The alternative pathway of complement is important in innate immunity, attacking not only microbes but all unprotected biological surfaces through powerful amplification. It is unresolved how host and nonhost surfaces are distinguished at the molecular level, but key components are domains 19-20 of the complement regulator factor H (FH), which interact with host (i.e., nonactivator surface glycosaminoglycans or sialic acids) and the C3d part of C3b. Our structure of the FH19-20:C3d complex at 2.3-Å resolution shows that FH19-20 has two distinct binding sites, FH19 and FH20, for C3b. We show simultaneous binding of FH19 to C3b and FH20 to nonactivator surface glycosaminoglycans, and we show that both of these interactions are necessary for full binding of FH to C3b on nonactivator surfaces (i.e., for target discrimination). We also show that C3d could replace glycosaminoglycan binding to FH20, thus providing a feedback control for preventing excess C3b deposition and complement amplification. This explains the molecular basis of atypical hemolytic uremic syndrome, where mutations on the binding interfaces between FH19-20 and C3d or between FH20 and glycosaminoglycans lead to complement attack against host surfaces. structure and function | X-ray crystallography | hemolysis | kidney diseases | human mutations P reviously unencountered microbes invading a human body must be rapidly recognized and eliminated. This is the function of innate immunity, which includes the alternative pathway (AP) of complement. AP components can attack targets with hydroxyl or amine groups (i.e., all biological surfaces). This is a powerful defense mechanism, because there is rapid amplification leading to efficient opsonization or target lysis by the membrane attack complex (MAC). The AP attack is, therefore, also potentially dangerous for the host if one's cells and acellular structures are not protected.The AP activation is based on spontaneous hydrolysis of C3 in plasma leading to production of C3b, which then randomly attaches onto any surface hydroxyl or amine group through a reactive thioester located on the C3d part [i.e., thioester domain (TED)] of C3b. If these surface-attached C3b molecules are not quickly inactivated to iC3b and C3d, C3b deposition is rapidly amplified by a positive enzymatic feedback loop, leading to opsonophagocytosis and formation of the lytic membrane attack complex. On host surfaces, which are naturally nonactivators of the AP, efficient down-regulation of bound C3b occurs in three ways: factor I-mediated cleavage of C3b to inactive iC3b, acceleration of the decay of the preformed C3 convertases, or inhibition of factor B binding to C3b. Factor H (FH) is required for all these. It also down-regulates C3b deposition on noncellular surfaces, such as the heparan sulfate-rich glomerular basement membrane. FH is, thus, essential for restricting AP attack against host surfaces while allowing AP attack against foreign surfaces (i.e., for target discrimination) (1). A long-standing central question in complemen...

To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19–20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a “superevasion site.”

Complement is a major innate immune surveillance system. One of its most important regulators is the plasma protein factor H (FH). FH inactivation by mutations or by autoantibodies is associated with a thrombotic microangiopathy disease, atypical hemolytic uremic syndrome. In this study, we report the characterization of blood samples from 19 anti-FH Ab-positive atypical hemolytic uremic syndrome patients collected at the acute phase of the disease. Analyses of the functional consequences and epitope mapping, using both fluid phase and solid phase approaches, were performed. The anti-FH Abs perturbed FH-mediated cell protection (100%), inhibited FH interaction with C3 (46%), and caused C3 consumption (47%). The Abs were directed against multiple FH epitopes located at the N and C termini. In all tested patients, high titers of FH-containing circulating immune complexes were detected. The circulating immune complex titers correlated with the disease stage better than did the Ab titers. Our results show that anti-FH autoantibodies induce neutralization of FH at acute phase of the disease, leading to an overall impairment of several functions of FH, extending the role of autoantibodies beyond the impairment of the direct cell surface protection.

Key Points• Sialic acids are critical for factor H-mediated complement regulation on endothelial cells, erythrocytes, and platelets. • Impaired ability of factor H mutants to simultaneously bind sialic acid and C3b on cells explains their association with aHUS.Uncontrolled activation of the complement system against endothelial and blood cells is central to the pathogenesis of atypical hemolytic uremic syndrome (aHUS). aHUS patients frequently carry mutations in the inhibitory complement regulator factor H (FH). Mutations cluster in domains [19][20], which are critical for recognizing self surfaces. On endothelial cells, binding of FH is generally attributed to heparan sulfate. This theory, however, is questioned by the puzzling observation that some aHUSassociated mutations markedly enhance FH binding to heparin and endothelial cells. In this article, we show that, instead of disturbed heparin interactions, the impaired ability of C-terminal mutant FH molecules to recognize sialic acid in the context of surface-bound C3b explains their pathogenicity. By using recombinant FH19-20 as a competitor for FH and measuring erythrocyte lysis and deposition of complement C3b and C5b-9 on endothelial cells and platelets, we now show that several aHUS-associated mutations, which have been predicted to impair FH19-20 binding to sialic acid, prevent FH19-20 from antagonizing FH function on cells. When sialic acid was removed, the wild-type FH19-20 also lost its ability to interfere with FH function on cells. These results indicate that sialic acid is critical for FH-mediated complement regulation on erythrocytes, endothelial cells, and platelets. The inability of C-terminal mutant FH molecules to simultaneously bind sialic acid and C3b on cells provides a unifying explanation for their association with aHUS. Proper formation of FH-sialic acid-C3b complexes on surfaces exposed to plasma is essential for preventing cell damage and thrombogenesis characteristic of aHUS.

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Complement is an essential humoral component of innate immunity; however, its inappropriate activation leads to pathology. Polymorphisms, mutations, and autoantibodies affecting factor H (FH), a major regulator of the alternative complement pathway, are associated with various diseases, including age-related macular degeneration, atypical hemolytic uremic syndrome, and C3 glomerulopathies. Restoring FH function could be a treatment option for such pathologies. In this article, we report on an engineered FH construct that directly combines the two major functional regions of FH: the N-terminal complement regulatory domains and the C-terminal surface-recognition domains. This minimal-size FH (mini-FH) binds C3b and has complement regulatory functions similar to those of the full-length protein. In addition, we demonstrate that mini-FH binds to the FH ligands C-reactive protein, pentraxin 3, and malondialdehyde epitopes. Mini-FH was functionally active when bound to the extracellular matrix and endothelial cells in vitro, and it inhibited C3 deposition on the cells. Furthermore, mini-FH efficiently inhibited complement-mediated lysis of host-like cells caused by a disease-associated FH mutation or by anti-FH autoantibodies. Therefore, mini-FH could potentially be used as a complement inhibitor targeting host surfaces, as well as to replace compromised FH in diseases associated with FH dysfunction.

Background: It is unknown why patients with autoantibodies against complement factor H (CFH) lack homologous CFHR1 protein.Results: The autoantibody epitope on CFH was identified, and the structure of the corresponding part of CFHR1 was solved.Conclusion: The autoantigenic epitope of CFH and its homologous site in CFHR1 are structurally different.Significance: A plausible explanation for formation of autoantibodies due to CFHR1 deficiency in autoimmune atypical hemolytic uremic syndrome was obtained.

Staphylococcus aureus is a major human pathogen causing more than a tenth of all septicemia cases and often superficial and deep infections in various tissues. One of the immune evasion strategies of S. aureus is to secrete proteins that bind to the central complement opsonin C3b. One of these, extracellular complement binding protein (Ecb), is known to interfere directly with functions of C3b. Because C3b is also the target of the physiological plasma complement regulator, factor H (FH), we studied the effect of Ecb on the complement regulatory functions of FH. We show that Ecb enhances acquisition of FH from serum onto staphylococcal surfaces. Ecb and FH enhance mutual binding to C3b and also the function of each other in downregulating complement activation. Both Ecb and the C-terminal domains 19–20 of FH bind to the C3d part of C3b. We show that the mutual enhancing effect of Ecb and FH on binding to C3b depends on binding of the FH domain 19 to the C3d part of C3b next to the binding site of Ecb on C3d. Our results show that Ecb, FH, and C3b form a tripartite complex. Upon exposure of serum-sensitive Haemophilus influenzae to human serum, Ecb protected the bacteria, and this effect was enhanced by the addition of the C-terminal domains 19–20 of FH. This finding indicates that the tripartite complex formation could give additional protection to bacteria and that S. aureus is thereby able to use host FH and bacterial Ecb in a concerted action to eliminate C3b at the site of infection.