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

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 us...

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Autores principales: McGuinness, Conall, Walsh, James C, Bayly-Jones, Charles, Dunstone, Michelle A, Christie, Michelle P, Morton, Craig J, Parker, Michael W, Böcking, Till
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9457685/
https://www.ncbi.nlm.nih.gov/pubmed/36000711
http://dx.doi.org/10.7554/eLife.74901
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author McGuinness, Conall
Walsh, James C
Bayly-Jones, Charles
Dunstone, Michelle A
Christie, Michelle P
Morton, Craig J
Parker, Michael W
Böcking, Till
author_facet McGuinness, Conall
Walsh, James C
Bayly-Jones, Charles
Dunstone, Michelle A
Christie, Michelle P
Morton, Craig J
Parker, Michael W
Böcking, Till
author_sort McGuinness, Conall
collection PubMed
description 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.
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spelling pubmed-94576852022-09-09 Single-molecule analysis of the entire perfringolysin O pore formation pathway McGuinness, Conall Walsh, James C Bayly-Jones, Charles Dunstone, Michelle A Christie, Michelle P Morton, Craig J Parker, Michael W Böcking, Till eLife Biochemistry and Chemical Biology 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. eLife Sciences Publications, Ltd 2022-08-24 /pmc/articles/PMC9457685/ /pubmed/36000711 http://dx.doi.org/10.7554/eLife.74901 Text en © 2022, McGuinness, Walsh et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biochemistry and Chemical Biology
McGuinness, Conall
Walsh, James C
Bayly-Jones, Charles
Dunstone, Michelle A
Christie, Michelle P
Morton, Craig J
Parker, Michael W
Böcking, Till
Single-molecule analysis of the entire perfringolysin O pore formation pathway
title Single-molecule analysis of the entire perfringolysin O pore formation pathway
title_full Single-molecule analysis of the entire perfringolysin O pore formation pathway
title_fullStr Single-molecule analysis of the entire perfringolysin O pore formation pathway
title_full_unstemmed Single-molecule analysis of the entire perfringolysin O pore formation pathway
title_short Single-molecule analysis of the entire perfringolysin O pore formation pathway
title_sort single-molecule analysis of the entire perfringolysin o pore formation pathway
topic Biochemistry and Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9457685/
https://www.ncbi.nlm.nih.gov/pubmed/36000711
http://dx.doi.org/10.7554/eLife.74901
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