Abstract
Staphylococcus aureus Panton-Valentine leukocidin is a pore-forming toxin targeting the human C5a receptor (hC5aR), enabling this pathogen to battle the immune response by destroying phagocytes through targeted lysis. The mechanisms that contribute to rapid cell lysis are largely unexplored. Here, we show that cell lysis may be enabled by a process of toxins targeting receptor clusters and present indirect evidence for receptor “recycling” that allows multiple toxin pores to be formed close together. With the use of live cell single-molecule super-resolution imaging, Förster resonance energy transfer and nanoscale total internal reflection fluorescence colocalization microscopy, we visualized toxin pore formation in the presence of its natural docking ligand. We demonstrate disassociation of hC5aR from toxin complexes and simultaneous binding of new ligands. This effect may free mobile receptors to amplify hyperinflammatory reactions in early stages of microbial infections and have implications for several other similar bicomponent toxins and the design of new antibiotics.—Haapasalo, K., Wollman, A. J. M., de Haas, C. J. C., van Kessel, K. P. M., van Strijp, J. A. G., Leake, M. C. Staphylococcus aureus toxin LukSF dissociates from its membrane receptor target to enable renewed ligand sequestration.
Highlights
We studied human embryonic kidney (HEK) cells modified to express monomeric green fluorescent protein-labeled human C5a receptor (hC5aR), exposed to Alexa Fluor dye-labeled S. aureus toxin components LukS and LukF and imaged using standard total internal reflection fluorescence (TIRF) realtime microscopy (Supplemental Fig. S1), allowing us to monitor the spatiotemporal dynamics of receptor and toxin molecules in the cell membrane
As C5a is the natural ligand for hC5aR and can outcompete binding of LukS on the receptor, we analyzed whether LukSF formation and disengagement of hC5aR would allow rebinding of C5a on the receptor
To detect C5a rebinding at higher LukF concentrations, we used a F(G130D) mutant that interacts with LukS but does not cause cell lysis
Summary
ABBREVIATIONS: CCR, C–C chemokine receptor; CD88, cluster of differentiation 88; Ecb, extracellular complement binding; F(G130D), G130D leukocidin component F; F(wt), wild-type Staphylococcus aureus leukocidin F; FRET, Forster resonance energy transfer; GFP, green fluorescent protein; hC5aR, human C5a receptor; HEK, human embryonic kidney; HSA, human serum albumin; Luk, leukocidin; mF, Staphylococcus aureus leukocidin F mutant; mF*, labeled Staphylococcus aureus leukocidin F mutant; mGFP, monomeric green fluorescent protein; MRSA, methicillin-resistant Staphylococcus aureus; mS, Staphylococcus aureus leukocidin S mutant; mS*, labeled Staphylococcus aureus leukocidin S mutant; MSD, mean squared displacement; PDB ID, Protein Data Bank identification; PE, phycoerythrin; PFT, pore-forming toxin; PMN, polymorphonuclear cell; pRSET, plasmid Restriction Enzyme T7 promoter; PSF, point spread function; PVL, Panton-Valentine leukocidin; RPMI, Roswell Park Memorial Institute; S(wt), wild-type Staphylococcus aureus leukocidin S; SSI, structural similarity index; TIRF, total internal reflection fluorescence A previous study using a rabbit animal model on necrotizing pneumonia suggests that PVL itself directly or indirectly causes tissue injury and by this way, induces local inflammation [14]
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