dunstone, Michelle A, Christie, Michelle P, Morton, Craig J, Parker, Michael W, Böcking, Till
Format: Article
Language:English
Subjects:
Animals
assembly kinetics
Bacterial Toxins - metabolism
Biochemistry and Chemical Biology
Cell membranes
Cholesterol
cholesterol dependent cytolysin
Clostridium perfringens - metabolism
Dyes
Experiments
Hemolysin Proteins - metabolism
Labeling
Liposomes - metabolism
Mammals - metabolism
membrane pore
Membranes
Microscopy
Oligomers
Proteins
single-molecule fluorescence
Stoichiometry
Structural Biology and Molecular Biophysics
Virulence factors
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Summary:The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by 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.
ISSN:2050-084X
2050-084X
DOI:10.7554/eLife.74901