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. Author Summary Staphylococcus aureus is a bacterial species naturally present in our external flora and environment, but it is also one of the main pathogens responsible for nosocomial infections in hospital, with strains having highly problematic multi-resistance to antibiotics. S. aureus is able to secrete various virulence factors, some of which can specifically target and lyse our immune cells, making us more vulnerable to this pathogen. Thus, leukotoxins bind to receptors on the cell surface, drastically change their conformation and form cytotoxic pores in the membrane. Studying the molecular mechanisms underlying the formation of these pores is technically challenging due to their requirement for specific receptors. Here, we tested a simple electrophysiological method enabling the real-time measurement of pore formation on model cells (Xenopus oocytes), which express the receptors of interest. We were thus able to elucidate the kinetics of pore formation, the limiting role of receptors in this process, and propose a complementary model to the standard model. We also demonstrated the ability of this method to detect pore formation of non-cognate pairs of subunits and suggest further applications to characterize pore-forming properties of other toxins, to identify new target receptors, or to screen therapeutic agents inhibiting the formation of pores.