Streptococcal inhibitor of complement (SIC) inhibits the membrane attack complex by preventing uptake of C567 onto cell membranes

Abstract

Streptococcal inhibitor of complement (SIC) was first described in 1996 as a putative inhibitor of the membrane attack complex of complement (MAC). SIC is a 31 000 MW protein secreted in large quantities by the virulent Streptococcus pyogenes strains M1 and M57, and is encoded by a gene which is extremely variable. In order to study further the interactions of SIC with the MAC, we have made a recombinant form of SIC (rSIC) in Escherichia coli and purified native M1 SIC which was used to raise a polyclonal antibody. SIC prevented reactive lysis of guinea pig erythrocytes by the MAC at a stage prior to C5b67 complexes binding to cell membranes, presumably by blocking the transiently expressed membrane insertion site on C7. The ability of SIC and clusterin (another putative fluid phase complement inhibitor) to inhibit complement lysis was compared, and found to be equally efficient. In parallel, by enzyme-linked immunosorbent assay both SIC and rSIC bound strongly to C5b67 and C5b678 complexes and to a lesser extent C5b-9, but only weakly to individual complement components. The implications of these data for virulence of SIC-positive streptococci are discussed, in light of the fact that Gram-positive organisms are already protected against complement lysis by the presence of their peptidoglycan cell walls. We speculate that MAC inhibition may not be the sole function of SIC.

Figure 1

Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by SDS–PAGE and Western blotting using polyclonal anti-C4bp. The position of the 70 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of HindIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic-like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).

Figure 2

Western blot analysis of recombinant and native SIC(a) Western blot showing reactivity of rabbit polyclonal antibody to native SIC and recombinant SIC: (1) rSIC; (2) E. coli protein extract negative control; (3) concentrated M1 culture supernatant. Lower MW bands are presumed to represent breakdown products of SIC and rSIC. (b) Coomassie stained 10% SDS–PAGE gel of purified native SIC. The lower band is a breakdown product of SIC. N-terminal sequencing of this band showed that 33 amino acids had been lost from the native protein. (c) Coomassie stained 10% SDS–PAGE gel of rSIC-containing column fractions, and (d) autoradiograph of a Western blot from an identical SDS–PAGE gel demonstrating that rSIC eluted mainly at 350 mm imidazole. rSIC was detected using HRP-conjugated monoclonal anti-V5 epitope and chemiluminescence. Non-reactive bands in panel (c) are residual E. coli proteins.

Figure 3

SIC inhibits the binding of C5b-7 complexes to cell membranes. Reactive lysis of guinea pig erythrocytes was initiated by addition of pre-formed C56 complex followed by either (a) 1/40 normal human serum in 10 mm EDTA, or (b) purified C7, C8 and C9. Cells preincubated with SIC were protected against lysis compared to control cells incubated with BSA. When added to cells after C5b-7 sites had been formed on their surfaces (c), SIC had very little effect upon haemolysis, showing that it works by blocking uptake of C5b-7 complexes. A comparison of the activity of SIC and clusterin (d) showed that SIC and clusterin were equally efficient at inhibiting haemolysis of GPE by C56 and 1/40 NHS/EDTA. Data are expressed as mean ± SEM of triplicate results from a typical experiment. Error bars are too small to be seen on some data points.

Figure 4

SIC binds to C5b-7, C5b-8 and C5b-9 complexes in ELISA. ELISA plates were coated with SIC (panels a and b), rSIC (panels c and d) or coating buffer only and intermediate or complete TCC complexes were added. Binding was detected with (i) polyclonal anti-C6 (a and c); or (ii) monoclonal anti-C5b neo-epitope (b and d). SIC and rSIC bind preferentially to C5b-7 complexes and to a lesser extent to C5b-8 and C5b-9 using anti-C6 to detect, but predominantly to C5b-8 using anti-C5b neo-epitope. Results displayed are the net ODs after deduction of background in coated wells plus antibodies but no sera.

Figure 5

SIC binds to purified C7 and C8 in ELISA. The ELISA plate was coated with individual purified components of the TCC (or SIC or buffer only as controls). SIC was added and binding detected with polyclonal anti-SIC showing that SIC binds slightly to purified C7 and C8 and to a lesser extent to C6.