Functional basis for complement evasion by staphylococcal superantigen-like 7

Abstract

The human pathogen Staphylococcus aureus has a plethora of virulence factors that promote its colonization and survival in the host. Among such immune modulators are staphylococcal superantigen-like (SSL) proteins, comprising a family of 14 small, secreted molecules that seem to interfere with the host innate immune system. SSL7 has been described to bind immunoglobulin A (IgA) and complement C5, thereby inhibiting IgA-FcαRI binding and serum killing of Escherichia coli. As C5a generation, in contrast to C5b-9-mediated lysis, is crucial for immune defence against staphylococci, we investigated the impact of SSL7 on staphylococcal-induced C5a-mediated effects. Here, we show that SSL7 inhibits C5a generation induced by staphylococcal opsonization, slightly enhanced by its IgA-binding capacity. Moreover, we demonstrate a strong protective activity of SSL7 against staphylococcal clearance in human whole blood. SSL7 strongly inhibited the C5a-induced phagocytosis of S. aureus and oxidative burst in an in vitro whole-blood inflammation model. Furthermore, we found that SSL7 affects all three pathways of complement activation and inhibits the cleavage of C5 by interference of its binding to C5 convertases. Finally, SSL7 effects were also demonstrated in vivo. In a murine model of immune complex peritonitis, SSL7 abrogated the C5a-driven influx of neutrophils in mouse peritoneum.

Fig. 1

SSL7 inhibits generation of C5a during staphylococcal opsonisation. (A,B) Staphylococci were treated with 10% human serum for 30 minutes at 37°C in the presence of 0 – 450 nM SSL7 or SSL10. In one case (“SSL7 after”), 450 nM SSL7 was added to serum supernate after opsonisation. C5a generation was measured by using supernates as stimuli for calcium mobilization in U937-C5aR cells. Results depict in (A) representative calcium mobilization plots and in (B) mean values ± SEM of three independent experiments and are expressed relative to cells treated with supernate without staphylococcal proteins. In (A), “background” and “control” represent cells stimulated with untreated serum and serum after opsonisation without SSL7, respectively. (C) Cell-bound C5 convertases were constructed on staphylococci by initial deposition of C3b and subsequent addition of soluble fD, fB, and properdin. C5 was subsequently added in the presence of 0 – 450 nM SSL7, and C5a generation in the absence of IgA was analysed by using supernates as stimuli for calcium mobilization in U937-C5aR cells. Results represent mean values ± SEM of three independent experiments and are expressed relative to cells treated with supernate without SSL7.

Fig. 2

SSL7 effects are not dependent on IgA binding. (A-C) ELISA experiments showing binding of IgA₁ (A), IgA₂ (B), or C5 (C) to wells coated with 450 nM SSL7 and SSL7-IgA mutant. Results represent mean values ± SEM of two independent experiments. (D) Staphylococci were opsonized with 10% human serum in the presence of 450 nM SSL7 or SSL7-IgA mutant. Supernates were examined for generated C5a by Western blotting. Representative of three experiments.

Fig. 3

SSL7 inhibits phagocytosis and generation of oxidative burst in whole human blood. (A,B) Phagocytosis of FITC-labeled staphylococci was measured in whole human blood in the presence of 0 – 450 nM SSL7, SSL7-IgA mutant, or SSL10. (A) Representative histograms depicting background neutrophil fluorescence, neutrophil phagocytosis of bacteria without (control) and with 450 nM SSL7 or SSL7-IgA mutant. (B) Results express phagocytosis of FITC-labeled staphylococci in whole human blood treated with staphylococcal proteins relative to control-treated blood and represent mean values ± SEM of three independent experiments. (C) After addition of 0 – 450 nM SSL7 or SSL10 and staphylococci to whole human blood, oxidative burst was measured over time. Results are expressed relative to control-treated cells and represent mean values ± SEM of three independent experiments.

Fig. 4

SSL7 affects binding of C5 to C5 convertases. (A,B) Complement ELISA experiments showing C3b (A) and C5b-9 (B) deposition after complement activation of the classical (5% serum), lectin (5% serum), or alternative (25% serum) pathway in the presence of 0 – 450 nM SSL7. Results are expressed relative to control-treated serum and represent mean values ± SEM of three independent experiments. (C) Binding of C5 to (C3b)₂ in the presence of 10 μg/ml CRIg (Complement Receptor of the Immunoglobulin superfamily recognizes C3b molecules), SSL7 (450 nM), or SSL7 (450 nM) and IgA (60 μg/ml). Results represent mean values ± SEM of two independent experiments.

Fig. 5

SSL7 inhibits C5a generation in vivo. (A and B) Classical complement pathway-mediated hemolysis of sheep (A) or rabbit (B) red blood cells in human (A) or mouse (B) serum preincubated with SSL7. Results represent mean values ± SEM of three independent experiments. NHS and NMS stand for normal human serum and normal mouse serum, respectively. (C) Staphylococci were treated with 30% mouse serum for 30 minutes at 37°C in the presence of 0 – 450 nM SSL7. C5a generation was measured by using supernates as stimuli for calcium mobilization in U937-C5aR cells. Results depict mean values ± SEM of four independent experiments and are expressed relative to cells treated with supernate without staphylococcal proteins. (D) In the reverse passive Arthus reaction peritonitis model, peritoneal cavity lavage was performed six hours after challenge of mice with immune complexes. SSL7 was injected 30 minutes before challenge with immune complexes or was used as a challenge itself. Collected neutrophils were subsequently quantified. P < 0.001 by analysis of variance (n = 10 mice per group).