Immune evasion of Borrelia burgdorferi by acquisition of human complement regulators FHL-1/reconectin and Factor H

To understand immune evasion mechanisms of Borrelia burgdorferi we compared serum-resistant B. afzelii and serum-sensitive B. garinii isolates for their capacity toacquire human complement regulators. Here we demonstrate that the two borrelial genospecies show different binding of the two important human complement regulators, FHL-1/reconectin and Factor H. All serum-resistant B. afzelii isolates bound FHL-1/reconectin and also Factor H, and all analyzed serum-sensitive B. garinii isolates showed no or a significantly lower binding activity. Using recombinant deletion mutants, the binding domains were localized to the C terminus of FHL-1/reconectin to short consensus repeats 5-7. The borrelial binding proteins were located in the surface of the bacteria as demonstrated by immunofluorescence staining of intact, serum-exposed bacteria and by enrichment of outer membrane proteins. The surface-attached complement regulators maintained complement regulatory activity as demonstrated in a cofactor assay. By ligand blotting two different borrelial binding proteins were identified that were responsible for the surface attachment of FHL-1/reconectin and Factor H. These borrelial complement regulators acquiring surface proteins (CRASP) were further characterized as either CRASP-1, a 27.5-kDa molecule which preferentially binds FHL-1/reconectin and which was present in all serum-resistant borreliae, or CRASP-2, a 20/21-kDa protein which interacts preferentially with Factor H and the expression of which was more restricted, being detected in four of the six isolates analyzed. In summary, we describe a new immune evasion mechanism of B. burgdorferi, as these bacteria acquire human complement regulators to control complement activation on their surface and to prevent formation of toxic activation products.     

Further Characterization of Complement Regulator-Acquiring Surface Proteins of Borrelia burgdorferi

The three genospecies Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii, all causative agents of Lyme disease, differ in their susceptibilities to human complement-mediated lysis. We recently reported that serum resistance of borrelias correlates largely with their ability to bind the human complement regulators FHL-1/reconectin and factor H. To date, two complement regulator-acquiring-proteins (CRASP-1 and CRASP-2) have been identified in serum-resistant B. afzelii isolates (P. Kraiczy, C. Skerka, M. Kirschfink, V. Brade, and P. F. Zipfel, Eur. J. Immunol. 31:1674-1684, 2001). Here, we present a comprehensive study of the CRASPs detectable in both serum-resistant and intermediate serum-sensitive B. afzelii and B. burgdorferi isolates. These CRASPs were designated according to the genospecies either as BaCRASPs, when derived from B. afzelii, or as BbCRASPs, for proteins identified in B. burgdorferi isolates. Each borrelial isolate expresses distinct CRASPs that can be differentiated by their mobility and binding phenotypes. A detailed comparison reveals overlapping and even identical binding profiles for BaCRASP-1 (27.5 kDa), BbCRASP-1 (25.9 kDa), and BbCRASP-2 (23.2 kDa), which bind FHL-1/reconectin strongly and interact weakly with factor H. In contrast, two B. afzelii proteins (BaCRASP-4 [19.2 kDa] and BaCRASP-5 [22.5 kDa]) and three B. burgdorferi proteins (BbCRASP-3 [19.8 kDa], BbCRASP-4 [18.5 kDa], and BbCRASP-5 [17.7 kDa]) bind factor H but not FHL-1/reconectin. Most CRASPs bind both human immune regulators at their C-terminal ends. Temperature-dependent up-regulation of CRASPs (BaCRASP-1, BaCRASP-2, and BaCRASP-5) is detected in low-passage borrelias cultured at 33 or 37 degrees C compared with those cultured at 20 degrees C. The characterization of the individual CRASPs on the molecular level is expected to identify new virulence factors and potential vaccine candidates.     

Borrelia burgdorferi regulates expression of complement regulator-acquiring surface protein 1 during the mammal-tick infection cycle

During the natural mammal-tick infection cycle, the Lyme disease spirochete Borrelia burgdorferi comes into contact with components of the alternative complement pathway. B. burgdorferi, like many other human pathogens, has evolved the immune evasion strategy of binding two host-derived fluid-phase regulators of complement, factor H and factor H-like protein 1 (FHL-1). The borrelial complement regulator-acquiring surface protein 1 (CRASP-1) is a surface-exposed lipoprotein that binds both factor H and FHL-1. Analysis of CRASP-1 expression during the mammal-tick infectious cycle indicated that B. burgdorferi expresses this protein during mammalian infection, supporting the hypothesized role for CRASP-1 in immune evasion. However, CRASP-1 synthesis was repressed in bacteria during colonization of vector ticks. Analysis of cultured bacteria indicated that CRASP-1 is differentially expressed in response to changes in pH. Comparisons of CRASP-1 expression patterns with those of other infection-associated B. burgdorferi proteins, including the OspC, OspA, and Erp proteins, indicated that each protein is regulated through a unique mechanism.     

FHL-1/reconectin: a human complement and immune regulator with cell-adhesive function

A novel regulator of the alternative complement pathway with functions similar to that of factor H has been identified in human plasma. The cDNA encoding this factor H-like protein 1 (FHL-1/reconectin) was isolated several years ago. Here, Peter Zipfel and Christine Skerka describe functional analyses revealing that this novel complement regulatory protein forms a molecular link between immune defense and cell adhesion.     

The yeast Candida albicans binds complement regulators factor H and FHL-1

The human facultative pathogenic yeast Candida albicans causes mucocutaneous infections and is the major cause of opportunistic fungal infections in immunocompromised patients. C. albicans activates both the alternative and classical pathway of the complement system. The aim of this study was to assay whether C. albicans binds human complement regulators in order to control complement activation at its surface. We observed binding of two central complement regulators, factor H and FHL-1, from normal human serum to C. albicans by adsorption assays, immunostaining, and fluorescence-activated cell sorter (FACS) analyses. Specificity of acquisition was further confirmed in direct binding assays with purified proteins. The surface-attached regulators maintained their complement regulatory activities and mediated factor I-dependent cleavage of C3b. Adsorption assays with recombinant deletion mutant proteins were used to identify binding domains. Two binding sites were localized. One binding domain common to both factor H and FHL-1 is located in the N-terminal short consensus repeat domains (SCRs) 6 and 7, and the other one located in C-terminal SCRs 19 and 20 is unique to factor H. These data indicate that by surface acquisition of host complement regulators, the human pathogenic yeast C. albicans is able to regulate alternative complement activation at its surface and to inactivate toxic complement activation products.     

Immune evasion of Borrelia burgdorferi: Insufficient killing of the pathogens by complement and antibody

The innate immune system and, in particular, the complement system play a key role in the elimination of micro-organisms after entrance in the human host. Like other pathogens, borreliae must develop strategies to inactivate host defence mechanisms. By investigating serum (NHS)-suscepti-bility of borreliae, we found that mainly B.afzelii isolates are serum-resistant, whereas the majority of B. burgdorferi s.s. isolates display an intermediate serum-sensitive phenotype. In contrast, B.garinii isolates are killed effectively by complement and therefore are classified as serum-sensitive. Up to now, we have identified two distinct proteins of 27.5 kDa and 20.7 kDa expressed on the outer surface of borreliae, which interact directly with FHL-1/reconectin and factor H, the two major regulators of the alternative complement pathway. These borrelial proteins are termed CRASPs (complement regulator-acquiring surface proteins). CRASPs are detectable only on serumresistant borreliae and, accordingly, binding of FHL-1/reconectin and factor H only occur with serum-resistant borrelial isolates. We conclude from these results that the control of complement activation on the borrelial surface is due to the interaction of borrelial CRASPs with host complement regulatory proteins. Thus, CRASPs represent an important mechanism of immune evasion on the part of borrelial isolates belonging mostly to the genospecies B.afzelii.By analysing the humoral adaptive immune response of patients, we detected sera that killed NHS-resistant borreliae. Borreliacidal activity is observed most frequently with sera of patients at stage III of the disease. The killing of NHS-resistant isolates by these immune sera always requires the combination of antibodies and complement. Bactericidal activity, however, is not detected in all immune sera at the different disease stages, although specific anti-Borrelia antibodies are present according to serological test results. This observation suggests that not all borrelial antigens are able to induce a borreliacidal immune response. In an extensive analysis of 24 immune sera, we identified up to 12 borrelial antigens, including OspC, which possess the greatest potential for the induction of borreliacidal antibody. The borreliacidal potential of anti-OspC antibodies was tested directly on an OspC-expressing borrelial wild-type isolate and a corresponding variant lacking OspC. In these studies, only the wild-type isolate expressing OspC on its surface proved positive for the lytic complement complex, thereby indicating the great importance of this antigen for the control of the infection. Additional studies are required to identify further “protective” antigens among these 12 proteins, all of which are candidates for infection control according to our studies involving patient immune sera. These antigens may include the recently detected CRASPs.     

Human keratinocytes produce the complement inhibitor factor H: synthesis is regulated by interferon-gamma

Locally synthesized complement is believed to play an important role in host defense and inflammation at organ level. In the epidermis, keratinocytes have so far been shown to synthesize two complement components, C3 and factor B. Here, we studied the synthesis of factor H by human keratinocytes. We also studied the regulation of factor H synthesis in keratinocytes by several cytokines, namely IL-1alpha, IL-2, IL-6, TGF-beta1, TNF-alpha and IFN-gamma. Human keratinocytes expressed factor H mRNA and constitutively released small amounts of factor H protein into the culture medium. This release was strongly upregulated by IFN-gamma but not by other cytokines tested. Western blot analysis revealed that IFN-gamma augments the synthesis of both molecular species, factor H (FH; 155kDa) and factor H-like protein-1 (FHL-1; 45kDa), of factor H. Factor H released in response to IFN-gamma was functionally active. In conclusion, we demonstrate that keratinocytes are capable of synthesizing factor H and that this synthesis is regulated by IFN-gamma.     

Assessment of the interaction of human complement regulatory proteins with group A Streptococcus. Identification of a high-affinity group A Streptococcus binding site in FHL-1

Group A Streptococcus (GAS), the most frequent bacterial cause of suppurative infections in humans, expresses on the cell surface M proteins with capacity to bind factor H, FHL-1 and C4b binding protein (C4BP). This has been interpreted as a mechanism developed by this pathogen to decrease phagocytosis by macrophages and polymorphonuclear cells. We report the analysis of the capacity to bind factor H, FHL-1 and C4BP of 69 clinical isolates from 19 different serotypes. We show that strains binding complement regulators (30/69) belong to specific M serotypes. Of these, M18 strains are relatively frequent and interact with all three complement regulators simultaneously. However, the most virulent M1 and M3 strains did not bind complement regulators in our assays. The relevance of the interaction between complement regulators and S. pyogenes was analyzed using different approaches with the conclusion that under physiological conditions only FHL-1 and C4BP bind to streptococci. We show that FHL-1 presents a higher binding affinity for S. pyogenes than factor H because it carries a hydrophobic, high-affinity, GAS binding site in addition to the heparin binding site in SCR7. Using synthetic peptides we provide evidence that the high-affinity GAS binding site in FHL-1 involves the hydrophobic tail (Ser-Phe-Thr-Leu) that distinguishes FHL-1 from factor H.     

Assessment of the regions within complement regulator-acquiring surface protein (CRASP)-2 of Borrelia burgdorferi required for interaction with host immune regulators FHL-1 and factor H

The ability of Borrelia burgdorferi sensu lato to survive and persist in a variety of vertebrate hosts is a multifactorial process. Several potential mechanisms of immune evasion have been identified. We have shown that some Borrelia species bind host-derived fluid-phase immune regulators FHL-1 and factor H to their surface via complement regulator-acquiring surface proteins (CRASPs). Factor H and FHL-1 serve as cofactors for factor I, a serine protease that cleaves C3b directly on the cell surface and thereby confers resistance of spirochetes to complement-mediated lysis. Among the CRASP molecules produced by B. burgdorferi, BbCRASP-2 represents a novel FHL-1 and factor H binding protein that is distinct from other borrelial CRASP molecules and is predominantly expressed by serum-resistant Borrelia strains. The aim of this study was to identify BbCRASP-2 determinants required for FHL-1 and factor H binding. A number of recombinant BbCRASP-2 mutants were generated by in vitro mutagenesis and tested for factor H and FHL-1 binding employing ELISA. Up to 8 amino acid substitutions in the proposed binding regions 2 and 3 of BbCRASP-2 resulted in reduced or complete loss of FHL-1 and/or factor H binding. These results suggest that the factor H/FHL-1 binding regions are discontinuous and long distance interaction is involved in binding of both immune regulators. Furthermore, putative coiled-coil structural elements as recently discussed to be important in the interaction of BbCRASP-1 with factor H seem to play a subordinate role for binding of BbCRASP-2 to FHL-1 and factor H. The elucidation of host–pathogen interactions will help to develop novel therapeutic strategies against Lyme disease/borreliosis.     

Identification and characterization of the factor H and FHL-1 binding complement regulator-acquiring surface protein 1 of the Lyme disease spirochete Borrelia spielmanii sp. nov

Borrelia spielmanii, one of the etiological agents of Lyme disease found in Europe, evades host complement-mediated killing by recruitment of the immune regulators factor H and FHL-1 from human serum. Serum-resistant and intermediate serum-resistant isolates express up to 3 distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. The present study describes identification and functional characterization of BsCRASP-1 as the dominant factor H and FHL-1 binding protein of B. spielmanii. BsCRASP-1 is a 27.7kDa outer surface lipoprotein, which after processing has a predicted mass of 24.9kDa. BsCRASP-1 is encoded by a single copy gene, cspA, that maps to a linear plasmid of approximately 55kb. Ligand affinity blot techniques revealed that both native and recombinant BsCRASP-1 from different isolates can strongly bind FHL-1, but only weakly factor H. Deletion mutants of recombinant BsCRASP-1 were generated and a high-affinity binding site for factor H and FHL-1 was mapped to its carboxy-terminal 10-amino-acid residue domain. Similarly, the dominant binding site of factor H and FHL-1 was localized to short consensus repeats (SCRs) 5-7. Factor H and FHL-1 maintained cofactor activity for factor I-mediated C3b inactivation when bound to full-length BsCRASP-1 but not to a deletion mutant lacking the carboxy-terminal 10-amino-acid residue domain. In conclusion, BsCRASP-1 binds the host immune regulators factor H and FHL-1, and is suggested to represent a key molecule of B. spielmanii for complement resistance. Thus, BsCRASP-1 most likely contributes to persistence of B. spielmanii and to pathogenesis of Lyme disease.     

Human pathogenic Borrelia spielmanii sp. nov. resists complement-mediated killing by direct binding of immune regulators factor H and factor H-like protein 1

Borrelia spielmanii sp. nov. has recently been shown to be a novel human pathogenic genospecies that causes Lyme disease in Europe. In order to elucidate the immune evasion mechanisms of B. spielmanii, we compared the abilities of isolates obtained from Lyme disease patients and tick isolate PC-Eq17 to escape from complement-mediated bacteriolysis. Using a growth inhibition assay, we show that four B. spielmanii isolates, including PC-Eq17, are serum resistant, whereas a single isolate, PMew, was more sensitive to complement-mediated lysis. All isolates activated complement in vitro, as demonstrated by covalent attachment of C3 fragments; however, deposition of the later activation products C6 and C5b-9 was restricted to the moderately serum-resistant isolate PMew and the serum-sensitive B. garinii isolate G1. Furthermore, serum adsorption experiments revealed that all B. spielmanii isolates acquired the host alternative pathway regulators factor H and factor H-like protein (FHL-1) from human serum. Both complement regulators retained their factor I-mediated C3b inactivation activities when bound to spirochetes. In addition, two distinct factor H and FHL-1 binding proteins, BsCRASP-1 and BsCRASP-2, were identified, which we estimated to be approximately 23 to 25 kDa in mass. A further factor H binding protein, BsCRASP-3, was found exclusively in the tick isolate, PC-Eq17. This is the first report describing an immune evasion mechanism utilized by B. spielmanii sp. nov., and it demonstrates the capture of human immune regulators to resist complement-mediated killing.     

Mapping of the domains required for decay acceleration activity of the human factor H-like protein 1 and factor H

The human factor H-like protein 1 (FHL-1) is composed of seven repetitive elements (short consensus repeats; SCR) that are identical in sequence to the seven N-terminal SCR of complement factor H. We show that the FHL-1 protein has decay acceleration activity in that it can dissociate C3/C5-convertases bound to the surface of sheep red blood cells. The same activity was also determined for factor H. However, compared to FHL-1, factor H was more efficient in decay acceleration, as about 100-fold less protein was required for a 50% inhibition of activity. The domain required for decay accelerating activity of FHL-1 and factor H was mapped by the use of recombinant fragments. FHL-1 and a series of truncated forms of the protein were expressed in the baculovirus system. Recombinant FHL-1 and all mutants which include SCR 1–4 were functionally active. These four N-terminal SCR are essential and sufficient for activity, as deletion mutants which lack SCR 1 or SCR 4 showed no activity. These results demonstrate that FHL-1 and factor H have identical and overlapping regulatory functions in the complement system and that the domain required for this activity is located in the overlapping region of both proteins within the N-terminal four SCR.     

Demonstration of factor H-like protein 1 binding to Treponema denticola, a pathogen associated with periodontal disease in humans

Treponema denticola is an important contributor to periodontal disease. In this study we investigated the ability of T. denticola to bind the complement regulatory proteins factor H and factor H-like protein 1 (FHL-1). The binding of these proteins has been demonstrated to facilitate evasion of the alternative complement cascade and/or to play a role in adherence and invasion. Here we demonstrate that T. denticola specifically binds FHL-1 via a 14-kDa, surface-exposed protein that we designated FhbB. Consistent with its FHL-1 binding specificity, FhbB binds only to factor H recombinant fragments spanning short consensus repeats (SCRs) 1 to 7 (H7 construct) and not to SCR constructs spanning SCRs 8 to 15 and 16 to 20. Binding of H7 to FhbB was inhibited by heparin. The specific involvement of SCR 7 in the interaction was demonstrated using an H7 mutant (H7AB) in which specific charged residues in SCR 7 were replaced by alanine. This construct lost FhbB binding ability. Analyses of the ability of FHL-1 bound to the surface of T. denticola to serve as a cofactor for factor I-mediated cleavage of C3b revealed that C3b is cleaved in an FHL-1/factor I-independent manner, perhaps by an unidentified protease. Based on the data presented here, we hypothesize that the primary function of FHL-1 binding by T. denticola might be to facilitate adherence to FHL-1 present on anchorage-dependent cells and in the extracellular matrix.     

Complement escape of human pathogenic bacteria by acquisition of complement regulators

Pathogenic micro-organisms employ a broad range of strategies to survive in and to persistently infect the human host. Far from being completely understood by which highly sophisticated means invading pathogens overcome the host's destructive immune defence, there is a growing body of evidence on particular mechanisms which play a pivotal role for immune evasion. This review focuses on evasion of medically and scientifically important bacteria by acquisition of host derived fluid-phase complement regulatory proteins, in particular factor H, FHL-1, and C4b binding protein. Expression of microbial surface molecules binding to human complement regulators and thus fixing them in a functionally active state allows pathogens to inhibit and finely regulate complement activation directly on their surface. Further studies on the utilization of host complement regulatory proteins will likely have a marked impact on a more efficient and specific clinical treatment.     

The opportunistic human pathogenic fungus Aspergillus fumigatus evades the host complement system

The opportunistic human pathogenic fungus Aspergillus fumigatus causes severe systemic infections and is a major cause of fungal infections in immunocompromised patients. A. fumigatus conidia activate the alternative pathway of the complement system. In order to assess the mechanisms by which A. fumigatus evades the activated complement system, we analyzed the binding of host complement regulators to A. fumigatus. The binding of factor H and factor H-like protein 1 (FHL-1) from human sera to A. fumigatus conidia was shown by adsorption assays and immunostaining. In addition, factor H-related protein 1 (FHR-1) bound to conidia. Adsorption assays with recombinant factor H mutants were used to localize the binding domains. One binding region was identified within N-terminal short consensus repeats (SCRs) 1 to 7 and a second one within C-terminal SCR 20. Plasminogen was identified as the fourth host regulatory molecule that binds to A. fumigatus conidia. In contrast to conidia, other developmental stages of A. fumigatus, like swollen conidia or hyphae, did not bind to factor H, FHR-1, FHL-1, and plasminogen, thus indicating the developmentally regulated expression of A. fumigatus surface ligands. Both factor H and plasminogen maintained regulating activity when they were bound to the conidial surface. Bound factor H acted as a cofactor to the factor I-mediated cleavage of C3b. Plasminogen showed proteolytic activity when activated to plasmin by urokinase-type plasminogen activator. These data show that A. fumigatus conidia bind to complement regulators, and these bound host regulators may contribute to evasion of a host complement attack.     

The hyphal and yeast forms of Candida albicans bind the complement regulator C4b-binding protein

Candida albicans, an important pathogenic yeast, activates all three pathways of the complement system. To understand how this yeast evades the effects of the activated system, we have analyzed the binding of the classical pathway inhibitor C4b-binding protein (C4BP) by C. albicans. Purified native as well as recombinant C4BP bound dose dependently to the yeast and hyphal forms, as shown by multiple methods, such as confocal microscopy, flow cytometry, a novel enzyme-linked immunosorbent assay, absorption from human serum, and direct binding assays with purified proteins. A prominent binding site was identified at the tip of the germ tube, a structure that is considered important for tissue penetration and pathogenesis. The binding site in C4BP was localized to the two N-terminal complement control protein domains by using recombinant deletion constructs and site-specific monoclonal antibodies. As the alternative pathway inhibitors factor H and FHL-1 also bind to C. albicans, the binding of all three plasma proteins was compared. Simultaneous binding of the classical regulator C4BP and the alternative pathway regulator factor H was demonstrated by confocal microscopy. In addition, FHL-1 competed for binding with C4BP, suggesting that these two related complement regulators bind to the same structures on the yeast surface. The surface-attached C4BP maintains its complement regulatory activities and inactivates C4b. The surface-attached human C4BP serves multiple functions relevant for immune evasion and likely pathogenicity. It inhibits complement activation at the yeast surface and, in addition, mediates adhesion of C. albicans to host endothelial cells.     

Acquisition of regulators of complement activation by Streptococcus pyogenes serotype M1

Opsonization of bacteria by complement proteins is an important component of the immune response. The pathogenic bacterium Streptococcus pyogenes has evolved multiple mechanisms for the evasion of complement-mediated opsonization. One mechanism involves the binding of human regulators of complement activation such as factor H (FH) and FH-like protein 1 (FHL-1). Acquisition of these regulatory proteins can limit deposition of the opsonin C3b on bacteria, thus decreasing the pathogen's susceptibility to phagocytosis. Binding of complement regulatory proteins by S. pyogenes has previously been attributed to the streptococcal M and M-like proteins. Here, we report that the S. pyogenes cell surface protein Fba can mediate binding of FH and FHL-1. We constructed mutant derivatives of S. pyogenes that lack Fba, M1 protein, or both proteins and assayed the strains for FH binding, susceptibility to phagocytosis, and C3 deposition. Fba expression was found to be sufficient for binding of purified FH as well as for binding of FH and FHL-1 from human plasma. Plasma adsorption experiments also revealed that M1(+) Fba(+) streptococci preferentially bind FHL-1, whereas M1(-) Fba(+) streptococci have similar affinities for FH and FHL-1. Fba was found to contribute to the survival of streptococci incubated with human blood and to inhibit C3 deposition on bacterial cells. Streptococci harvested from log-phase cultures readily bound FH, but binding was greatly reduced for bacteria obtained from stationary-phase cultures. Bacteria cultured in the presence of the protease inhibitor E64 maintained FH binding activity in stationary phase, suggesting that Fba is removed from the cell surface via proteolysis. Western analyses confirmed that E64 stabilizes cell surface expression of Fba. These data indicate that Fba is an antiopsonic, antiphagocytic protein that may be regulated by cell surface proteolysis.     

FHR-1, an additional human plasma protein,binds to complement regulator-acquiring surface proteins of Borrelia burgdorferi

The Borrelia burgdorferi strain LW2, a causative agent of Lyme disease, expresses up to five distinct complement regulator-acquiring surface proteins (CRASPs) that bind the human immune regulators factor H and/or FHL-1. In the present study, we identify FHR-1, a member of the human factor H protein family, as an additional ligand for CRASP-3, CRASP-4, and CRASP-5 but not for CRASP-1 and CRASP-2. A comparative analysis of the binding characteristics revealed unique and distinct binding profiles of the three host immune regulators to CRASPs. FHR-1 binds to CRASP-3, CRASP-4, and CRASP-5; factor H binds to all five CRASPs, and FHL-1 binds preferentially to CRASP-1 and CRASP-2. On the pathogen site, CRASP-3 interacts predominantly with factor H; CRASP-4 shows a preference for FHR-1, and CRASP-5 binds strongly and with equal intensity FHR-1 and factor H. Thus, expression of several CRASPs with distinct binding properties for host proteins allows the pathogen to attach functionally distinct host proteins to its surface.