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. 2015 Jun 5;290(23):14542-55.
doi: 10.1074/jbc.M114.623876. Epub 2015 Apr 20.

Pneumococcal Adhesins PavB and PspC Are Important for the Interplay with Human Thrombospondin-1

Ulrike Binsker  1 , Thomas P Kohler  2 , Krystin Krauel  3 , Sylvia Kohler  1 , Hansjörg Schwertz  3 , Sven Hammerschmidt  1
Affiliations

Pneumococcal Adhesins PavB and PspC Are Important for the Interplay with Human Thrombospondin-1

Ulrike Binsker et al. J Biol Chem. .

Abstract

The human matricellular glycoprotein thrombospondin-1 (hTSP-1) is released by activated platelets and mediates adhesion of Gram-positive bacteria to various host cells. In staphylococci, the adhesins extracellular adherence protein (Eap) and autolysin (Atl), both surface-exposed proteins containing repeating structures, were shown to be involved in the acquisition of hTSP-1 to the bacterial surface. The interaction partner(s) on the pneumococcal surface was hitherto unknown. Here, we demonstrate for the first time that pneumococcal adherence and virulence factor B (PavB) and pneumococcal surface protein C (PspC) are key players for the interaction of Streptococcus pneumoniae with matricellular hTSP-1. PavB and PspC are pneumococcal surface-exposed adhesins and virulence factors exhibiting repetitive sequences in their core structure. Heterologously expressed fragments of PavB and PspC containing repetitive structures exhibit hTSP-1 binding activity as shown by ELISA and surface plasmon resonance studies. Binding of hTSP-1 is charge-dependent and inhibited by heparin. Importantly, the deficiency in PavB and PspC reduces the recruitment of soluble hTSP-1 by pneumococci and decreases hTSP-1-mediated pneumococcal adherence to human epithelial cells. Platelet activation assays suggested that PavB and PspC are not involved in the activation of purified human platelets by pneumococci. In conclusion, this study indicates a pivotal role of PavB and PspC for pneumococcal recruitment of soluble hTSP-1 to the bacterial surface and binding of pneumococci to host cell-bound hTSP-1 during adhesion.

Keywords: MSCRAMMs; adhesin; bacterial pathogenesis; extracellular matrix protein; platelet; pneumococci; thrombospondin.

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Figures

FIGURE 1.
FIGURE 1.
PavB and PspC of S. pneumoniae recruit soluble human thrombospondin-1 to the bacterial cell surface. A and B, concentration-dependent binding of soluble hTSP-1 to S. pneumoniae D39Δcps, S. pneumoniae serotype 35A, and isogenic mutants deficient for PavB, PspC or both. Bacteria (2 × 108) were incubated with increasing concentrations of hTSP-1 (0–12.5 μg/ml) in 100 μl of PBS. Binding of surface-associated hTSP-1 was measured by flow cytometry using a specific polyclonal mouse anti-hTSP-1 antibody and secondary AlexaFluor® 488-conjugated anti-mouse IgG. Binding of hTSP-1 is shown as the geometric mean fluorescence intensity (GMFI) multiplied by the percentage of gated events (GMFI × % gated events). The mean values of at least three independent experiments are shown with error bars corresponding to S.D. **, p < 0.01; ***, p < 0.001 versus S. pneumoniae (S.p.) D39Δcps or serotype 35A.
FIGURE 2.
FIGURE 2.
Heterologously expressed PavB and PspC fragments. A and C, schematic model of PavB of S. pneumoniae TIGR4 and PspC subtypes PspC2 (S. pneumoniae ATCC33400) and PspC3 (S. pneumoniae D39 or serotype 35A) and heterologously expressed His6-tagged fragments of both surface proteins. SP, signal peptide; SSURE, streptococcal surface repeats; LPNTG, sortase anchoring motif; R, repeat domain; P, proline-rich sequence; CBD, choline-binding domain. B, SDS-PAGE of heterologously expressed PavB fragments (SSURE2, SSURE2+3, SSURE1–5) stained with CBB R250 and corresponding immunoblots. Proteins were detected using a specific polyclonal mouse anti-SSURE2+3 antibody or a monoclonal mouse anti-penta-His antibody and an alkaline phosphatase-coupled secondary anti-mouse antibody. D, SDS-PAGE of heterologously expressed PspC2 (SH2, SH3, SM1) and PspC3 (SH13) fragments stained with CBB R250 and corresponding immunoblots. Proteins were detected using a specific polyclonal mouse anti-SH2 antibody or a monoclonal mouse anti-penta-His antibody and an alkaline phosphatase-coupled secondary anti-mouse antibody.
FIGURE 3.
FIGURE 3.
Repetitive structures of PavB and PspC are involved in the interaction with hTSP-1. A and B, dose-dependent binding of soluble pneumococcal proteins PavB (A) or PspC (B) to immobilized hTSP-1. Human TSP-1 (0.1 μg in 100 μl/well) was coated on microtiter plates (MaxisorpTM, Nunc) and, after blocking, incubated with increasing molecular ratios of heterologously expressed PavB or PspC fragments. Bound pneumococcal proteins were detected using a polyclonal mouse anti-SSURE2+3 (PavB) antibody or a polyclonal mouse anti-SH2 (PspC) antibody and a peroxidase-coupled secondary anti-mouse antibody. Results are illustrated as mean values ± S.D. of at least three independent experiments. C and D, concentration-dependent binding of soluble hTSP-1 to immobilized, heterologously expressed PavB and PspC proteins. Pneumococcal proteins were immobilized on microtiter plates (PolysorpTM, Nunc) in equimolar amounts related to SSURE2 or SH3 (each 0.5 μg in 50 μl/well). Binding of hTSP-1 was detected after incubating the immobilized proteins with increasing concentrations of hTSP-1 (0–50 μg/ml) using a specific polyclonal mouse anti-hTSP-1 antibody and a peroxidase-coupled secondary anti-mouse antibody. Results are illustrated as mean values ± S.D. of at least four independent experiments.
FIGURE 4.
FIGURE 4.
Kinetics of the interaction between pneumococcal adhesins and hTSP-1. A and B, interactions of heterologously expressed pneumococcal proteins PavB and PspC with immobilized hTSP-1 were analyzed by surface plasmon resonance spectroscopy under different buffer conditions. Sensorgrams show the dose-dependent binding of the pneumococcal hTSP-1-binding proteins. A CM5-sensorchip was coated with native hTSP-1 (∼3000 response units), and the PavB and PspC proteins in PBS, Hepes, Hepes supplemented with 1 mm MgCl2 and 2 mm CaCl2 containing 0.05% Tween® 20 (pH 7.4) were used as analytes at a flow rate of 10 μl/min. The association and dissociation was observed, each for 300 s. Values of the control flow cells were subtracted from each sensorgram.
FIGURE 5.
FIGURE 5.
Interaction between pneumococcal adhesins and hTSP-1 is charge-dependent and inhibited by heparin. A–F, human TSP-1 (0.1 μg in 100 μl/well) was immobilized on microtiter plates (MaxisorpTM, Nunc) and incubated with a constant molecular ratio of heterologously expressed PavB or PspC fragments (related to hTSP-1) in the presence of increasing concentrations of sodium chloride (0–1.0 m), heparin (0–5 mg/ml), or chondroitin sulfate A (0–5 mg/ml). Bound pneumococcal proteins were detected using a polyclonal mouse anti-SSURE2+3 (PavB) antibody or a polyclonal mouse anti-SH2 (PspC) antibody and a peroxidase-coupled secondary anti-mouse antibody. The mean values of at least three independent experiments are shown with error bars corresponding to S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus buffer.
FIGURE 6.
FIGURE 6.
PavB and PspC contribute to pneumococcal adherence to A549 cells via hTSP-1. A and B, dose-dependent binding of hTSP-1-FITC to A549 cells was analyzed by flow cytometry. Approximately 2 × 105 cells were incubated with different concentrations of FITC labeled hTSP-1 (0–50 μg/ml) after preincubation with 1 μm MnCl2. Binding of hTSP-1 is shown as a histogram and geometric mean fluorescence intensity (GMFI) multiplied by the percentage of gated events (GMFI × % gated events). The mean values of three independent experiments are shown with error bars corresponding to S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, A549 cells (2 × 105) were preincubated with 1 μm MnCl2 and increasing concentrations of hTSP-1 followed by an infection with S. pneumoniae D39Δcps or D39ΔcpsΔpavBΔpspC using a multiplicity of infection of 25 bacteria per epithelial cell. Adherent bacteria were detected using a polyclonal rabbit anti-pneumococci antibody followed by incubation with secondary AlexaFluor® 488-labeled goat anti-rabbit IgG. A549 cells were stained with AlexaFluor® 488-coupled phalloidin. Pneumococcal adherence of 100 cells was quantified 1.5 h post infection by immune fluorescence microscopy. Results are presented as percentage related to the multiplicity of infection for three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus S. pneumoniae D39Δcps.
FIGURE 7.
FIGURE 7.
Influence of S. pneumoniae and pneumococcal proteins on platelet activation. A and B, 1 × 109 platelets were incubated with the recombinant pneumococcal proteins PavB SSURE1–5 (25 μg/ml), PspC SH13 (10 μg/ml), PspC SH2 (10 μg/ml), or 1 × 108 pneumococcal deletion mutants in the absence or presence of exogenous hTSP-1 (25 μg/ml). Convulxin (100 ng/ml) was used as a positive control. Platelet activation was analyzed by flow cytometry using a specific mouse anti-human CD62P (P-selectin)-PE Cy5-conjugated antibody (left panels) or a mouse PAC-1 (anti-αIIbβIII)-FITC-labeled antibody (right panels) and expressed as percentage of labeled platelets. Results are presented as mean values ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01 versus PBS. S.p., S. pneumoniae.
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