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.     

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.     

Recruitment of complement factor H-like protein 1 promotes intracellular invasion by group A streptococci

Numerous microbial pathogens exploit complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FHL-1 and FH binding protein expressed on the surface of the human pathogenic bacterium, Streptococcus pyogenes, a common agent of pharyngeal, skin, and soft-tissue infections. In the present study, we demonstrate that Fba and FHL-1 work in concert to promote invasion of epithelial cells by S. pyogenes. Fba fragments were expressed as recombinant proteins and assayed for binding of FHL-1 and FH by Western blotting, enzyme-linked immunosorbent assay, and surface plasmon resonance. A binding site for FHL-1 and FH was localized to the N-terminal half of Fba, a region predicted to contain a coiled-coil domain. Deletion of this coiled-coil domain greatly reduced FHL-1 and FH binding. PepSpot analyses identified a 16-amino-acid segment of Fba which overlaps the coiled-coil domain that binds both FHL-1 and FH. To localize the Fba binding site in FHL-1 and FH, surface plasmon resonance was used to assess the interactions between the streptococcal protein and a series of recombinant FH deletion constructs. The Fba binding site was localized to short consensus repeat 7 (SCR 7), a domain common to FHL-1 and FH. SCR 7 contains a heparin binding site, and heparin was found to inhibit FHL-1 binding to Fba. FHL-1 promoted entry of Fba(+) group A streptococci into epithelial cells in a dose-dependent manner but did not affect invasion by an isogenic fba mutant. To our knowledge, this is the first report of a bacterial pathogen exploiting a soluble complement regulatory protein for entry into host cells.     

Molecular analyses of the interaction of Borrelia hermsii FhbA with the complement regulatory proteins factor H and factor H-like protein 1

Borrelia hermsii, the primary etiological agent of tick-borne relapsing fever in North America, binds the complement regulatory protein factor H (FH) as a means of evading opsonophagocytosis and the alternative complement pathway. The ability of FH-binding protein A (FhbA) to bind FH-like protein 1 (FHL-1) has not been assessed previously. In this study, using a whole-cell absorption assay, we demonstrated that B. hermsii absorbs both FH and FHL-1 from human serum. Consistent with this, affinity ligand binding immunoblot analyses revealed that FH constructs spanning short consensus repeats 1 to 7 and 16 to 20 bind to FhbA. To investigate the molecular basis of the interaction of FhbA with FH/FHL-1, recombinant FhbA truncated proteins were generated and tested for FH/FHL-1 binding. Binding required determinants located in both the N- and C-terminal domains of FhbA, suggesting that long-range intramolecular interactions are involved in the formation and presentation of the FH/FHL-1-binding pocket. To identify specific FhbA residues involved in binding, random mutagenesis was performed. These analyses identified a loop region of FhbA that may serve as a contact point for FH/FHL-1. The data presented here expand our understanding of the pathogenic mechanisms of the relapsing fever spirochetes and of the molecular nature of the interaction between FH/FHL-1 and FhbA.     

Immunological and molecular analyses of the Borrelia hermsii factor H and factor H-like protein 1 binding protein, FhbA: demonstration of its utility as a diagnostic marker and epidemiological tool for tick-borne relapsing fever

It has been demonstrated that Borrelia hermsii, a causative agent of relapsing fever, produces a factor H (FH) and FH-like protein 1 (FHL-1) binding protein. The binding protein has been designated FhbA. To determine if FH/FHL-1 binding is widespread among B. hermsii isolates, a diverse panel of strains was tested for the FH/FHL-1 binding phenotype and FhbA production. Most isolates (23/24) produced FhbA and bound FH/FHL-1. Potential variation in FhbA among isolates was analyzed by DNA sequence analyses. Two genetically distinct FhbA types, designated fhbA1 and fhbA2, were delineated, and type-specific PCR primers were generated to allow for rapid differentiation. Pulsed-field gel electrophoresis and hybridization analyses demonstrated that all isolates that possess the gene carry it on a 200-kb linear plasmid (lp200), whereas isolates that lack the gene lack lp200 and instead carry an lp170. To determine if FhbA is antigenic during infection and to assess the specificity of the response, recombinant FhbA1 (rFhbA1) and rFhbA2 were screened with serum from infected mice and humans. FhbA was found to be expressed and antigenic and to elicit a potentially type-specific FhbA response. To localize the epitopes of FhbA1 and FhbA2, truncations were generated and screened with infection serum. The epitopes were determined to be conformationally defined. Collectively, these analyses indicate that FH/FHL-1 binding is a widespread virulence mechanism for B. hermsii and provide insight into the genetic and antigenic structure of FhbA. The data also have potential implications for understanding the epidemiology of relapsing fever in North America and can be applied to the future development of species-specific diagnostic tools.     

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.     

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.     

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.     

A novel monoclonal antibody against FbaA can inhibit the binding of the complement regulatory protein factor H to group A streptococcus

Some microbial pathogens utilize human complement regulatory proteins, such as factor H (FH) and factor H-like protein 1 (FHL-1), for immune evasion. FbaA is an FHL-1 and FH binding protein expressed on the surface of group A streptococcus (GAS), a common agent of pharyngeal, skin, and soft tissue infections. In this study, we prepared monoclonal antibodies (MAbs) against FbaA, assayed them for specificity, and located their binding domains in FbaA. We found an MAb called FbaA MAb2, which demonstrated the highest affinity to GAS among all of the MAbs. Based on the binding with component peptides, the detected epitope, which was specific for FbaA MAb2, was the amino acid residues 95 to 118 of FbaA; on the other hand, it did not bind with the truncated protein of the internally deleted residues of the segment from 95 to 118 of FbaA. Furthermore, the predominant amino acids specific for FbaA MAb2 screened by phage display epitope library were I, T, P, D, and L, corresponding to the amino acid residues 101, 103, 105, 106, and 110 of FbaA, respectively. The binding location of FbaA with FH and FHL-1 was a 16-amino-acid region corresponding to amino acid residues 97 to 112 of FbaA, which overlapped the FbaA MAb2 binding domain, as confirmed by competitive inhibition enzyme-linked immunosorbent assay and immunofluorescence microscopy. Based on the results of the invasion assay, FbaA MAb2 can inhibit the binding of FH to GAS.     

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.     

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.     

Multiple functions of the leucine-rich repeat protein LrrA of Treponema denticola

The gene lrrA, encoding a leucine-rich repeat protein, LrrA, that contains eight consensus tandem repeats of 23 amino acid residues, has been identified in Treponema denticola ATCC 35405. A leucine-rich repeat is a generally useful protein-binding motif, and proteins containing this repeat are typically involved in protein-protein interactions. Southern blot analysis demonstrated that T. denticola ATCC 35405 expresses the lrrA gene, but the gene was not identified in T. denticola ATCC 33520. In order to analyze the functions of LrrA in T. denticola, an lrrA-inactivated mutant of strain ATCC 35405 and an lrrA gene expression transformant of strain ATCC 33520 were constructed. Characterization of the mutant and transformant demonstrated that LrrA is associated with the extracytoplasmic fraction of T. denticola and expresses multifunctional properties. It was demonstrated that the attachment of strain ATCC 35405 to HEp-2 cell cultures and coaggregation with Tannerella forsythensis were attenuated by the lrrA mutation. In addition, an in vitro binding assay demonstrated specific binding of LrrA to a portion of the Tannerella forsythensis leucine-rich repeat protein, BspA, which is mediated by the N-terminal region of LrrA. It was also observed that the lrrA mutation caused a reduction of swarming in T. denticola ATCC 35405 and consequently attenuated tissue penetration. These results suggest that the leucine-rich repeat protein LrrA plays a role in the attachment and penetration of human epithelial cells and coaggregation with Tannerella forsythensis. These properties may play important roles in the virulence of T. denticola.     

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.     

Defective complement control of factor H (Y402H) and FHL-1 in age-related macular degeneration

The common variant in the human complement Factor H gene (CFH), with Tyr402His, is linked to age-related macular degeneration (AMD), a prevalent disorder leading to visual impairment and irreversible blindness in elderly patients. Here we show that the risk variant CFH 402His displays reduced binding to C reactive protein (CRP), heparin and retinal pigment epithelial cells. This reduced binding can cause inefficient complement regulation at the cell surface, particularly when CRP is recruited to injured sites and tissue. In addition, we identify the Factor H-like protein 1 (FHL-1), an alternative splice product of the CFH gene as an additional protein that includes the risk residue 402, and thus confers risk for AMD. FHL-1 is expressed in the eye and the FHL-1 402His risk variant shows similar reduced cell binding and likely reduced complement regulatory functions on the cell surface. CFH and FHL-1 may act in concert in the eye and the reduced surface binding may result in inappropriate local complement control, which in turn can lead to inflammation, disturbance of local physiological homeostasis and progression to cell damage. As a consequence, these processes may lead to AMD pathogenesis.     

Tuf of Streptococcus pneumoniae is a surface displayed human complement regulator binding protein

Streptococcus pneumoniae is a Gram-positive bacterium, causing acute sinusitis, otitis media, and severe diseases such as pneumonia, bacteraemia, meningitis and sepsis. Here we identify elongation factor Tu (Tuf) as a new Factor H binding protein of S. pneumoniae. The surface protein PspC which also binds a series of other human immune inhibitors, was the first identified pneumococcal Factor H binding protein of S. pneumoniae. Pneumococcal Tuf, a 55 kDa pneumococcal moonlighting protein which is displayed on the surface of pneumococci, is also located in the cytoplasm and is detected in the culture supernatant. Tuf binds the human complement inhibitors Factor H, FHL-1, CFHR1 and also the proenzyme plasminogen. Factor H and FHL-1 bound to Tuf, retain their complement regulatory activities. Similarly, plasminogen bound to Tuf was accessible for the activator uPA and activated plasmin cleaved the synthetic chromogenic substrate S-2251 as well as the natural substrates fibrinogen and the complement proteins C3 and C3b. Taken together, Tuf of S. pneumoniae is a new multi-functional bacterial virulence factor that helps the pathogen in complement escape and likely also in ECM degradation.     

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.     

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.     

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.