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

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