Structural basis for inhibition of complement C5 by the SSL7 protein fromStaphylococcus aureus

Abstract

Staphylococcus aureus secretes the SSL7 protein as part of its immune evasion strategy. The protein binds both complement C5 and IgA, yet it is unclear whether SSL7 cross-links these two proteins and, if so, what purpose this serves the pathogen. We have isolated a stable IgA-SSL7-C5 complex, and our crystal structure of the C5-SSL7 complex confirms that binding to C5 occurs exclusively through the C-terminal β-grasp domain of SSL7 leaving the OB domain free to interact with IgA. SSL7 interacts with C5 >70 Å from the C5a cleavage site without inducing significant conformational changes in C5, and efficient inhibition of convertase cleavage of C5 is shown to be IgA dependent. Inhibition of C5a production and bacteriolysis are all shown to require C5 and IgA binding while inhibition of hemolysis is achieved by the C5 binding SSL7 β-grasp domain alone. These results provide a conceptual and structural basis for the development of a highly specific complement inhibitor preventing only the formation of the lytic membrane attack complex without affecting the important signaling functions of C5a. Complement activation triggers cleavage of the three homologous 185-to 200-kDa proteins C3, C4, and C5. Three activation pathways converge on C3 cleavage to C3b (6). Activation by the alternative pathway (AP) results from spontaneous hydrolysis of an internal thioester bond in C3 or by deposition of properdin on an appropriate surface (7), whereas the classical pathway (CP) and lectin-mediated pathways are activated by surface-bound immune complexes or mannan binding lectins/ficolins, respectively. Activation of the CP or the lectin pathway generates the surface-bound C3 convertase (a proteolytic enzyme cleaving C3) C4b2a, whereas the AP generates the C3 convertase C3bBb. Both may recruit an additional C3b molecule to form the CP C5 convertase C4b2a3b or the AP C5 convertase C3bBb3b (8, 9), which cleaves C5 to generate the large fragment C5b and the anaphylatoxin C5a. This binds with high affinity to the C5a receptor (CD88) on myeloid cells triggering G protein (Gα I and Gα 16 )-mediated cell activation, chemotaxis, respiratory burst, and release of proinflammatory mediators (10). C5b quickly associates with C6, C7, C8, and multiple molecules of C9 to form the membrane attack complex (MAC) that results in rapid cell lysis (11). Elevated levels of C5a are implicated in a wide range of inflammatory disorders, such as rheumatoid arthritis, ischemia/reperfusion injury, sepsis, and fibrotic conditions (12). MAC deposition through C5b on erythrocytes results in destruction of these cells in the hemolytic disease paroxysmal nocturnal hemoglobinuria (PNH) (13). In addition, excessive MAC formation is linked with the pathophysiology of conditions such as antibody-mediated transplant rejection (14), inflammatory neuropathies (15) and multiple sclerosis (16).Given its importance to innate immune clearance, pathogens have developed many strategies to prevent complement activation (17). The Staphylococcal Superantigen-Like protein 7 (SSL7)...

Crystal Structure of C5b-6 Suggests Structural Basis for Priming Assembly of the Membrane Attack Complex

“…The two chains of C5b remain covalently linked via a disulfide bond. Although the structure of C5 (20,24,26) is known, as well as the homologous C3 and its proteolytic product, C3b, the structure of C5b has not previously been reported.…”

“…The structure was solved initially by placing the major domains, using a molecular replacement method implemented in the program PHASER from CCP4 (18). Search models were based on C3b (Protein Data Bank code 3IDH) (19), C5 (Protein Data Bank code 3PRX) (20), and C6 (Protein Data Bank code 3T5O) (14). Template 1 contained the first seven domains of C5 overlaid onto the equivalent domains of C3b; template 2 comprised the MAC-perforin (MACPF) and selected auxiliary domains of C6; and templates 3-5 comprised C5d, the C1r/C1s, Uegf, and BMP-1 (bone morphogenetic protein-1) (CUB) domain, and C345C from the structure of C5.…”

Streptococcus pneumoniae Phosphoglycerate Kinase Is a Novel Complement Inhibitor Affecting the Membrane Attack Complex Formation

“…Although the MAC formation directly kills the Gram-negative bacteria either by lysis or disturbance of metabolic processes, the role of MAC deposition-mediated lysis or attenuation of the Gram-positive organisms is perceived to be ineffective. Nevertheless, to date two MAC inhibitors have been identified in Gram-positive bacteria that include streptococcal inhibitor of complement (SIC) from Group A streptococci and staphylococcal superantigen-like protein 7 (SSL7) from S. aureus (20,21,38). In addition, several other MAC inhibitors are known that include the recently identified terminal pathway inhibitor CspA and the CD59-like protein from Gram-negative bacteria Borrelia burgdorferi, schistosome C inhibitory protein type 1 (SCIP-1) from Schistosoma mansoni, paramyosin from S. mansoni and Trichinella spiralis, and the galactose-specific adhesin from Entamoeba histolytica (39 -43).…”

“…Interestingly, the deficiency of MAC components in patients was associated with increased susceptibility to bacterial infections not only with Neisseria but also some Grampositive species (19). Moreover, despite the currently perceived non-essential role of MAC in Gram-positive bacterial killing, many Gram-positive bacteria such as Streptococcus pyogenes (Group A streptococci) and Staphylococcus aureus not only express proteins that inhibit MAC formation but also recruit the host inhibitors of MAC (20,21).…”

Specificity of Staphylococcal Superantigen-Like Protein 10 toward the Human IgG1 Fc Domain

“…sAgs bind the MHC class II and TCR. SSL7 binds to the Ca2/Ca3 region of IgA Fc through its N-terminal OB-fold domain (19,22) and complement factor C5 via the Cterminal b-grasp domain, making it a potent inhibitor of end-stage complement and IgA-FcaRI-mediated activities (18,23).…”