Characterization of the stoichiometry of the complex formed by Staphylococcal toxin LukSF and human C5a receptor

Karita Haapasalo-Tuomainen; Adam Wollman; Carla De Haas; Piet Aerts; Esther Van'T Veld; Karin Strijbis; Richard Wubbolts; Kok Van Kessel; Mark Leake; Jos Van Strijp

doi:10.1016/j.imbio.2016.06.039

Characterization of the stoichiometry of the complex formed by Staphylococcal toxin LukSF and human C5a receptor

Karita Haapasalo-Tuomainen, Adam Wollman, Carla De Haas, Piet Aerts, Esther Van'T Veld, Karin Strijbis, Richard Wubbolts, Kok Van Kessel, Mark Leake, Jos Van Strijp

Research output: Contribution to journal › Article › Academic

Abstract

Staphylococcus aureus causes diseases ranging from superficial skin and soft tissue infections (SSTI) to severe invasive diseases like osteomyelitis and necrotizing pneumonia. Panton Valentine Leukocidin (PVL) is a powerful leukocidal toxin produced by multiple S. aureus isolates. It is a pro-phage encoded bi-component, β-barell pore-forming toxin (β-PFT) comprising the protein subunits LukS-PV and LukF-PV. Binding of LukS-PV to the surface of target cells induces secondary binding of LukF-PV, initiating the assembly of lytic pore-forming hetero-octamers. The stoichiometric analysis of LukS/LukF complex suggests that the leukocidin exists as an octamer consisting of 4-plus-4 subunits. In this complex only LukS is known to interact with the human C5a receptor (C5aR, CD88) that is a G-protein coupled seven-transmembrane receptor (GPCR). However, binding of LukS alone on the receptor is not sufficient for the lysis of the cell but requires simultaneous interaction between both of the leukocidin subunits and C5aR. Here, multiple possible subunit and receptor combinations are theoretically possible and the exact stoichiometry between LukS, LukF and C5aR is not yet known. The aim of this study is to determine the stoichiometry of the complexes formed by Staphylococcal bi-component toxin LukSF and human C5aR. By using novel techniques of fluorescence microscopy such as total-internal-reflection fluorescence (TIRF) combined with single molecule photobleaching analysis we were able to obtain super-resolution pictures and identify single molecules within LukSF-C5aR complex on living eukaryotic cells. Our results suggest that in addition to determination of subunit stoichiometry this technique can be used to measure the kinetics of the PFT assembly on receptors within lipid membranes of living cells. These studies may facilitate our understanding of these crucial virulence factors and their interactions with host cells.

Original language	English
Pages (from-to)	1141
Number of pages	1
Journal	Immunobiology
Volume	221
Issue number	10
DOIs	https://doi.org/10.1016/j.imbio.2016.06.039
Publication status	Published - 1 Oct 2016
Externally published	Yes

Keywords

complement component C5a receptor
endogenous compound
guanine nucleotide binding protein
leukocidin
Staphylococcus toxin
virulence factor
bleaching
chemical binding
chemical reaction kinetics
cytolysis
eukaryotic cell
fluorescence microscopy
host cell
human
lipid membrane
stoichiometry
target cell

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10.1016/j.imbio.2016.06.039

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@article{4287739e9d36478abd427c9411616020,

title = "Characterization of the stoichiometry of the complex formed by Staphylococcal toxin LukSF and human C5a receptor",

abstract = "Staphylococcus aureus causes diseases ranging from superficial skin and soft tissue infections (SSTI) to severe invasive diseases like osteomyelitis and necrotizing pneumonia. Panton Valentine Leukocidin (PVL) is a powerful leukocidal toxin produced by multiple S. aureus isolates. It is a pro-phage encoded bi-component, β-barell pore-forming toxin (β-PFT) comprising the protein subunits LukS-PV and LukF-PV. Binding of LukS-PV to the surface of target cells induces secondary binding of LukF-PV, initiating the assembly of lytic pore-forming hetero-octamers. The stoichiometric analysis of LukS/LukF complex suggests that the leukocidin exists as an octamer consisting of 4-plus-4 subunits. In this complex only LukS is known to interact with the human C5a receptor (C5aR, CD88) that is a G-protein coupled seven-transmembrane receptor (GPCR). However, binding of LukS alone on the receptor is not sufficient for the lysis of the cell but requires simultaneous interaction between both of the leukocidin subunits and C5aR. Here, multiple possible subunit and receptor combinations are theoretically possible and the exact stoichiometry between LukS, LukF and C5aR is not yet known. The aim of this study is to determine the stoichiometry of the complexes formed by Staphylococcal bi-component toxin LukSF and human C5aR. By using novel techniques of fluorescence microscopy such as total-internal-reflection fluorescence (TIRF) combined with single molecule photobleaching analysis we were able to obtain super-resolution pictures and identify single molecules within LukSF-C5aR complex on living eukaryotic cells. Our results suggest that in addition to determination of subunit stoichiometry this technique can be used to measure the kinetics of the PFT assembly on receptors within lipid membranes of living cells. These studies may facilitate our understanding of these crucial virulence factors and their interactions with host cells.",

keywords = "complement component C5a receptor, endogenous compound, guanine nucleotide binding protein, leukocidin, Staphylococcus toxin, virulence factor, bleaching, chemical binding, chemical reaction kinetics, cytolysis, eukaryotic cell, fluorescence microscopy, host cell, human, lipid membrane, stoichiometry, target cell",

author = "Karita Haapasalo-Tuomainen and Adam Wollman and {De Haas}, Carla and Piet Aerts and {Van'T Veld}, Esther and Karin Strijbis and Richard Wubbolts and {Van Kessel}, Kok and Mark Leake and {Van Strijp}, Jos",

year = "2016",

month = oct,

day = "1",

doi = "10.1016/j.imbio.2016.06.039",

language = "English",

volume = "221",

pages = "1141",

journal = "Immunobiology",

issn = "0171-2985",

publisher = "Elsevier GmbH",

number = "10",

}

TY - JOUR

T1 - Characterization of the stoichiometry of the complex formed by Staphylococcal toxin LukSF and human C5a receptor

AU - Haapasalo-Tuomainen, Karita

AU - Wollman, Adam

AU - De Haas, Carla

AU - Aerts, Piet

AU - Van'T Veld, Esther

AU - Strijbis, Karin

AU - Wubbolts, Richard

AU - Van Kessel, Kok

AU - Leake, Mark

AU - Van Strijp, Jos

PY - 2016/10/1

Y1 - 2016/10/1

N2 - Staphylococcus aureus causes diseases ranging from superficial skin and soft tissue infections (SSTI) to severe invasive diseases like osteomyelitis and necrotizing pneumonia. Panton Valentine Leukocidin (PVL) is a powerful leukocidal toxin produced by multiple S. aureus isolates. It is a pro-phage encoded bi-component, β-barell pore-forming toxin (β-PFT) comprising the protein subunits LukS-PV and LukF-PV. Binding of LukS-PV to the surface of target cells induces secondary binding of LukF-PV, initiating the assembly of lytic pore-forming hetero-octamers. The stoichiometric analysis of LukS/LukF complex suggests that the leukocidin exists as an octamer consisting of 4-plus-4 subunits. In this complex only LukS is known to interact with the human C5a receptor (C5aR, CD88) that is a G-protein coupled seven-transmembrane receptor (GPCR). However, binding of LukS alone on the receptor is not sufficient for the lysis of the cell but requires simultaneous interaction between both of the leukocidin subunits and C5aR. Here, multiple possible subunit and receptor combinations are theoretically possible and the exact stoichiometry between LukS, LukF and C5aR is not yet known. The aim of this study is to determine the stoichiometry of the complexes formed by Staphylococcal bi-component toxin LukSF and human C5aR. By using novel techniques of fluorescence microscopy such as total-internal-reflection fluorescence (TIRF) combined with single molecule photobleaching analysis we were able to obtain super-resolution pictures and identify single molecules within LukSF-C5aR complex on living eukaryotic cells. Our results suggest that in addition to determination of subunit stoichiometry this technique can be used to measure the kinetics of the PFT assembly on receptors within lipid membranes of living cells. These studies may facilitate our understanding of these crucial virulence factors and their interactions with host cells.

AB - Staphylococcus aureus causes diseases ranging from superficial skin and soft tissue infections (SSTI) to severe invasive diseases like osteomyelitis and necrotizing pneumonia. Panton Valentine Leukocidin (PVL) is a powerful leukocidal toxin produced by multiple S. aureus isolates. It is a pro-phage encoded bi-component, β-barell pore-forming toxin (β-PFT) comprising the protein subunits LukS-PV and LukF-PV. Binding of LukS-PV to the surface of target cells induces secondary binding of LukF-PV, initiating the assembly of lytic pore-forming hetero-octamers. The stoichiometric analysis of LukS/LukF complex suggests that the leukocidin exists as an octamer consisting of 4-plus-4 subunits. In this complex only LukS is known to interact with the human C5a receptor (C5aR, CD88) that is a G-protein coupled seven-transmembrane receptor (GPCR). However, binding of LukS alone on the receptor is not sufficient for the lysis of the cell but requires simultaneous interaction between both of the leukocidin subunits and C5aR. Here, multiple possible subunit and receptor combinations are theoretically possible and the exact stoichiometry between LukS, LukF and C5aR is not yet known. The aim of this study is to determine the stoichiometry of the complexes formed by Staphylococcal bi-component toxin LukSF and human C5aR. By using novel techniques of fluorescence microscopy such as total-internal-reflection fluorescence (TIRF) combined with single molecule photobleaching analysis we were able to obtain super-resolution pictures and identify single molecules within LukSF-C5aR complex on living eukaryotic cells. Our results suggest that in addition to determination of subunit stoichiometry this technique can be used to measure the kinetics of the PFT assembly on receptors within lipid membranes of living cells. These studies may facilitate our understanding of these crucial virulence factors and their interactions with host cells.

KW - complement component C5a receptor

KW - endogenous compound

KW - guanine nucleotide binding protein

KW - leukocidin

KW - Staphylococcus toxin

KW - virulence factor

KW - bleaching

KW - chemical binding

KW - chemical reaction kinetics

KW - cytolysis

KW - eukaryotic cell

KW - fluorescence microscopy

KW - host cell

KW - human

KW - lipid membrane

KW - stoichiometry

KW - target cell

U2 - 10.1016/j.imbio.2016.06.039

DO - 10.1016/j.imbio.2016.06.039

M3 - Article

SN - 0171-2985

VL - 221

SP - 1141

JO - Immunobiology

JF - Immunobiology

IS - 10

ER -

Characterization of the stoichiometry of the complex formed by Staphylococcal toxin LukSF and human C5a receptor

Abstract

Keywords

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