Group A streptococcal phagocytosis resistance is independent of complement factor H and factor H-like protein 1 binding

Factor H (FH) and factor H-like protein 1 (FHL-1) regulate complement activation through the alternative pathway. Several extracellular bacterial pathogens, prime targets for the complement system, bind FH and FHL-1, thereby acquiring a potential mechanism for minimizing complement deposition on their surface. For group A streptococci (GAS), surface-bound antiphagocytic M proteins mediate the interaction. To study the role of the FH-FHL-1 interaction for complement deposition and opsonophagocytosis of GAS, we first constructed a set of truncated M5 protein variants and expressed them on the surface of a homologous M-negative GAS strain. Binding experiments with the resulting strains demonstrated that the major FH-FHL-1 binding is located in a 42-amino-acid region within the N-terminal third of M5. Measurement of bacteria-bound complement factor C3 after incubation in plasma showed that the presence of this region had little impact upon complement deposition through the alternative pathway. Moreover, streptococci expressing M5 proteins lacking the major FH and FHL-1 binding sequence resisted phagocytosis in human blood as efficiently as bacteria expressing the wild-type protein. Consequently, the data suggest that the binding of the regulators of the alternative pathway is of limited importance for GAS phagocytosis resistance.     

M41型A群链球菌的Ⅰ型胶原样蛋白与人低密度脂蛋白的相互作用

【目的】检测M41型A群链球菌(GAS)ATCC12373中Ⅰ型胶原样蛋白(Scl1)与人低密度脂蛋白(LDL)的相互作用。【方法】克隆了M41型GAS ATCC12373的Ⅰ型胶原样蛋白(Scl1)及其V区(Scl1-V)基因,并表达、纯化重组蛋白rScl1(C176)和rScl1-V(C176V)。通过重组蛋白与人血浆的亲和色谱层析、Western blot及酶联免疫吸附试验(ELISA)检测C176、C176V与LDL的相互作用;通过GAS与LDL的ELISA试验和人血浆与GAS的共孵育试验,检测GAS与LDL的相互作用。【结果】结果证明C176和C176V可以与LDL特异性结合;表达Scl1的M41型GAS可以与LDL相结合。【结论】M41型GAS的Scl1可以与LDL特异性结合。     

Binding of the low-density lipoprotein by streptococcal collagen-like protein Scl1 of Streptococcus pyogenes

Several bacterial genera express proteins that contain collagen-like regions, which are associated with variable (V) non-collagenous regions. The streptococcal collagen-like proteins, Scl1 and Scl2, of group A Streptococcus (GAS) are members of this 'prokaryotic collagen' family, and they too contain an amino-terminal non-collagenous V region of unknown function. Here, we use recombinant rScl constructs, derived from several Scl1 and Scl2 variants, and affinity chromatography to identify Scl ligands present in human plasma. First, we show that Scl1, but not Scl2, proteins from different GAS serotypes bind the same ligand identified as apolipoprotein B (ApoB100), which is a major component of the low-density lipoprotein (LDL). Scl1 binding to purified ApoB100 and LDL is specific and concentration-dependent. Furthermore, the non-collagenous V region of the Scl1 protein is responsible for LDL/ApoB100 binding because only those rScls, constructed by domain swapping, which contain the V region from Scl1 proteins, were able to bind to ApoB100 and LDL ligands, and this binding was inhibited by antibodies directed against the Scl1-V region. Electron microscopy images of Scl1-LDL complexes showed that the globular V domain of Scl1 interacted with spherical particles of LDL. Importantly, live M28-type GAS cells absorbed plasma LDL on the cell surface and this binding depended on the surface expression of the Scl1.28, but not Scl2.28, protein. Phylogenetic analysis showed that the non-collagenous globular domains of Scl1 and Scl2 evolved independently to form separate lineages, which differ in amino acid sequence, and these differences may account for the variations in binding patterns of Scl1 and Scl2 proteins. Present studies provide insight into the structure-function relationship of the Scl proteins and also underline the importance of lipoprotein binding by GAS.     

Complement factor H allotype 402H is associated with increased C3b opsonization and phagocytosis of Streptococcus pyogenes

The main virulence factor of group A streptococcus (GAS), M protein, binds plasma complement regulators factor H (FH) and FH-like protein 1 (FHL-1) leading to decreased opsonization. The M protein binding site on FH is within domain 7 in which also the age-related macular degeneration (AMD)-associated polymorphism Y402H is located. We studied if FH allotypes 402H and 402Y have different binding affinities to GAS. Plasma-derived FH allotype 402H and its recombinant fragment FH5-7(402H) showed decreased binding to several GAS strains. Growth of GAS in human blood taken from FH(402H) homozygous individuals was decreased when compared with blood taken from FH(402Y) homozygous individuals. The effect of the allotype 402H can be explained by combining the previous M protein mutagenesis data and the recently published crystal structure of FH6-8. In conclusion the data indicate that the AMD-associated allotype 402H leads to diminished binding of FH to GAS and increased opsonophagocytosis of the bacteria in blood. These results suggest that the homozygous presence of the allele 402H could be associated with decreased risk for severe GAS infections offering an explanation for the high frequency of the allele despite its association with visual impairment.     

Y402H polymorphism of complement factor H affects binding affinity to C-reactive protein

Complement factor H (FH) is an important regulator of the alternative complement pathway. The Y402H polymorphism within the seventh short consensus repeat of FH was recently shown to be associated with age-related macular degeneration, the most common cause of irreversible blindness in the Western world. We examined the effects of this polymorphism on various FH functions. FH purified from sera of age-related macular degeneration patients homozygous for the FH(402H) variant showed a significantly reduced binding to C-reactive protein (CRP), an acute phase protein, as compared with FH derived from unaffected controls homozygous for the FH(402Y) variant. Strongly reduced binding to CRP was also observed with a recombinant fragment of FH (short consensus repeat 5-7) containing the same amino acid change. Because the interaction of CRP and FH promotes complement-mediated clearance of cellular debris in a noninflammatory fashion, we propose that the reduced binding of FH(402H) to CRP could lead to an impaired targeting of FH to cellular debris and a reduction in debris clearance and enhanced inflammation along the macular retinal pigmented epithelium-choroid interface in individuals with age-related macular degeneration.     

Species-specific interaction of Streptococcus pneumoniae with human complement factor H

Streptococcus pneumoniae naturally colonizes the nasopharynx as a commensal organism and sometimes causes infections in remote tissue sites. This bacterium is highly capable of resisting host innate immunity during nasopharyngeal colonization and disseminating infections. The ability to recruit complement factor H (FH) by S. pneumoniae has been implicated as a bacterial immune evasion mechanism against complement-mediated bacterial clearance because FH is a complement alternative pathway inhibitor. S. pneumoniae recruits FH through a previously defined FH binding domain of choline-binding protein A (CbpA), a major surface protein of S. pneumoniae. In this study, we show that CbpA binds to human FH, but not to the FH proteins of mouse and other animal species tested to date. Accordingly, deleting the FH binding domain of CbpA in strain D39 did not result in obvious change in the levels of pneumococcal bacteremia or virulence in a bacteremia mouse model. Furthermore, this species-specific pneumococcal interaction with FH was shown to occur in multiple pneumococcal isolates from the blood and cerebrospinal fluid. Finally, our phagocytosis experiments with human and mouse phagocytes and complement systems provide additional evidence to support our hypothesis that CbpA acts as a bacterial determinant for pneumococcal resistance to complement-mediated host defense in humans.     

The group A streptococcal collagen‐like protein‐1, Scl1, mediates biofilm formation by targeting the extra domain A‐containing variant of cellular fibronectin expressed in wounded tissue

Wounds are known to serve as portals of entry for group A Streptococcus (GAS). Subsequent tissue colonization is mediated by interactions between GAS surface proteins and host extracellular matrix components. We recently reported that the streptococcal collagen‐like protein‐1, Scl1, selectively binds the cellular form of fibronectin (cFn) and also contributes to GAS biofilm formation on abiotic surfaces. One structural feature of cFn, which is predominantly expressed in response to tissue injury, is the presence of a spliced variant containing extra domain A (EDA/EIIIA). We now report that GAS biofilm formation is mediated by the Scl1 interaction with EDA‐containing cFn. Recombinant Scl1 proteins that bound cFn also bound recombinant EDA within the C‐C’ loop region recognized by the α₉β₁ integrin. The extracellular 2‐D matrix derived from human dermal fibroblasts supports GAS adherence and biofilm formation. Altogether, this work identifies and characterizes a novel molecular mechanism by which GAS utilizes Scl1 to specifically target an extracellular matrix component that is predominantly expressed at the site of injury in order to secure host tissue colonization.     

High‐density lipoprotein acts as an opsonin to enhance phagocytosis of group A streptococcus by U937 cells

We have previously demonstrated that high‐density lipoprotein (HDL) can specifically bind to streptococcal collagen‐like protein 1 (Scl1) of M41‐type group A Streptococcus (GAS). However, the pathological or physiological significance of Scl1?HDL interaction is unknown. Here, the hypothesis that HDL acts as an opsonin to enhance phagocytosis of HDL‐bound GAS by monocytes given that some scavenger receptors can mediate the endocytosis of HDL was tested by using FITC‐labeled bacteria, human U937 monocytes and HDL for phagocytic assays. HDL (10 µg/mL) was found to significantly enhance internalization of M41‐type (ATCC 12373) GAS by U937 cells after 60 min incubation, compared with an HDL‐free group. The internalized GAS were dead after 60 min incubation with U937 cells regardless of presence and absence of HDL. Although very‐low‐density lipoprotein (VLDL) could specifically bind to ATCC 12373 strain, it did not promote phagocytosis of GAS. Additionally, LDL, HDL and VLDL did not enhance phagocytosis of CMCC 32198 strain because this strain did not bind to these lipoproteins. A physiological concentration of HDL (1000 µg/mL) had a similar effect. Anti‐CD36 antibody completely abolished opsonic phagocytosis whereas anti‐CD4 antibody did not, indicating that CD36 is the major scavenger receptor mediating the uptake of HDL‐opsonized GAS by U937 cells. Furthermore, because rScl1 competitively blocked the interaction of ATCC 12373 strain with HDL recombinant Scl1 (rScl1) derived from M41‐type GAS, it significantly decreased opsonophagocytosis of ATCC 12373 strain but not of CMCC 32198 strain. Therefore, our findings suggest that HDL may be an opsonin that enhances CD36‐dependent opsonophagocytosis of GAS by U937 cells.

     

Characterization of the immune response to collagen-like proteins Scl1 and Scl2 of serotype M1 and M28 group A Streptococcus

Group A Streptococcus (GAS) infections may trigger autoimmune sequelae thought to involve streptococcal antibodies cross-reactive to human antigens. Here, the antigenicity of the streptococcal collagen-like (CL) proteins, Scl1 and Scl2, that exhibit structural similarity to human collagen, was analyzed. Antibodies to Scl1.1 protein were detected in human sera from healthy individuals previously infected with M1 GAS and from patients with various GAS infections, as well as in sera from mice infected with M1 GAS. Linear B-cell epitope mapping identified immunoreactive peptides corresponding to the CL region of Scl1.1. Humoral responses to Scl1.28 and Scl2.28 were also detected in pediatric patients and mice infected with M28 GAS.     

Haemophilus influenzae interacts with the human complement inhibitor factor H

Pathogenic microbes acquire human complement inhibitors to circumvent the innate immune system. In this study, we identify two novel host-pathogen interactions, factor H (FH) and factor H-like protein 1 (FHL-1), the inhibitors of the alternative pathway that binds to Hib. A collection of clinical Haemophilus influenzae isolates was tested and the majority of encapsulated and unencapsulated bound FH. The isolate Hib 541 with a particularly high FH-binding was selected for detailed analysis. An increased survival in normal human serum was observed with Hib 541 as compared with the low FH-binding Hib 568. Interestingly, two binding domains were identified within FH; one binding site common to both FH and FHL-1 was located in the N-terminal short consensus repeat domains 6-7, whereas the other, specific for FH, was located in the C-terminal short consensus repeat domains 18-20. Importantly, both FH and FHL-1, when bound to the surface of Hib 541, retained cofactor activity as determined by analysis of C3b degradation. Two H. influenzae outer membrane proteins of approximately 32 and 40 kDa were detected with radiolabeled FH in Far Western blot. Taken together, in addition to interactions with the classical, lectin, and terminal pathways, H. influenzae interferes with the alternative complement activation pathway by binding FH and FHL-1, and thereby reducing the complement-mediated bactericidal activity resulting in an increased survival. In contrast to incubation with active complement, H. influenzae had a reduced survival in FH-depleted human serum, thus demonstrating that FH mediates a protective role at the bacterial surface.     

Domains of group A streptococcal M protein that confer resistance to phagocytosis, opsonization and protection: implications for vaccine development

Streptococcus pyogenes (group A streptococcus) colonizes skin and throat tissues resulting in a range of benign and serious human diseases. Opsonization and phagocytosis are important defence mechanisms employed by the host to destroy group A streptococci. Antisera against the cell-surface M protein, of which over 150 different types have been identified, are opsonic and contribute to disease protection. In this issue of Molecular Microbiology, Sandin and colleagues have comprehensively analysed the regions of M5 protein that contribute to phagocytosis resistance and opsonization. Human plasma proteins bound to M5 protein B- and C-repeats were shown to block opsonization, an observation that needs to be carefully considered for the development of M protein-derived vaccines. While safe and efficacious human group A streptococcal vaccines are not commercially available, candidate M protein-derived vaccines have shown promise in murine vaccine models and a recent phase 1 human clinical trial.     

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or ‘double’ tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the ‘double’ tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.     

Scl1-dependent internalization of group A Streptococcus via direct interactions with the alpha2beta(1) integrin enhances pathogen survival and re-emergence

The molecular pathogenesis of infections caused by group A Streptococcus (GAS) is not fully understood. We recently reported that a recombinant protein derived from the collagen-like surface protein, Scl1, bound to the human collagen receptor, integrin α₂β₁. Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin α₂β₁ and affects the biological outcome of host–pathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the α₂β₁ integrin, because (i) both adherence and internalization of the scl1- inactivated mutant were significantly decreased, and were restored by in-trans complementation of Scl1 expression, (ii) GAS internalization was reduced by pre-treatment of HEp-2 cells with anti-α₂ integrin-subunit antibody and type I collagen, (iii) recombinant α₂-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the α₂β₁ integrin as the only collagen-binding integrin. Next, we determined that internalized GAS re-emerges from epithelial cells into the extracellular environment. Taken together, our data describe a new molecular mechanism used by GAS involving the direct interaction between Scl1 and integrins, which increases the overall capability of the pathogen to survive and re-emerge.     

Identification of the streptococcal M protein binding site on membrane cofactor protein (CD46)

Adherence of group A streptococcus (GAS) to keratinocytes is mediated by an interaction between human CD46 (membrane cofactor protein) with streptococcal cell surface M protein. CD46 belongs to a family of proteins that contain structurally related short consensus repeat (SCR) domains and regulate the activation of the complement components C3b and/or C4b. CD46 possesses four SCR domains and the aim of this study was to characterize their interaction with M protein. Following confirmation of the M6 protein-dependent interaction between GAS and human keratinocytes, we demonstrated that M6 protein binds soluble recombinant CD46 protein and to a CD46 construct containing only SCRs 3 and 4. M6 protein did not bind to soluble recombinant CD46 chimeric proteins that had the third and/or fourth SCR domains replaced with the corresponding domains from another complement regulator, CD55 (decay-accelerating factor). Homology-based molecular modeling of CD46 SCRs 3 and 4 revealed a cluster of positively charged residues between the interface of these SCR domains similar to the verified M protein binding sites on the plasma complement regulators factor H and C4b-binding protein. The presence of excess M6 protein did not inhibit the cofactor activity of CD46 and the presence of excess C3b did not inhibit the ability of CD46 to bind M6 protein by ELISA. In conclusion, 1) adherence of M6 GAS to keratinocytes is M protein dependent and 2) a major M protein binding site is located within SCRs 3 and 4, probably at the interface of these two domains, at a site distinct from the C3b-binding and cofactor site of CD46.     

Binding of complement regulators factor H and C4b binding protein to group A streptococcal strains isolated from tonsillar tissue and blood

Group A streptococcus (GAS) is the most common pathogen causing bacterial pharyngitis. We isolated streptococcal strains from tonsils removed from patients with tonsillar disease (n=202) and studied their ability to bind the complement regulators factor H (FH) and C4b binding protein (C4BP) using 125 I-labeled proteins. Blood isolates of GAS (n=10) were obtained from patients with bacteraemia. Streptococci were isolated from 21% of the tonsillitis patients. The emm and T types of the GAS strains were determined. Of the 26 GAS strains studied, only six could bind FH and/or C4BP above the threshold levels. The fraction of the offered radioactive protein bound ranged between 6-12% for FH and 19-56% for C4BP. The clinical course of the tonsillar disease was not related to the binding of FH or C4BP by GAS. The binding strains were mostly of the T4M4 or T28M28 type. From the invasive strains (n=10), three bound FH (binding level: 8-11%) and two C4BP (36-39%). The binding correlated only partially to M-protein (emm) type suggesting that the binding was not exclusively due to M-protein. The results indicate that complement regulator binding by GAS is only partially related to pathogenicity and not a universal property of all group A streptococci.     

Factor H Binds to the Hypervariable Region of Many Streptococcus pyogenes M Proteins but Does Not Promote Phagocytosis Resistance or Acute Virulence

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.     

Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen responsible for mild to severe, life-threatening infections. GAS expresses a wide range of virulence factors, including the M family proteins. The M proteins allow the bacteria to evade parts of the human immune defenses by triggering the formation of a dense coat of plasma proteins surrounding the bacteria, including IgGs. However, the molecular level details of the M1-IgG interaction have remained unclear. Here, we characterized the structure and dynamics of this interaction interface in human plasma on the surface of live bacteria using integrative structural biology, combining cross-linking mass spectrometry and molecular dynamics (MD) simulations. We show that the primary interaction is formed between the S-domain of M1 and the conserved IgG Fc-domain. In addition, we show evidence for a so far uncharacterized interaction between the A-domain and the IgG Fc-domain. Both these interactions mimic the protein G-IgG interface of group C and G streptococcus. These findings underline a conserved scavenging mechanism used by GAS surface proteins that block the IgG-receptor (FcγR) to inhibit phagocytic killing. We additionally show that we can capture Fab-bound IgGs in a complex background and identify XLs between the constant region of the Fab-domain and certain regions of the M1 protein engaged in the Fab-mediated binding. Our results elucidate the M1-IgG interaction network involved in inhibition of phagocytosis and reveal important M1 peptides that can be further investigated as future vaccine targets.     

Adaptation of the group A Streptococcus adhesin Scl1 to bind fibronectin type III repeats within wound‐associated extracellular matrix: implications for cancer therapy

The human‐adapted pathogen group A Streptococcus (GAS) utilizes wounds as portals of entry into host tissue, wherein surface adhesins interact with the extracellular matrix, enabling bacterial colonization. The streptococcal collagen‐like protein 1 (Scl1) is a major adhesin of GAS that selectively binds to two fibronectin type III (FnIII) repeats within cellular fibronectin, specifically the alternatively spliced extra domains A and B, and the FnIII repeats within tenascin‐C. Binding to FnIII repeats was mediated through conserved structural determinants present within the Scl1 globular domain and facilitated GAS adherence and biofilm formation. Isoforms of cellular fibronectin that contain extra domains A and B, as well as tenascin‐C, are present for several days in the wound extracellular matrix. Scl1‐FnIII binding is therefore an example of GAS adaptation to the host's wound environment. Similarly, cellular fibronectin isoforms and tenascin‐C are present in the tumor microenvironment. Consistent with this, FnIII repeats mediate GAS attachment to and enhancement of biofilm formation on matrices deposited by cancer‐associated fibroblasts and osteosarcoma cells. These data collectively support the premise for utilization of the Scl1‐FnIII interaction as a novel method of anti‐neoplastic targeting in the tumor microenvironment.     

Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway

Human fibrinogen (Fg) binds to surface proteins expressed by many pathogenic bacteria and has been implicated in different host-pathogen interactions, but the role of bound Fg remains unclear. Here, we analyse the role of Fg bound to Streptococcus pyogenes M protein, a major virulence factor that confers resistance to phagocytosis. Studies of the M5 system showed that a chromosomal mutant lacking the Fg-binding region was completely unable to resist phagocytosis, indicating that bound Fg plays a key role in virulence. Deposition of complement on S. pyogenes occurred via the classical pathway even under non-immune conditions, but was blocked by M5-bound Fg, which reduced the amount of classical pathway C3 convertase on the bacterial surface. This property of M protein-bound Fg may explain its role in phagocytosis resistance. Previous studies have shown that many M proteins do not bind Fg, but interfere with complement deposition and phagocytosis by recruiting human C4b-binding protein (C4BP), an inhibitor of the classical pathway. Thus, all M proteins may share ability to recruit a human plasma protein, Fg or C4BP, which inhibits complement deposition via the classical pathway. Our data identify a novel function for surface-bound Fg and allow us to propose a unifying mechanism by which M proteins interfere with innate immunity.     

The factor H variant associated with age-related macular degeneration (His-384) and the non-disease-associated form bind differentially to C-reactive protein, fibromodulin, DNA, and necrotic cells

Recently, a polymorphism in the complement regulator factor H (FH) gene has been associated with age-related macular degeneration. When histidine instead of tyrosine is present at position 384 in the seventh complement control protein (CCP) domain of FH, the risk for age-related macular degeneration is increased. It was recently shown that these allotypic variants of FH, in the context of a recombinant construct corresponding to CCPs 6-8, recognize polyanionic structures differently, which may lead to altered regulation of the alternative pathway of complement. We show now that His-384, corresponding to the risk allele, binds C-reactive protein (CRP) poorly compared with the Tyr-384 form. We also found that C1q and phosphorylcholine do not compete with FH for binding to C-reactive protein. The interaction with extracellular matrix protein fibromodulin, which we now show to be mediated, at least in part, by CCP6-8 of FH, occurs via the polypeptide of fibromodulin and not through its glycosaminoglycan modifications. The Tyr-384 variant of FH bound fibromodulin better than the His-384 form. Furthermore, we find that CCP6-8 is able to interact with DNA and necrotic cells, but in contrast the His-384 allotype binds these ligands more strongly than the Tyr-384 variant. The variations in binding affinity of the two alleles indicate that complement activation and local inflammation in response to different targets will differ between His/His and Tyr/Tyr homozygotes.