Streptococcal inhibitor of complement-mediated lysis (SIC): an anti-inflammatory virulence determinant

Abstract

Since the late 1980s, a worldwide increase of severe Streptococcus pyogenes infections has been associated with strains of the M1 serotype, strains which all secrete the streptococcal inhibitor of complement-mediated lysis (SIC). Previous work has shown that SIC blocks complement-mediated haemolysis, inhibits the activity of antibacterial peptides and has affinity for the human plasma proteins clusterin and histidine-rich glycoprotein; the latter is a member of the cystatin protein family. The present work demonstrates that SIC binds to cystatin C, high-molecular-mass kininogen (HK) and low-molecular-mass kininogen, which are additional members of this protein family. The binding sites in HK are located in the cystatin-like domain D3 and the endothelial cell-binding domain D5. Immobilization of HK to cellular structures plays a central role in activation of the human contact system. SIC was found to inhibit the binding of HK to endothelial cells, and to reduce contact activation as measured by prolonged blood clotting time and impaired release of bradykinin. These results suggest that SIC modifies host defence systems, which may contribute to the virulence of S. pyogenes strains of the M1 serotype.

Fig. 1.

Cystatin C and kininogens in human plasma interact with immobilized SIC. SIC (circles) or the albumin binding protein PAB of F. magnus (triangles) were immobilized on microtitre plates and incubated with plasma in dilution series starting at 1 : 20. When binding of cystatin C was investigated, the plasma was supplemented with 10 μg cystatin C ml^–1 prior to dilution. After washing, bound cystatin C was detected with specific cystatin C antibodies (closed symbols), and kininogens with antibodies recognizing both HK and LK (open symbols). Bound antibodies were visualized after incubation with peroxidase-labelled secondary antibodies. Results from one of three representative experiments are shown.

Fig. 2.

Binding of HRG, kininogens and cystatin C to protein SIC in an indirect ELISA. (a) Microtitre plates were coated with dilution series of protein SIC and incubated with HRG (▪), HK (▴), LK (▾) or IgG (□) (all at 2 μg ml⁻¹). Binding was detected by specific antibodies to HRG and the kininogens followed by a peroxidase-labelled secondary antibody. In the case of IgG, a peroxidase-labelled anti-Ig antibody was used for detection. (b) Binding of cystatin C (2 μg ml⁻¹) to dilution series of coated protein SIC (•), M1 protein (▸), protein H (○), human serum albumin (▿), human IgG (◂) or fibrinogen (▵) was tested. Bound cystatin C was detected as described in Methods. Results from one of three representative experiments are shown.

Fig. 3.

Binding of radiolabelled SIC to immobilized cystatin C and HK. Various amounts of cystatin C, HK and human serum albumin were applied in slots to a PVDF membrane and probed with ¹²⁵I-labelled SIC (2×10⁵ c.p.m. ml⁻¹) for 3 h.

Fig. 4.

SIC binds to domains D3 and D5 of HK. Microtitre plates were coated with dilution series of SIC and incubated with domain D3 (•) or domain D5 (○) of HK (both at 2 μg ml⁻¹). After washing, bound proteins were detected with a polyclonal antiserum against HK. Bound antibodies were detected with peroxidase-labelled secondary antibodies. Results from one of at least three representative experiments are shown.

Fig. 5.

SIC inhibits kaolin-induced plasma clotting. Human plasma samples were preincubated with various concentrations of SIC (•) or protein PAB of F. magnus (○) for 60 min and subsequently analysed by the aPTT test.

Fig. 6.

SIC blocks BK generation. Twenty-five microlitres of human plasma was incubated with 25 μl SIC of various concentrations for 30 min. After the addition of the aPTT reagent kaolin (50 μl), the amount of BK in 2 μl of the reaction mixtures was determined by a competitive ELISA. Plasma not incubated with SIC or kaolin (‘no aPTT reagent’) and plasma activated by kaolin in the absence of SIC (‘plus aPTT reagent’) were negative and positive controls, respectively. Bars, sem.

Fig. 7.

SIC interferes with HK binding to endothelial cells. Biotin–HK (20 nM) was added to endothelial cells (HUVEC) in HEPES-Tyrode's buffer containing 50 μM Zn²⁺ in the presence of protein SIC (•, 1–200 μM), CM–papain (▿, 1–30 μM) or cystatin C (○, 1–100 μM). The proportion of biotin–HK bound to HUVECs was determined in the presence of each competitor protein after non-specific binding was subtracted. Non-specific binding was determined by the level of binding seen in the presence of a 50-fold molar excess of unlabelled HK. Data presented are the mean±sem of three experiments. The absence of error bars at some data points indicates a small deviation of values.

Fig. 8.

Cystatin C binding by SIC is not affected by cathepsin B complex formation. Microtitre plates were coated with SIC and then incubated with 0.2 ml pH 6.5 buffer containing cathepsin B (40 μg ml⁻¹), cystatin C (20 μg ml⁻¹) or cathepsin B applied to wells that had already been incubated with cystatin C for 60 min at 25 °C. After incubation for 15 min, the contents in the wells were subjected to chromogenic assay using the substrate Bz-Arg-pNA (grey bars), and cystatin C bound to the wells coated with SIC was detected by specific antibodies followed by peroxidase-labelled secondary antibodies (black bars). No cathepsin B activity over background levels was detected, verifying that virtually all cystatin C is available for cathepsin B binding despite preincubation with SIC and, conversely, that cathepsin B–cystatin C complexes are bound by SIC. Results of one representative experiment out of four are shown.