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

2021 ◽  
Author(s):  
Conall Mc Guinness ◽  
James Walsh ◽  
Charles Bayly-Jones ◽  
Michelle Dunstone ◽  
Craig Morton ◽  
...  
Keyword(s):  
Single Molecule ◽  
Pore Formation ◽  
Membrane Insertion ◽  
Formation Pathway ◽  
Perfringolysin O ◽  
Assembly Pathway ◽  
Bacterial Virulence Factor ◽  
Membrane Bound ◽  
Single Molecule Analysis ◽  
Kinetics Of

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.

scholarly journals Investigation of the pore-forming mechanism of a cytolytic δ-endotoxin from Bacillus thuringiensis

2003 ◽  
Vol 374 (1) ◽  
pp. 255-259 ◽  
Author(s):  
Boonhiang PROMDONKOY ◽  
David J. ELLAR
Keyword(s):  
Bacillus Thuringiensis ◽  
Blood Cells ◽  
Pore Formation ◽  
Response Curve ◽  
Effect Of Temperature ◽  
Membrane Insertion ◽  
Free Solution ◽  
Forming Mechanism ◽  
Close Proximity ◽  
Membrane Bound

Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose–response curve, indicating that toxin aggregation or oligomerization is required for pore formation. Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore. Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules. When toxin-treated red blood cells at 4 °C were transferred to a toxin-free solution at 37 °C, no significant increase in haemolysis was observed. This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore. From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer. Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers.

scholarly journals Regulation of a Coupled MARCKS–PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module

Biochemistry ◽  
2016 ◽  
Vol 55 (46) ◽  
pp. 6395-6405 ◽  
Author(s):  
Brian P. Ziemba ◽  
G. Hayden Swisher ◽  
Glenn Masson ◽  
John E. Burke ◽  
Roger L. Williams ◽  
...  
Keyword(s):  
Single Molecule ◽  
Lipid Kinase ◽  
Membrane Bound ◽  
Single Molecule Analysis

scholarly journals Characterization of the stoichiometry of the complex formed by Staphylococcal LukSF and human C5aR receptor in living cells

2017 ◽  
Author(s):  
Karita Haapasalo ◽  
Adam J.M Wollman ◽  
Carla de Haas ◽  
Kok van Kessel ◽  
Jos van Strijp ◽  
...  
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Pore Formation ◽  
Membrane Receptors ◽  
Host Cell Membrane ◽  
C5a Receptor ◽  
Protein Subunits ◽  
Kinetics Of ◽  
Panton Valentine Leukocidin

SUMMARYStaphylococcus aureusPanton Valentine Leukocidin (PVL) is a pore-forming toxin comprising protein subunits LukS and LukF. Binding of LukS to human C5a receptor (hC5aR) on leukocytes induces secondary binding of LukF and assembly of lytic complexes. Previous analysis suggests that PVL consists of 4-plus-4 LukS/LukF subunits but the exact stoichiometry between LukS, LukF and hC5aR is not yet known. In this study we determine the stoichiometry and spatiotemporal dynamics of functional LukS/LukF-hC5aR complexes in living eukaryotic cells. By using rapid total internal reflection fluorescence (TIRF) and single-molecule photobleaching analysis we found that tetrameric LukS-hC5aR complexes are formed within a cluster of receptors. Upon binding to hC5aR each LukS subunit binds LukF leading to lytic pore formation and simultaneous dissociation of receptors from the complex. Our findings corroborate a hetero-octamer model but provide a new view on the kinetics of crucial virulence factor assembly on integrated host cell membrane receptors.

scholarly journals Single-molecule imaging of cholesterol-dependent cytolysin assembly

2021 ◽  
Author(s):  
Michael J Senior ◽  
Carina Monico ◽  
Eve E Weatherill ◽  
Robert Gilbert ◽  
Alejandro Heuck ◽  
...  
Keyword(s):  
Temporal Resolution ◽  
Single Molecule ◽  
Single Channel ◽  
Single Molecule Imaging ◽  
Single Channel Recording ◽  
Single Molecule Tracking ◽  
Perfringolysin O ◽  
Assembly Pathway

We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway of the Cholesterol-Dependent Cytolysin, Perfringolysin O. This enables quantification of the stoichiometry of PFO complexes during assembly with millisecond temporal resolution and 20 nanometre spatial precision. Our results support a model of overall stepwise irreversible assembly, dominated by monomer addition, but with infrequent assembly from larger partial complexes. Furthermore, our results suggest a dominant proportion of inserted, but non-conductive intermediates in assembly.

scholarly journals Experiment-friendly kinetic analysis of single molecule data in and out of equilibrium

2016 ◽  
Author(s):  
Sonja Schmid ◽  
Markus Götz ◽  
Thorsten Hugel
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Thermodynamic Quantities ◽  
Kinetic Rate ◽  
Complete Dataset ◽  
Likelihood Approach ◽  
Single Molecule Analysis ◽  
Kinetics Of

We present a simple and robust technique to extract kinetic rate models and thermodynamic quantities from single molecule time traces. SMACKS (Single Molecule Analysis of Complex Kinetic Sequences) is a maximum likelihood approach that works equally well for long trajectories as for a set of short ones. It resolves all statistically relevant rates and also their uncertainties. This is achieved by optimizing one global kinetic model based on the complete dataset, while allowing for experimental variations between individual trajectories. In particular, neither a priori models nor equilibrium have to be assumed. The power of SMACKS is demonstrated on the kinetics of the multi-domain protein Hsp90 measured by smFRET (single molecule Förster resonance energy transfer). Experiments in and out of equilibrium are analyzed and compared to simulations, shedding new light on the role of Hsp90’s ATPase function. SMACKS pushes the boundaries of single molecule kinetics far beyond current methods.

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