Real-time analysis of pore formation by bi-component staphylococcal leukotoxins using the two-electrode voltage-clamp technique
Abstract
Pore forming toxins (PFTs) are cytotoxins secreted in water-soluble form by pathogenic bacteria. They have the ability to form pores in the membrane of host cells, ultimately leading to cell death by lytic activity. Staphylococcus aureus produces a variety of bi-component PFTs, the leukocidins, which target and lyse particular leukocytes, erythrocytes and endothelial cells through specific interactions with membrane receptors. Most of these receptors belong to the family of complement or chemokine receptors that are G protein-coupled receptors (GPCRs). Gamma-hemolysins (Hlgs) are the major leukocidins secreted by S. aureus , and form receptor-dependent hetero-octameric pores through mechanisms that are not fully elucidated. Studying these molecular mechanisms is technically challenging due to the requirement of specific receptors in a lipid bilayer environment. In the present article, we developed a simple and highly sensitive method allowing cell surface expression of a large diversity of target receptors and recording in real-time, currents generated by neo-formed pores. This method is based on the heterologous expression of receptors in Xenopus oocytes and on the two-electrode voltage-clamp technique with electrophysiological robots. Using this approach, we characterized the concentration dependent-kinetics of pore formation, determined the receptor density as a limiting factor, showed specific response to non-cognate pairing of PFTs, observed cell surface binding of F subunits preceding pore formation and propose a hybrid model of subunit oligomerization. This method could be easily implemented for the in vitro characterization of various PFTs on a wide diversity of membrane receptors, to decipher early mechanisms of pore formation or to screen therapeutic agents blocking the cytotoxicity of receptor-dependent PFTs.
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BibTeX
@misc{lemel2026realtime,
title = {{Real-time analysis of pore formation by bi-component staphylococcal leukotoxins using the two-electrode voltage-clamp technique}},
author = {Laura Lemel and Simon Harris and Guillaume Audic and Laure Bellard and Jean-Daniel Savoie and Claire M Grison and Sébastien Granier and Justine Magnat and Normand Voyer and Thierry Vernet and Isabel D Alves and Anne-Marie Di Guilmi and Christophe J Moreau},
howpublished = {HAL},
year = {2026},
month = aug,
doi = {10.64898/2026.08.03.742423},
url = {https://hal.science/hal-05843754v1},
note = {CC BY 4.0},
}
RIS
TY - UNPB
TI - Real-time analysis of pore formation by bi-component staphylococcal leukotoxins using the two-electrode voltage-clamp technique
AU - Laura Lemel
AU - Simon Harris
AU - Guillaume Audic
AU - Laure Bellard
AU - Jean-Daniel Savoie
AU - Claire M Grison
AU - Sébastien Granier
AU - Justine Magnat
AU - Normand Voyer
AU - Thierry Vernet
AU - Isabel D Alves
AU - Anne-Marie Di Guilmi
AU - Christophe J Moreau
PY - 2026
DA - 2026/08/04
PB - HAL
DO - 10.64898/2026.08.03.742423
UR - https://hal.science/hal-05843754v1
AB - Pore forming toxins (PFTs) are cytotoxins secreted in water-soluble form by pathogenic bacteria. They have the ability to form pores in the membrane of host cells, ultimately leading to cell death by lytic activity. Staphylococcus aureus produces a variety of bi-component PFTs, the leukocidins, which target and lyse particular leukocytes, erythrocytes and endothelial cells through specific interactions with membrane receptors. Most of these receptors belong to the family of complement or chemokine receptors that are G protein-coupled receptors (GPCRs). Gamma-hemolysins (Hlgs) are the major leukocidins secreted by S. aureus , and form receptor-dependent hetero-octameric pores through mechanisms that are not fully elucidated. Studying these molecular mechanisms is technically challenging due to the requirement of specific receptors in a lipid bilayer environment. In the present article, we developed a simple and highly sensitive method allowing cell surface expression of a large diversity of target receptors and recording in real-time, currents generated by neo-formed pores. This method is based on the heterologous expression of receptors in Xenopus oocytes and on the two-electrode voltage-clamp technique with electrophysiological robots. Using this approach, we characterized the concentration dependent-kinetics of pore formation, determined the receptor density as a limiting factor, showed specific response to non-cognate pairing of PFTs, observed cell surface binding of F subunits preceding pore formation and propose a hybrid model of subunit oligomerization. This method could be easily implemented for the in vitro characterization of various PFTs on a wide diversity of membrane receptors, to decipher early mechanisms of pore formation or to screen therapeutic agents blocking the cytotoxicity of receptor-dependent PFTs.
ER -