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.     

Binding of complement regulatory protein factor H enhances serum resistance of Borrelia spielmanii sp. nov.

The European Borrelia species, B. spielmanii, has recently been shown to be associated with Lyme borreliosis/disease infections in humans. In order to elucidate complement susceptibility of diverse B. spielmanii species, we analyzed and compared two isolates obtained from patients with Lyme borreliosis for their ability to escape from complement-mediated bacteriolysis. Growth inhibition assays revealed striking differences between the two B. spielmanii isolates tested with regard to their complement sensitivity. B. spielmanii isolate A14S displayed a resistant phenotype, whereas isolate PSigII was highly sensitive to complement-mediated lysis. In addition, we demonstrate that B. spielmanii isolate A14S can bind complement regulator factor H at higher concentrations to the cell surface as compared to serum-sensitive isolate PSigII. The data presented here provide strong evidence for a correlation between factor H binding and serum resistance among the two isolates tested that can contribute to immune evasion of B. spielmanii from complement-mediated killing.     

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.     

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.     

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.     

Impact of the SpeB protease on binding of the complement regulatory proteins factor H and factor H-like protein 1 by Streptococcus pyogenes

Microbial pathogens often exploit human complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FH and FHL-1 binding protein expressed on the surface of the human pathogenic bacterium Streptococcus pyogenes, a common agent of pharyngeal, skin, and soft-tissue infections. Fba has been shown to contribute to phagocytosis resistance, intracellular invasion, and virulence in mice. Here, we look at the role of Fba in recruitment of FH and FHL-1 by five serotype M1 isolates of streptococci. Inactivation of fba greatly inhibited binding of FH and FHL-1 by all isolates, indicating that Fba is a major FH and FHL-1 binding factor of serotype M1 streptococci. For three isolates, FH binding was significantly reduced in stationary-phase cultures and correlated with high levels of protease activity and SpeB (an extracellular cysteine protease) protein in culture supernatants. Analysis of a speB mutant confirmed that SpeB accounts for the loss of Fba from the cell surface, suggesting that the protease may modulate FH and FHL-1 recruitment during infection. Comparisons of fba DNA sequences revealed that the FH and FHL-1 binding site in Fba is conserved among the M1 isolates. Although the ligand binding site is not strictly conserved in Fba from a serotype M49 isolate, the M49 Fba protein was found to bind both FH and FHL-1. Collectively, these data indicate that binding of FH and FHL-1 is a conserved function of Fba while modulation of Fba function by SpeB is variable.     

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.     

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.     

Comprehensive analysis of the factor h binding capabilities of borrelia species associated with lyme disease: delineation of two distinct classes of factor h binding proteins

Some Lyme disease spirochete isolates can bind complement regulatory protein factor H (fH), a process that may allow evasion of complement-mediated killing. Here we demonstrate significant differences in the fH binding capabilities of species of the Borrelia burgdorferi sensu lato complex. The percentages of B. burgdorferi, B. afzelii, and B. garinii bacteria that bound fH in either enzyme-linked immunosorbent assays or affinity ligand binding immunoblot assays were 100, 83, and 29%, respectively. The fH binding protein profiles were examined and found to exhibit variability among isolates and to form two distinct classes. Differences in fH binding ability may contribute to the differences in pathogenesis and clinical course observed upon infection with different species of the B. burgdorferi sensu lato complex.     

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.     

Relapsing fever spirochetes Borrelia recurrentis and B. duttonii acquire complement regulators C4b-binding protein and factor H

Relapsing fever is a rapidly progressive and severe septic disease caused by certain Borrelia spirochetes. The disease is divided into two forms, i.e., epidemic relapsing fever, caused by Borrelia recurrentis and transmitted by lice, and the endemic form, caused by several Borrelia species, such as B. duttonii, and transmitted by soft-bodied ticks. The spirochetes enter the bloodstream by the vector bite and live persistently in plasma even after the development of specific antibodies. This leads to fever relapses and high mortality and clearly indicates that the Borrelia organisms utilize effective immune evasion strategies. In this study, we show that the epidemic relapsing fever pathogen B. recurrentis and an endemic relapsing fever pathogen, B. duttonii, are serum resistant, i.e., resistant to complement in vitro. They acquire the host alternative complement pathway regulator factor H on their surfaces in a similar way to that of the less serum-resistant Lyme disease pathogen, B. burgdorferi sensu stricto. More importantly, the relapsing fever spirochetes specifically bind host C4b-binding protein, a major regulator of the antibody-mediated classical complement pathway. Both complement regulators retained their functional activities when bound to the surfaces of the spirochetes. In conclusion, this is the first report of complement evasion by Borrelia recurrentis and B. duttonii and the first report showing capture of C4b-binding protein by spirochetes.     

The tick salivary protein Salp15 inhibits the killing of serum-sensitive Borrelia burgdorferi sensu lato isolates

Borrelia burgdorferi, the agent of Lyme disease, is transmitted by ticks. During transmission from the tick to the host, spirochetes are delivered with tick saliva, which contains the salivary protein Salp15. Salp15 has been shown to protect spirochetes against B. burgdorferi-specific antibodies. We now show that Salp15 from both Ixodes ricinus and Ixodes scapularis protects serum-sensitive isolates of Borrelia burgdorferi sensu lato against complement-mediated killing. I. ricinus Salp15 showed strong protective effects compared to those of I. scapularis Salp15. Deposition of terminal C5b to C9 (one molecule each of C5b, C6, C7, and C8 and one or more molecules of C9) complement complexes, part of the membrane attack complex, on the surface of B. burgdorferi was inhibited in the presence of Salp15. In the presence of normal human serum, serum-sensitive Borrelia burgdorferi requires protection against complement-mediated killing, which is provided, at least in part, by the binding to the tick salivary protein Salp15.     

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.     

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.     

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.     

Complement evasion by Borrelia burgdorferi: serum-resistant strains promote C3b inactivation

The most characteristic features of the Lyme disease pathogens, the Borrelia burgdorferi sensu lato (s.l.) group, are their ability to invade tissues and to circumvent the immune defenses of the host for extended periods of time, despite elevated levels of borrelia-specific antibodies in serum and other body fluids. Our aim in the present study was to determine whether B. burgdorferi is able to interfere with complement (C) at the level of C3 by accelerating C3b inactivation and thus to inhibit the amplification of the C cascade. Strains belonging to different genospecies (Borrelia garinii, B. burgdorferi sensu stricto, and Borrelia afzelii) were compared for their sensitivities to normal human serum and abilities to promote factor I-mediated C3b degradation. B. burgdorferi sensu stricto and B. afzelii strains were found to be serum resistant. When the spirochetes were incubated with radiolabeled C3b, factor I-mediated degradation of C3b was observed in the presence of C-resistant B. afzelii (n = 3) and B. burgdorferi sensu stricto (n = 1) strains but not in the presence of C-sensitive B. garinii (n = 7) strains or control bacteria (Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis). Immunoblotting and radioligand binding analyses showed that the C-resistant strains had the capacity to acquire the C inhibitors factor H and factor H-like protein 1 (FHL-1) from growth medium and human serum. A novel surface protein with an apparent molecular mass of 35 kDa was found to preferentially bind to the N terminus region of factor H. Thus, the serum-resistant B. burgdorferi s.l. strains can circumvent C attack by binding the C inhibitors factor H and FHL-1 to their surfaces and promoting factor I-mediated C3b degradation.     

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.     

Identification and functional characterization of complement regulator-acquiring surface protein 1 of the Lyme disease spirochetes Borrelia afzelii and Borrelia garinii

Complement regulator-acquiring surface protein 1 (CRASP-1) is the dominant factor-H-like protein 1 (FHL-1)- and factor-H-binding protein of Borrelia burgdorferi and is suggested to contribute to persistence of the pathogen. The prototype CRASP-1 of B. burgdorferi sensu stricto (CRASP-1Bb) has been formerly characterized. As shown recently, serum-resistant Borrelia afzelii strains express a unique FHL-1 and factor H-binding protein, designated CRASP-1Ba. Here, we describe for the first time the isolation and functional characterization of the gene encoding the full-length CRASP-1Ba of 28 kDa, which, upon processing, is predicted to be 26.4 kDa. CPASP-1Ba of B. afzelii spirochetes is associated with a genetic locus encoding the orthologous gbb54 gene family that maps to the linear plasmid of approximately 54 kb. Ligand affinity blotting techniques demonstrate that both native and recombinant CRASP-1Ba molecules strongly bind to FHL-1 and much more weakly to factor H. The FHL-1 and factor-H-binding site in CRASP-1Ba is shown to be localized to a 12-amino-acid residue domain at the C terminus of the protein. For comparison, the corresponding cspA-like gene(s) of a serum-sensitive Borrelia garinii strain has also been cloned and characterized. Most notably, two CRASP-1-related B. garinii proteins were identified; however, both molecules bind only weakly to FHL-1 and not at all to factor H. The present identification of the binding site of CRASP-1Ba represents an important step forward in our understanding of the pathogenesis of Lyme disease and may be helpful to design therapeutic regimens to interfere with complement evasion strategies of human pathogenic Borrelia strains.