Staphylococcus aureus toxin LukSF dissociates from its membrane receptor target to enable renewed ligand sequestration

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.

Keywords: bacterial toxin; immune response; pore formation; single molecule; super-resolution.

Figure 1

Toxin functionality on PMN and HEK cells. A) PMN cell permeability in the presence of unlabeled LukSK281CY113H and LukFK288C (mS + mF; number of biologic replicates, n = 2), Alexa Fluor mS* or mF*, and F(wt) or S(wt) (n = 1), compared with PMN cell permeability of S(wt) and F(wt) (n = 3). B) Inhibition of 3 µg/ml FITC-labeled FITC-S(wt) (n = 3) and mS* (n = 1) binding to PMN cell by mS. Permeability dose dependencies for A and B are shown with a polynomial spline fit; statistical significance indicated between low (0.3 and 0.001 nM) and high (300 nM) toxin concentrations using Student’s t test. Error bars indicate sd. C) Column indicating binding responses for mF* on hC5aR cells (n = 2). ***P < 0.001, statistically significant difference between mS* binding on HEK-hC5aR cells compared with HEK-CCR2 and mF* binding on these cells. D) Permeability of hC5aR-transfected HEK cells using unlabeled mS and mF and Alexa Fluor mS* and mF* compared with S(wt) and F(wt) (n = 2). E) Inhibition of 3 µg/ml mS* binding by mS on HEK-hC5aR cells (n = 3). CCR2-transfected HEK cells used as negative controls for toxin binding and lysis in C and D (n = 2) or 1 representative experiment in C. Dose dependency shown with polynomial spline fit. Statistical significance calculated between low (0.3 and 0.001 nM) and high (300 nM) toxin concentrations using Student’s t test. F) Permeability response of hC5aR-transfected HEK cells following incubation with unlabeled mS and Alexa Fluor maleimide mF* or toxins F(wt) and S(wt) (n = 3). Statistical significance calculated between 15 and 0 min time points using Student’s t test. Error bars indicate sd. Percentages of mean fluorescence intensity are shown as relative to the maximum intensity in each individual experiment (B, C, E). Permeability of the cells was analyzed after 30 min incubation at +37°C, whereas the inhibition assays were analyzed after 45 min incubation at +4°C (Supplemental Movie S3).

Figure 2

Standard TIRF microscopy of LukS/F with hC5aR on HEK cells. A, left) TIRF image of hC5aR-mGFP on the surface of a HEK cell before addition of toxin; A, right) zoom-ins of yellow, dashed square (left) immediately following 2 min incubation with Alexa Fluor 647-labeled LukSK281CY113H [mS*(Alexa647)]. B) Equivalent images of the same cell of B after >15 min incubation with LukFK288C (mF). C, upper) TIRF image of colocalization of Alexa Fluor 594- and Alexa Fluor 647 mF* and mS* [mF*(Alexa594) and mS*(Alexa647)] with hC5aR-mGFP on HEK cells; C, lower) zoom-in of yellow, dashed square (upper) with colocalized foci indicated (arrows).

Figure 3

Single-molecule TIRF microscopy of hC5aR, LukS, and LukF in live cells. A) Images of HEK cells treated with LukSK281CY113H (mS) and Alexa Fluor-labeled LukFK288C (mF*) showing brightfield (left), hC5aR-mGFP (green), and mF* (red). B–D) Probability distribution for stoichiometry of hC5aR in the absence and presence of Alexa Fluor mS* and mF* (B), and of mS* foci (C), indicating tetramer periodicity (D) from Fourier spectral analysis. E) A random tetramer overlap model cannot account for mS* experimental stoichiometry data (R² < 0), but a tetramer-multimer model results in excellent agreement (R² = 0.85). F) hC5aR and mS* stoichiometry as a function of incubation time. G) Proportion of immobile and mobile colocalized foci in the presence and absence of mS and mF. Error bars show sem from n = 5–15 image subregions (n = 20–30 cells,∼1000–10,000 foci).

Figure 4

Relative stoichiometry of hC5aR, LukS, and LukF in fixed cells. A) Micrographs of fixed hC5aR-mGFP HEK cells treated with LukSK281CY113H (mS) and LukFK288C (mF) showing hC5aR-mGFP (left), Alexa Fluor 647 (Aöexa647, middle), and merge (right) on Alexa Fluor mS* with zoom-in (lower panels) showing colocalized foci. B) Proportion of colocalized foci treated with mS, mS* + mF, and mS + mF* for hC5aR. Error bars show sem from n = 4 image subregions (n ∼ 1000 foci). C) Heatmap of correlation between hC5aR and mS stoichiometry (red, dashed line indicates 4 mS per hC5aR molecule); R² ∼ 0.15 (n ∼ 1000 foci from ∼ 10 cells). D, E) FRET images and efficiencies. The FRET experiment was performed in live and fixed sortase-tagged FITC-hC5aR-expressing cells. Live cells (number of biologic replicates, n = 2) were incubated in the presence of Cy3 mS* for 1 h at +4°C and washed, after which, unlabeled mF was added. FRET was analyzed before (mS*) or after (mS* + mF) addition of mF. FRET from fixed cells (n = 3) was analyzed in the presence of mS* or unlabeled mS and Cy3 mF* (n = 2). Statistical significance between cells with only mS and both of the toxin components, mS and mF, was analyzed using Student’s t test. Error bars indicate sd.

Figure 5

LukSF dissociation and rebinding of C5a on hC5aR-expressing cells. A) Inhibition of anti-CD88 binding on hC5aR-expressing HEK cells using increasing concentrations (x axis) of S(wt) and C5a; F(wt) is used as a negative control for inhibition of anti-CD88 binding (number of biologic repeats, n = 2). The values are normalized against the maximum binding observed with only anti-CD88. B) Disengagement of hC5aR from LukSF was observed as an increase in PE-conjugated anti-CD88 binding (right y axis, indicated with bars) on S(wt)-precoated cells using increasing but sublytic concentrations (x axis, indicated with dots) of F(wt). The values are normalized against the maximum binding observed with S(wt)-incubated cells with only anti-CD88. Minimal cell lysis (percent of lysed cells, left y axis) detected in F(wt) concentrations below 3 nM (n = 3). C) Rebinding of constant amount of NT647-labeled C5a on hC5aR upon LukSF formation, analyzed by incubating S(wt)-precoated cells with increasing concentrations (x axis) of LukF mutant F(G130D) that associates with LukS but does not lead to cell lysis (n = 2). The values are normalized against the maximum binding observed with only NT647-C5a. D) Effect of LukSF-mediated cell lysis on complement activation and C5a formation on full blood measured by using C5b-9 as a marker for complement activation in plasma (n = 3). Maximal C5a formation is observed by the incubation of full blood with live S. aureus bacteria. Ecb (B, C), F(wt) (A), or S(wt) (D) is used as a negative control in the assays. Percentages of mean fluorescence intensities is shown as relative to the maximal intensity in each individual experiment (A–C). Statistical significances are calculated using Student’s t test. Error bars indicate sd.

Figure 6

Model for LukSF receptor binding and the mechanism of LukSF-induced inflammation. A). LukS [Protein Data Bank identification (PDB ID): 1T5R] binds on hC5aR (structure based on angiotensin receptor data PDB ID: 4YAY as a soluble monomer on the cell membrane. Each LukS monomer binds 1 hC5aR molecule via the receptor-interacting residues R73, Y184, Y250, and T244 (marked with blue dots) within a cluster of ∼4–5 hC5aR homo-oligomers Upon binding to hC5aR, LukS exposes residues for LukF (PDB ID: 1LKF) binding (interface indicated by dashed ellipse). In these tight clusters, each LukF can bind to 2 LukS monomers via 2 interfaces. B) Binding of LukF on LukS and formation of the octameric pore (PDB ID: 3B07) causes dissociation of the receptors from the complex because of leakage of the cell membrane and possibly also because the receptor-binding region (marked with a circle) is buried between the monomers in the complex. C) The detached hC5aR molecule can be reused by its ligands LukS or C5a anaphylatoxin (PDB ID: 1KJS). D) Zoom-out of A–C, illustrating the putative mechanism of LukSF-induced inflammation. RBC, red blood cell.