Protein A is the von Willebrand factor binding protein onStaphylococcus aureus by Jörg Hartleib, Nicola Köhler, Richard B. Dickinson, Gursharan S. Chhatwal, Jan J. Sixma, Orla M. Hartford, Timothy J. Foster, Georg Peters, Beate E. Kehrel, and Mathias Herrmann Blood Volume 96(6):2149-2156 September 15, 2000 ©2000 by American Society of Hematology

Interaction of vWF with soluble S. aureuscomponents or with SPA Interaction of vWF with soluble S. aureuscomponents or with SPA.(A) Western ligand and Western blot analyses of whole-cell staphylococcal lysates. Interaction of vWF with soluble S. aureuscomponents or with SPA.(A) Western ligand and Western blot analyses of whole-cell staphylococcal lysates. S. aureus Cowan 1, Newman, and NCTC 8325-4 lysates (2 μL) (lanes 1, 2, and 3, respectively) were separated on 7.5% SDS-PAGE, blotted on nitrocellulose, and incubated either with [DIG]-vWF in PBS (left), with polyclonal [DIG]-anti-SPA-Abs in PBS (right), or with PBS alone (center). Binding was detected using anti-DIG-Fab fragments and subsequent exposure with a chromogenic substrate (5 minutes). (B) Western ligand analysis of vWF binding to lysates of S. aureus wild-type (lanes 1, 2, and 3, respectively) and their respective Δspa deletion mutants (lanes 1′-3′). Experimental conditions identical to those in A (left). For enhanced detection sensitivity, gels were intentionally overloaded (20 μL lysate/lane), and blots were overexposed (30 minutes). (C) SDS-PAGE of rhvWF and Western ligand analysis of SPA binding. rhvWF expressed from Chinese hamster ovary cells (40 μg) was subjected to SDS-PAGE (7.5% gels), and gels were either stained with Coomassie blue (left) or used for blotting of vWF onto nitrocellulose (right). Membranes were subsequently blocked and incubated with [DIG]-rSPA, and rSPA binding was demonstrated in a color reaction described in A. Jörg Hartleib et al. Blood 2000;96:2149-2156 ©2000 by American Society of Hematology

SPR analysis of vWF interaction with rSPA-coupled CM5 sensor surfaces (solid lines) or uncoupled surfaces (dashed lines).(A) After instrument equilibration, vWF (250 μg/mL in HEPES buffer) was injected (➁) followed by HEPES buffer (➀) and surface regenerati... SPR analysis of vWF interaction with rSPA-coupled CM5 sensor surfaces (solid lines) or uncoupled surfaces (dashed lines).(A) After instrument equilibration, vWF (250 μg/mL in HEPES buffer) was injected (➁) followed by HEPES buffer (➀) and surface regeneration with glycine buffer (③) as described in “Materials and methods,” and RU was determined. (inset) Response measured as a function of time using different vWF concentrations (250/300/350/400/450/500 μg vWF/mL, lower through upper line) calculated by subtracting RUvWF-CM5-RUCM5. (B) Response curves after the injection of HSA (upper 2 lines) and BSA (lower 2 lines) followed by the injection of HEPES buffer. (C) Response after 4 injections of pooled human IgG (1060 nmol/L) (➁) followed by the injection of vWF (250 μg/mL) (➀). After regeneration of the surfaces, vWF (250 μg/mL) was injected (③), and surfaces were equilibrated and regenerated as in A. (D) Interaction of vWF and V8 protease-treated vWF with SPA. After immobilization of SPA (9300 RU), either vWF (100 μg/mL, 30 μL) (➁) or vWF digested with protease V8 (4 IU, 30 minutes at 37°C) (➀) was injected. After binding, surfaces were regenerated as described in A. Shown is the difference plot of signal obtained on [RUvWF-CM5 − RUCM5]. Jörg Hartleib et al. Blood 2000;96:2149-2156 ©2000 by American Society of Hematology

Flow cytometric analysis of binding of vWF to S. aureus Flow cytometric analysis of binding of vWF to S. aureus.Microorganisms (120 000 cells/mL) were incubated with FITC-vWF (150 μg/mL, 10 minutes at 21°C), then analyzed for binding by flow cytometry as described in “Materials and methods.” (A) Binding of vWF t... Flow cytometric analysis of binding of vWF to S. aureus.Microorganisms (120 000 cells/mL) were incubated with FITC-vWF (150 μg/mL, 10 minutes at 21°C), then analyzed for binding by flow cytometry as described in “Materials and methods.” (A) Binding of vWF to 3 S. aureus strains (▴, Cowan 1; ▪, Newman; ●, 8325-4). (B) Inhibition of vWF binding to S. aureus strains by the addition of excess unlabeled vWF over FITC-vWF (200 μg/mL) (symbol denotation as in A). (C, D) Inhibition of vWF binding toS. aureus Cowan 1 (C) and Newman (D) by the addition of polyclonal anti-SPA (▪) and IgG (●) antibodies. Shown are means ± SD (n = 3). RF indicates relative fluorescence. Jörg Hartleib et al. Blood 2000;96:2149-2156 ©2000 by American Society of Hematology

Binding of vWF to Δspa mutants and pSPA7235-complemented Δspa mutants Binding of vWF to Δspa mutants and pSPA7235-complemented Δspa mutants.Experimental conditions as described in Figure 3. Binding of vWF to Δspa mutants and pSPA7235-complemented Δspa mutants.Experimental conditions as described in Figure 3. (A) S. aureus NCTC 8325-4. (B) S. aureus Newman. ▪, wild-type strains; ●, Δspa mutants; ▴, complemented mutants. RF indicates relative fluorescence. Jörg Hartleib et al. Blood 2000;96:2149-2156 ©2000 by American Society of Hematology

Adhesion of S. aureus to vWF surfaces Adhesion of S. aureus to vWF surfaces.PMMA coverslips were preadsorbed with indicated concentrations of vWF, then incubated with [3H]-thymidine–labeled cells (4 × 106 CFU/mL) of either S. aureus 8325-4 (A), Cowan 1 (B), the respective Δspa mutants, or the p... Adhesion of S. aureus to vWF surfaces.PMMA coverslips were preadsorbed with indicated concentrations of vWF, then incubated with [3H]-thymidine–labeled cells (4 × 106 CFU/mL) of either S. aureus 8325-4 (A), Cowan 1 (B), the respective Δspa mutants, or the pSPA7235-complemented Δspa mutant in PBS/HSA (60 minutes at 37°C). ▪, wild-type strains; ●, Δspa mutants; ♦, complemented mutant. Shown are mean values ± SD of 3 determinations performed in quintuplicate. Jörg Hartleib et al. Blood 2000;96:2149-2156 ©2000 by American Society of Hematology

Adhesion of S. aureus Cowan 1 to vWF-PMMA in the presence of IgG Adhesion of S. aureus Cowan 1 to vWF-PMMA in the presence of IgG.Experimental conditions as in Figure 5. Adhesion of S. aureus Cowan 1 to vWF-PMMA in the presence of IgG.Experimental conditions as in Figure 5. After adsorption of vWF (50 μg/mL), adhesion assays were performed in the presence of increasing concentrations of human pooled IgG (Venimmun) (solid line). Control assays were performed with PMMA pretreated with PBS alone (dashed line). Shown are mean values ± SD of a quintuplicate determination. Jörg Hartleib et al. Blood 2000;96:2149-2156 ©2000 by American Society of Hematology

Adhesion of S. aureus 8325-4 and of DU 5875 to vWF adsorbed PMMA under defined flow conditions.PMMA slides were preincubated with vWF (50 μg/mL), mounted on a flow chamber, and perfused with S. aureus cells (5 × 107 CFU) (shear rate, 110/s). Adhesion of S. aureus 8325-4 and of DU 5875 to vWF adsorbed PMMA under defined flow conditions.PMMA slides were preincubated with vWF (50 μg/mL), mounted on a flow chamber, and perfused with S. aureus cells (5 × 107 CFU) (shear rate, 110/s). Adherent microorganisms visualized by light microscopy were determined over indicated time periods using automated image analysis. Shown are mean values of 10 fields ± SD. ▪, S. aureus 8325-4; ●, DU 5875. Jörg Hartleib et al. Blood 2000;96:2149-2156 ©2000 by American Society of Hematology