Convertase inhibitory properties of Staphylococcal extracellular complement-binding protein

Abstract

The human pathogen Staphylococcus aureus secretes several complement evasion molecules to combat the human immune response. Extracellular complement-binding protein (Ecb) binds to the C3d domain of C3 and thereby blocks C3 convertases of the alternative pathway and C5 convertases via all complement pathways. Inhibition of C5 convertases results in complete inhibition of C5a generation and subsequent neutrophil migration. Here, we show that binding of Ecb to the C3d domain of C3b is crucial for inhibition of C5 convertases. Ecb does not interfere with substrate binding to convertases but prevents formation of an active convertase enzyme.

FIGURE 1.

Ecb binding to the C3d domain of C3b is essential for C5 convertase inhibition. C5a generation in the presence of Ecb or its C3d-binding mutant is shown. Bacteria were incubated with human serum, and C5a activity in supernatants was analyzed by calcium mobilization of neutrophils. A, C5a generation via the CP/LP in ΔfD serum. B, C5a generation via the AP in human serum in the presence of MgEGTA. Both panels represent the mean ± S.E. (error bars) of three separate experiments.

FIGURE 2.

Ecb binds to C3b on the bacterial surface and inhibits C3b-containing convertases. A, C4b-coated bacteria were opsonized with C2 and C3 in the presence or absence of His-Ecb or His-CHIPS (both at 10 μg/ml). B, protein binding to the bacterial surface and C3b deposition on the bacterial surface was measured. C3-dependent binding of His-Ecb (10 μg/ml) is shown. C, C5a generation via the CP is shown. C4b-coated bacteria were incubated with C2, C3, and C5 in the presence or absence of Ecb. C5a generation was analyzed by calcium mobilization using U937-C5aR cells. D, C3b deposition via the AP is shown. Preopsonized bacteria were incubated with fB, fD, and C3 in the presence or absence of Ecb (10 μg/ml). E, C5a generation via the AP is shown. Opsonized bacteria were incubated with fB, fD, C3, and C5 in the presence of Ecb (10 μg/ml). C5a generation in the supernatant was tested by calcium mobilization. A, B, D, and E represent the mean ± S.E. (error bars) of three separate experiments. C is a representative of three separate experiments.

FIGURE 3.

Ecb does not inhibit C5 binding to convertases. A, C3b₂ binding to C5. C5 was captured on anti-C5a-coated microtiter plates. Binding of C3b₂ to C5 in the presence or absence of Ecb or CRIg-S (both at 10 μg/ml) was analyzed. B, C5 binding to decayed AP convertases in the presence of Ecb or CRIg-S (both at 10 μg/ml). C, C5 binding to decayed CP convertases in the presence or absence of Ecb (10 μg/ml). A is a representative of three separate experiments; B and C represent the mean ± S.E. (error bars) of three experiments.

FIGURE 4.

Ecb stabilizes fB binding to C3b and prevents C3bBb formation. A, fB binding to C3b via Biacore. C3b was covalently coupled to the surface of a CM5 biosensor chip where after Ecb or buffer was injected, and fB binding was analyzed. B, C3b deposition on the Biacore surface in the presence or absence of Ecb. The panels are representatives of three separate flow paths.

FIGURE 5.

Ecb prevents fB conversion on the zymosan surface. A, fB stabilization on zymosan in the presence or absence of Ecb (10 μg/ml). Zymosan was opsonized with human serum whereupon Ecb was added during decay. fB binding was detected by immunoblotting. B, fluid phase fB conversion. C3b, fB, and fD were incubated with or without Ecb or Ecb N63E/R75E/N82E (10 μg/ml), and Bb formation was analyzed by immunoblotting. C, fB conversion on zymosan. Preopsonized zymosan was incubated with fB and fD in the presence or absence of Ecb or Ecb N63E/R75E/N82E (10 μg/ml). Bb formation was analyzed by immunoblotting. All figures are representatives of three separate experiments.

FIGURE 6.

Convertase inhibition by S. aureus. A, schematic representation of the covalent attached C3b on the target surface (left), the active C3 convertase (C3bBb, center), and the binding of C3 to the convertase (right). B, schematic representation of SCIN bound to the AP C3 convertase (left) and Ecb bound to the convertase (right).