Real-time analysis of pore formation by bi-component staphylococcal leukotoxins using the two-electrode voltage-clamp technique
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…
Pore forming toxins, or PFTs, are cytotoxins produced by certain bacteria in a water-soluble form. These toxins can create pores in the membranes of host cells, causing cell death through lytic activity. Staphylococcus aureus is known to produce bi-component PFTs, specifically the leukocidins, which target and lyse specific types of leukocytes, erythrocytes, and endothelial cells by interacting with membrane receptors.
Most of these receptors are G protein-coupled receptors (GPCRs), such as complement or chemokine receptors.
Gamma-hemolysins, also known as Hlgs, are the primary leukocidins secreted by S. aureus. These toxins form receptor-dependent hetero-octameric pores, though the exact mechanisms are not yet fully understood. Due to the necessity of specific receptors within a lipid bilayer environment, studying these molecular mechanisms is technically challenging.
To address this issue, researchers developed a novel, sensitive method for cell surface expression of a wide variety of target receptors and real-time recording of currents generated by newly formed pores.
This method involves the heterologous expression of receptors in Xenopus oocytes, coupled with the two-electrode voltage-clamp technique and the use of electrophysiological robots. By employing this approach, researchers were able to characterize the concentration-dependent kinetics of pore formation, determine receptor density as a limiting factor, observe specific responses to non-cognate pairing of PFTs, and detect cell surface binding of F subunits preceding pore formation.
Additionally, the team proposed a hybrid model of subunit oligomerization to explain these findings.
The newly developed method can easily be implemented for in vitro characterization of various PFTs on a wide range of membrane receptors. This technique can be used to decipher early mechanisms of pore formation and screen therapeutic agents capable of blocking the cytotoxicity of receptor-dependent PFTs.
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