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* Infectious Immunology Group, Institute for Immunology, University of Heidelberg, Heidelberg, Germany;
Institute of Medical Microbiology and Infection Control, University Hospital of Frankfurt, Frankfurt, Germany;
Molecular Immunobiology Group and Department of Infection Biology, Leibniz-Institute for Natural Products Research, Jena, Germany; and
Metschnikoff Laboratory, Max-Planck-Institute for Immunobiology, Freiburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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A further strategy of bacteria to resist hosts’ innate immunity, which constitutes the first barriers to infection, is their potential to acquire fluid phase complement regulators, particularly those of the alternative complement pathway such as factor H (FH),5 to the spirochetal surface. Bound FH controls complement activation by accelerating the decay of the C3 convertase of the alternative pathway and by inactivating newly formed C3b (7, 8) as shown for several important human pathogens, e.g., Candida albicans, Neisseria gonorrhoeae, Streptococcus pyogenes, and Streptococcus pneumoniae (9, 10, 11, 12, 13, 14). FH represents the main human fluid phase regulator of the alternative pathway of complement activation and belongs to the factor H protein family, which consists of seven structurally related proteins in humans including FH-like protein 1 (FHL-1) and the FH-related proteins (FHRs) (15). All FH protein family members are composed of short consensus repeats (SCRs) (15, 16). In contrast to FH and FHL-1, the precise function(s) of the FHR proteins is currently unknown. For B. hermsii, surface-bound FH was shown to participate as a cofactor for factor I-mediated cleavage of C3b (17, 18, 19). Furthermore, for the closely related spirochete Borrelia burgdorferi, the causal agent of Lyme disease, a strong correlation between the serum resistance of a given isolate and its expression profile of FH-binding outer surface lipoproteins, termed complement regulator-acquiring surface proteins (CRASP), was reported (20, 21, 22, 23, 24, 25, 26, 27, 28). Moreover, it was suggested that the dominant FH binding molecule of serum-resistant B. burgdorferi strains, BbCRASP-1, is necessary to resist killing by human serum (29).
Some bacteria, such as Porphyromonas gingivalis, Pseudomonas aeruginosa, and Clostridium perfringens, produce their own proteolytic enzymes that digest the extracellular matrix to facilitate invasion (30). Others, like B. burgdorferi and Borrelia crocidurae, make use the hosts’ fibrinolytic system to invade tissues (31, 32, 33, 34). Accordingly, spirochetes bind the host plasminogen that is subsequently processed via urokinase-type plasminogen activator (uPA) to active plasmin, a broad-spectrum serine protease, leading to extracellular matrix degradation (31, 33, 35, 36, 37). B. burgdorferi organisms bind host plasminogen via a multitude of outer surface proteins (Osp), such as OspA and OspC, a 70-kDa protein, and several low molecular weight proteins (33, 35, 38, 39). Thus, the fact that relapsing fever spirochetes, including B. hermsii, also disseminate from the blood to many distinct organs suggests the involvement of plasminogen-binding proteins in these processes.
By screening a B. hermsii expression library we have now identified a novel 21.5 kDa outer surface lipoprotein termed BhCRASP-1. We demonstrate for the first time that BhCRASP-1 displays dual binding specificities both for members of the FH complement regulator protein family and for plasminogen/plasmin and that the two host proteins bind to distinct, nonoverlapping BhCRASP-1 domains.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B. hermsii (ATCC35209) strain HS1 and YOR isolates (provided by T. Schwan, Rocky Mountain Laboratories) and the Lyme disease spirochetes B. burgdorferi isolate B31 and mutant B313 were cultivated in Barbour-Stoenner-Kelly (BSK)-H complete medium (PAN Biotech) supplemented with 5% rabbit serum (Cell Concept) at 30°C. B313 mutant spirochetes harbor plasmids cp32-1, cp32-2, cp32-3, cp32-4, cp26, and lp7 exclusively and therefore lack expression of BbCRASP-1 to BbCRASP-4 (27). Bacteria were harvested by centrifugation and washed with PBS. The density of spirochetes was determined using dark-field microscopy and a Kova counting chamber (Hycor Biomedical). Escherichia coli DH5
and MC1061 were grown at 37°C in 2xYT or Luria-Bertani medium, respectively.
Cloning of BhCRASP-1, construction of expression plasmids, and production of recombinant proteins
A B. hermsii genomic DNA expression library was prepared and screened using recombinant FH deletion constructs as previously described (22). Briefly, bacterial colonies were plated onto Luria-Bertani agar plates and transferred to nitrocellulose filters. Membranes were incubated with supernatant of Sf9 cells infected with recombinant FHL-1 or various recombinant deletion constructs of FH (FH15–20, FH8–20, FH19–20) for 12 h at 4°C. After three washings with TBS containing 0.2% Tween 20, filters were incubated with antisera to SCR1–4 (8) and a mAb (VIG8) specific for SCR20 (40) in the presence of 1% MC1061 cell lysate, followed by incubation with the appropriate peroxidase-conjugated secondary Ab. B. hermsii genomic DNA fragments cloned in pUEX1 or pGEX-2T plasmid derivatives were sequenced by using the BigDye terminator cycle sequencing kit (PE Applied Biosystems) in accordance with the manufacturer’s recommendation.
The gene encoding BhCRASP-1 was amplified by PCR amplification using plasmid pGEMbh, the primers BhBam and BhR (Table I), and a Mastercycler gradient (Eppendorf). Denaturation was conducted at 94°C for 30s, annealing at 50°C for 30s, and extension at 68°C for 30s, respectively. After digestion with BamHI and EcoRI, the amplified DNA fragment was ligated in-frame into the vector pGEX-2T, which included the glutathione S-transferase gene at the N terminus of the recombinant protein. The resulting plasmid was used to transform JM109 host cells. Expression of the GST-BhCRASP-1 fusion protein in E. coli JM109, affinity purification, and endoproteinase thrombin cleavage of the fusion protein were performed as recommended by the manufacturer (Amersham Bioscience).
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-12 primer and the 130Bam and 195Bam primers, respectively (Table I). The amplified DNA fragments were digested with BamHI and ligated in-frame with the His6 tag encoding sequence into the pQE30Xa vector (Qiagen), resulting in plasmids BhCRASP-121–173, BhCRASP-151–185, and BhCRASP-176–185. These plasmids were used for transformation of the JM109 host cells. Expression of the respective fusion proteins and affinity purification were performed as recommended by the manufacturer. Expression of recombinant proteins of FH, FHL-1, and FHR-1
Deletions constructs of FH (FH1–2, FH1–3, FH1–4, FH1–5, FH1–6, FH8–20, FH15–20, and FH19–20), FHL-1, and FHR-1 were expressed in Spodoptera frugiperda Sf9 insect cells infected with a recombinant baculovirus. The cloning, expression, and purification of various deletion constructs have been described previously (8, 41).
Construction of a shuttle vector for transformation with BhCRASP-1
The BhCRASP-1-encoding cspA gene, including its native promoter region, was amplified by PCR using BhF and BhR primers containing the respective restriction sites. Highly purified DNA obtained from the B. hermsii strain HS1 was used as the DNA template for PCR. The resulting amplicon was digested with SacI and SphI and cloned into pKFSS1 at the corresponding restriction sites yielding the shuttle vector pBH. The shuttle vector was transformed into E. coli JM109 and purified plasmids were subjected to nucleotide sequencing to verify that no mutations were introduced during PCR. E. coli transformants were grown in Luria-Bertani broth containing 50 µg of streptomycin (Sigma-Aldrich) per milliliter and the expression of BhCRASP-1 was checked by ligand affinity blot analysis of whole cell lysates (data not shown) as described (27).
Characterization of B. burgdorferi B313 transformants
The transformation of B. burgdorferi B313 and the characterization of transformants were previously described (27). Several clones were selected and expanded for 7 days. The analysis of genes harbored by B313 transformants was determined by PCR using specific primers (Table I). PCR was conducted for 25 cycles using following parameters: denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min. The expression of the BhCRASP-1 of posttransformation B. burgdorferi B313 was determined by Western blotting using mAb BH-1.
Serum susceptibility testing for Borrelia strains
The serum susceptibility of B. hermsii HS1 mutants B313 and B313 containing shuttle vector pBH was assessed using a growth inhibition assay (42). Briefly, cells grown to mid-logarithmic phase were harvested, washed, and resuspended in fresh Barbour-Stoenner-Kelly medium. Spirochetes (1.25 x 107 B. hermsii and 2.5 x 107 B. burgdorferi B313 or B313/pBH organisms) diluted in a final volume of 100 ml in Barbour-Stoenner-Kelly medium containing 240 µg/ml phenol red were incubated with 50% normal human serum (NHS) or 50% heat-inactivated NHS in microtiter plates for 72 h at 33°C (Costar). B313 and B313/pBH were incubated with 25% NHS or 25% heat-inactivated NHS. Barbour-Stoenner-Kelly medium instead of human serum was included in all assays as control. Growth of spirochetes was monitored by measuring the indicator color shift of the medium at 562/630 nm in an ELISA reader (PowerWave 200' Bio-Tek Instruments). For calculation of the growth curves, the MikroWin version 3.0 software (Mikrotek) was used.
Serum adsorptions assays using intact borrelial cells
To determine whether B. hermsii HS1 can bind FH, FHR-1, and plasminogen, a whole cell absorption assay was performed as previously described. Borreliae (2 x 109 cells) were grown to mid-log phase, harvested by centrifugation (5000 x g for 30 min at 4°C) and resuspended in 100 µl of veronal-buffered saline supplemented with 1 mM Mg2+, 0.15 mM Ca2+, and 0.1% gelatin (pH 7.4). To inhibit complement activation, NHS was incubated with 0.34 M EDTA for 15 min at room temperature. The cell suspension was then incubated in 1.5 ml NHS-EDTA for 1 h at room temperature with gentle agitation. After three washes with PBSA (0.15 M NaCl, 0.03 M phosphate, and 0.02% sodium azide, pH 7.2) containing 0.05% Tween 20, the proteins bound to the cells were eluted by incubation with 0.1 M glycine-HCl (pH 2.0) for 15 min. Borrelial cells were removed by centrifugation at 14,000 x g for 20 min at 4°C and the supernatant was analyzed by Western blotting and probed with mAb VIG8 for FH and FHR-1 or 10-V-1 (Calbiochem) for plasminogen.
Immunofluorescence analysis
Spirochetes were grown to mid-log phase, harvested by centrifugation at 5000 x g for 10 min, washed, and resuspended in 300 µl of 30 mM Tris, 60 mM NaCl (pH 7.4). Cells (2 x 108) were incubated for 1 h with a mAb directed either against BhCRASP-1 (BH-1) or the periplasmic flagellin protein (LA21). After incubation with the Abs, spirochetes were gently washed three times in Tris buffer containing 0.2% BSA and collected by centrifugation at 5000 x g for 10 min. Pellets were then resuspended in 100 µl of Tris buffer containing 0.2% BSA. Aliquots (10 µl) were spotted on coverslips and allowed to air dry for 3 h. After fixation with acetone, samples were dried for 15 min at room temperature and incubated for 60 min in a humidified chamber with a 1/200 dilution of Cy3-conjugated rabbit anti-mouse IgG (Dianova) and a 1/1000 dilution of the DNA-binding dye 4',6'-diamidino-2-phenylindole (Roth) for counterstaining. Slides were then washes four times with 0.2% BSA in Tris buffer before being sealed with Mowiol mounting medium (Calbiochem) and covered with glass slides. Organisms were visualized at a magnification of x1000 using a Nikon Eclipse 90i microscope.
SDS-PAGE, ligand affinity blot, Western and slot blot analyses, and ELISA
Borrelial whole cell lysates (15 µg) or purified recombinant proteins (500 ng) were subjected to 10% Tris/Tricine SDS-PAGE under reducing conditions and transferred to nitrocellulose as previously described (24). Alternatively, recombinant proteins (1 µg/lane) were transferred onto nitrocellulose membranes using the Bio-DOT SF blotting apparatus (Bio-Rad). After the transfer of proteins onto nitrocellulose, nonspecific binding sides were blocked using 5% (w/v) dried milk in TBS (50 mM Tris-HCl 200 mM NaCl, and 0.1% Tween 20) (pH 7.4), for 6 h at room temperature. Subsequently, membranes were rinsed four times in TBS and incubated at 4°C overnight with NHS, recombinant proteins, or human plasminogen (Cell Systems). After four washings with 50 mM Tris-HCl 150 mM NaCl, and 0.2% Tween 20 (TBST) (pH 7.5), membranes were incubated for 3 h with a polyclonal rabbit antiserum recognizing the N terminus (anti-SCR1–4), the mAb B22 directed against the SCR5 of FH and FHL-1, the mAb VIG8 directed against the C terminus of FH and FHR-1, or the plasminogen-specific mAb 10-V-1. Following four washes with TBST, blot strips were incubated with a secondary peroxidase-conjugated anti-rabbit IgG Ab or anti-mouse IgG Ab (DakoCytomation) for 60 min at room temperature. Detection of bound Abs was performed using 3,3',5,5'-tetramethylbenzidine as the substrate.
For Western blot analysis, membranes were incubated for 60 min at room temperature with either mAb or immune sera. Following four washes with TBST, membranes were incubated with a secondary peroxidase-conjugated anti-mouse IgG Ab (DakoCytomation) for 60 min at room temperature and bound Abs were detected using 3,3',5,5'-tetramethylbenzidine as substrate.
For ELISA using nondenatured recombinant proteins, the wells of microtiter plates (Maxisorp; Nunc) were coated for 2 h at room temperature with BhCRASP-1 or the deletion mutants thereof (100 µl; 1 µg/ml). The wells were washed three times with PBS and blocked by incubation with PBS plus 0.1% gelatin. FH (1 µg/ml) or plasminogen (10 µg/ml) was added to the wells and after a 2-h incubation and three washes with PBS a 5000-fold dilution of goat anti-FH (Calbiochem) or a 3000-fold dilution of goat anti-plasminogen (Acris) serum was added, respectively. For the detection of specific Abs, peroxidase-labeled rabbit anti-goat IgG (Dianova) Abs (1/2000) were used as conjugates. Substrate reaction was performed with o-phenyldiamine dihydrochloride (Sigma-Aldrich) at room temperature. For competition binding tests with FH and plasminogen, BhCRASP-1-coated microtiter plates were used. To analyze the ability of plasminogen to inhibit the binding of FH to immobilized BhCRASP-1 (1 µg/ml), FH (0.1 µg/ml) was mixed with different amounts of plasminogen (0.001–100 µg/ml) and these mixtures were added to the wells coated with BhCRASP-1. Bound FH was detected as described above. The ability of different amounts of FH (0.001–100 µg/ml) to inhibit the binding of plasminogen (5 µg/ml) to immobilized BhCRASP-1 was analyzed accordingly.
In situ protease treatment of spirochetes
Whole cells of B. hermsii strain HS1 were treated with proteases by modification of a method described previously (43). Briefly, freshly harvested cells were washed twice with PBS-MgCl and, after centrifugation at 5000 rpm for 10 min, the sedimented spirochetes were resuspended in 100 µl of this buffer. To 5 x 106 intact borrelial cells (final volume of 0.5 ml), proteinase K in distilled water (Sigma-Aldrich) was added to a final concentration of 12.5–100 µg/ml. Following incubation for 1 or 2 h at room temperature, proteinase K was inhibited by adding 5 µl of PMSF (Sigma-Aldrich) (50 mg/ml in isopropanol). The cells were then washed twice with PBS-magnesium, resuspended in 20 µl of the same buffer, and lysed by sonication five times using a Branson B-12 sonifier (Heinemann). Whole cell protein preparations (10 µl) were separated by using Tris/Tricine SDS-PAGE via 4% stacking and 10% separating gels as described previously (23).
Surface plasmon resonance analysis
Protein-protein interactions were analyzed by surface plasmon resonance technique using a Biacore 3000 instrument as described earlier (22, 44). Briefly, the borrelial recombinant protein BhCRASP-1 (20 µg/ml; dialyzed against 10 mM acetate buffer (pH 5.5)) was coupled via a standard amine-coupling procedure to the flow cell of a sensor chip (CM5; Biacore) until a level of resonance units >4000 was reached. A control cell was prepared in the same way but without injecting a protein. FH, FHL-1, and the deletion construct FH1–6 were dialyzed against running buffer (75 mM PBS (pH 7.4)). Each ligand (FH, 333 nM; FHL-1, FH8–20, and FH15–20, 1 µM each) was injected separately into the flow cell coupled with BhCRASP-1 or the deletion mutants and into a control cell using a flow rate of 5 µl/min at 25°C. Each interaction was analyzed at least three times.
The binding kinetics were determined by using a lower density of the immobilized ligand (<1000 resonance units) at 22°C in 75 mM PBS (pH 7.4) and by using a natural logarithmic Langmuir 1:1 binding model and the simultaneous Ka/Kd fitting routine of the BIAevaluation 3.1 software (Biacore). The equilibrium constants were calculated from the rate constants.
Functional assay for cofactor activity of FH
The cofactor activity of FH was analyzed on immobilized recombinant BhCRASP-1 by measuring the factor I-mediated conversion of C3b to iC3b. Briefly, recombinant BhCRASP-1 (20 µg/ml) immobilized on a microtiter plate was incubated with an excess of purified FH. After washing, purified C3b (Calbiochem) and purified factor I (Sigma-Aldrich) were added and the mixture was incubated for 15 min at 37°C. iC3b generation was quantified by ELISA applying a neoepitope-specific mouse monoclonal anti-iC3b IgG (Quidel) as the capture Ab and biotinylated rabbit anti-C3c IgG (DakoCytomation) as the detector Ab. The reaction was visualized by the addition of streptavidin-peroxidase followed by o-phenylenediamine with H2O2 as the substrate. Purified iC3b (Calbiochem) was used as a standard. Control experiments included BbCRASP-1, BbCRASP-3, or buffer instead of BhCRASP-1 as well as soluble and immobilized FH, respectively, in the identical system.
Chromogenic substrate assays for plasmin and plasminogen activators
Intact B. hermsii spirochetes were incubated with 10 µl of plasminogen (1 mg/ml; Chromogenix) with or without 50 mM tranexamic acid for 30 min at 34°C in Eppendorf tubes if not otherwise indicated. Following two washes, B. hermsii was resuspended in 50 µl of assay buffer (30 mM Tris, 60 mM NaCl (pH 7.4)) and transferred to microtiter plates, and 50 µl of uPA (2,5 µg/ml; Chemicon International) as well as 50 µl of the plasmin substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (S-2251; Sigma-Aldrich) was added (0.4 mg/ml). Control reactions without B. hermsii consisted of buffer alone (followed by uPA) and a sham preparation to control for possible residual unbound plasminogen not subsequently removed by washing (this reaction received plasminogen in buffer at the same concentration as that used in tubes with B. hermsii, followed by uPA). Control reactions with B. hermsii consisted of plasminogen alone (no uPA) and uPA alone (no previous plasminogen incubation) at the same concentrations as in the experimental reaction mixture. All samples received the chromogenic substrate S-2251 and were subjected to the same manipulations. The absorbance change at 405 nm was followed for several hours directly in the plates and the background activity of OD450 = 0.1 (B. hermsii plus substrate) was subtracted. Similarly, BhCRASP-1 (0.2 µg/ml) was coated to microtiter plates and, after blocking, 10 µl of plasminogen (1 mg/ml) with or without 50 mM tranexamic acid was added and incubated for 10 min at 37°C. Following three washes with 200 µl of buffer, 50 µl of uPA (2.5 µg/ml) and 50 µl of substrate S-2251 were added (0.4 mg/ml). The absorbance change at 405 nm was followed as indicated above.
Nucleotide sequence deposition
The cspA gene sequence reported in this paper has been deposited in the EMBL/GenBank databases under the accession number AM408562.
Statistical analysis
To determine the statistical significance of the observed absorbance values, BIAS version 8.1 software was used. Values of p < 0.05 were considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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To identify the FH binding proteins of B. hermsii, a genomic DNA expression library derived from B. hermsii strain HS1 was screened for FH binding clones. The sequence of one clone that strongly bound FH revealed an open reading frame of 555 bp encoding a putative lipoprotein with a calculated molecular mass of 21.5 kDa. The encoding gene was designated cspA. Pulse-field gel electrophoresis and hybridization analysis revealed that the cspA gene encoding BhCRASP-1 represents a single genetic locus that maps to a plasmid of 
200 kb. Hybridization analyses using a cspA PCR-generated probe with HaeIII- and BamHI-digested DNA yielded fragments of 3 and 8 kb, respectively (data not shown). After cleavage of the leader peptide, the predicted molecular mass of BhCRASP-1 is 19.5 kDa. The N terminus of BhCRASP-1 shows significant homology to the signal peptides of other bacterial lipoproteins (45, 46). This motif includes two lysine residues near the N terminus, a hydrophobic region, and a sequence with significant similarity to the consensus signal peptidase II cleavage sequence Leu(Ala, Ser)–4-Leu(Val, Phe, Ile)–3- Ile(Val, Gly)–2-Ala(Ser, Gly)–1-Cys+1. Using LipoP for prediction of the lipoproteins of Gram-negative bacteria (47), a unique cleavage side for signal peptidase II was found between aa 19 and 20, suggesting lipidation at cysteine residue 20 of BhCRASP-1. The amino acid sequence exhibited 83% identity with the recently identified FHBP19/FhbA protein of B. hermsii YOR (18). A mAb, BH-1, with specificity for BhCRASP-1 was shown to be nonreactive with FHBP-19/FhbA and with the deletion mutant BhCRASP-176–185, suggesting that the specific epitope recognized by mAb BH-1 includes amino acids residing in the N-terminal domain of BhCRASP-1 (data not shown).
Surface exposure and protease sensitivity of BhCRASP-1
To determine whether BhCRASP-1 is surface exposed, an immunofluorescence assay was performed using the mAb BH-1, specific for BhCRASP-1. B. hermsii was incubated sequentially with mAb BH-1 and the rabbit anti-mouse Cy3-conjugated Ab (Fig. 1A, upper panels). Epifluorescence microscopy revealed that B. hermsii expressed BhCRASP-1 on its outer surface in a patch-like manner. The mouse mAb LA21 directed against the periplasmic FlaB protein was used in these experiments as an internal control to confirm that the fragile spirochetal outer membrane was not damaged (Fig. 1A, lower panels). Controls incubated with the secondary Ab alone were negative (not shown).
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12.5 µg/ml. The band intensity observed for FlaB was not changed, indicating that periplasmic flagella are not affected by proteolytic digestion. Thus, the susceptibility of BhCRASP-1 to proteolytic digestion indicates that this protein is exposed at the outer surface of B. hermsii. Interaction of BhCRASP-1 with serum proteins
To test the binding of recombinant BhCRASP-1 to the serum proteins FH, FHL-1, and FHR-1 or to plasminogen, slot blot analysis was used. Of the three members of the factor H family analyzed, FH and FHR-1 bound to BhCRASP-1 whereas no binding was observed for FHL-1 (Fig. 2A). Using BbCRASP-1 derived from B. burgdorferi as a control, binding to FHL-1 and FH but not to FHR-1 could be detected. OspA, OspB, and BSA did not bind to any of the three proteins. Furthermore, plasminogen bound to recombinant BhCRASP-1 and OspA, whereas no binding was observed for the control proteins OspB and BSA (Fig. 2B).
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Localization of the FH/FHR-1 and the plasminogen-binding domains of BhCRASP-1
To localize the binding sites for FH/FHR-1 and plasminogen on BhCRASP-1, a number of BhCRASP-1 deletion mutants with N- and C-terminal truncations were constructed (Fig. 3). Protein expression was confirmed by using Coomassie blue staining, and all of the recombinant proteins exhibited the predicted size and reacted with the BhCRASP-1 immune serum (data not shown). Screening for FH/FHR-1 binding, using ELISA revealed that, of the protein preparations tested, only the full-length form of BhCRASP-1 bound to FH and FHR-1 (Fig. 4A). No binding to FH was detected with any of the other deletion mutants of BhCRASP-1. Thus, the binding of FH/FHR-1 required determinants located in both the C- and N-terminal domains of BhCRASP-1, suggesting that long-range intramolecular interactions are involved in the formation and presentation of the FH/FHR-1 binding pocket.
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Activation of bound plasminogen by host-derived plasminogen activators
To determine whether plasminogen bound to the outer surface of B. hermsii was converted to its enzymatically active form, plasmin, by either endogenously or exogenously supplied plasminogen activator(s), B. hermsii spirochetes were incubated with plasminogen. After the transfer of extensively washed spirochetes to microtiter plates, human uPA and the chromogenic plasmin substrate S-2251 were added. As shown in Fig. 5A, degradation of the chromogenic substrate demonstrates that plasminogen bound to the surface of B. hermsii is converted to enzymatically active plasmin in the presence of exogenous uPA. No or only marginal plasmin activity was seen in the presence of tranexamic acid, indicating that the previous binding of plasmin(ogen) to the spirochete is a prerequisite for optimal cleavage by plasminogen activators. Spirochetes treated with plasminogen alone (without subsequent activation with uPA) or with uPA alone (without previous incubation with plasminogen) showed only marginal, if any, degradation of S-2251. No plasmin was formed in the absence of plasminogen activators, indicating that spirochetes do not express endogenous plasminogen activators. Similar findings were observed using BhCRASP-1-coated microtiter plates. In contrast to intact spirochetes, plasmin activity bound to BhCRASP-1 was reduced by 
50% in the presence of tranexamic acid (Fig. 5B).
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To precisely map the binding domain of FH that binds to the recombinant BhCRASP-1 of B. hermsii, various deletion constructs of FH and FHL-1 were used for ligand affinity assays. As shown in Fig. 6A, BhCRASP-1 strongly bound to FH (lane 7 from left) as well as to the deletion constructs FH8–20 (lane 8), FH15–20 (lane 9), and FH19–20 (lane 10), but not to the deletion constructs SCR1–2, SCR1–3, SCR1–4, SCR1–5, SCR1–6, FHL-1 (SCR1–7) (lanes 1–6), and deletion construct FH15–19 (lane 11). These data indicate that SCR20 of FH is critical for interaction with BhCRASP-1. In addition, FHR-1 but not FHL-1 bound to immobilized BhCRASP-1 using ligand affinity blotting (Fig. 2A), supporting the notion that the SCR20 of FH is primarily involved in binding BhCRASP-1. As indicated in the schematic representation of FH, FHL-1, and FHR-1 (Fig. 6B), domain SCR20 of FH displays 97% sequence similarity to the SCR5 of FHR-1 (48).
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The cofactor activity of FH was analyzed on immobilized recombinant BhCRASP-1 protein by measuring factor I-mediated conversion of C3b to iC3b (21). Recombinant BhCRASP-1 immobilized on a microtiter plate was incubated with excess of purified FH or buffer alone. As controls, functional activity of FH bound to B. burgdorferi BbCRASP-1 or BbCRASP-3 was tested for C3b-inactivating capacity (21, 22). BhCRASP-1-bound FH was more efficient in mediating C3b conversion than FH bound to either BbCRASP-1 or BbCRASP-3 under similar conditions (Fig. 8). As previously shown, FH bound to BbCRASP-1 is up to 10-fold more efficient in factor I-mediated C3b conversion as compared with BbCRASP-3 (21, 22). Incubation of immobilized proteins in the absence of FH served as negative controls and had no effect on C3b conversion (data not shown).
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Preliminary experiments indicated that the B. hermsii strain HS1 was not suitable for genetic manipulation. We thus transformed the serum-sensitive B. burgdorferi mutant strain B313 lacking the FH/FHL-1 binding proteins BbCRASP-1 to BbCRASP-4 with the shuttle vector pBH containing the entire cspA gene to assess the role of BhCRASP-1 for complement resistance. Transformants were selected by limiting dilution and characterized for the presence of the cspA gene of B. hermsii by PCR analysis (Fig. 9A). B313/pBH, but not parental strain B313, showed the expected amplicon product. Lack of plasmids lp54, lp28-3, and cp32-9 harboring ospA and cspA (BbCRASP-1), cspZ (BbCRASP-2), and erpP (BbCRASP-3) in B313 was confirmed by PCR, respectively (Fig. 9A). Next, the expression of BhCRASP-1 was determined by Western blotting with the specific mAb (BH-1). As shown in Fig. 9B, B. hermsii HS1 and the transformant B313/pBH, but not the mutant strain B313, showed a specific band. Furthermore, a growth inhibition assay (42) was used to compare the susceptibility of B. hermsii HS1, B313, and B313/pBH to human serum. Resistance to complement-mediated killing was indicated by a continuous growth of spirochetes in the presence of human serum and a subsequent reduction of A562/A630 ratios, whereas the inhibition of sensitive cells was indicated by lack of changes in absorbance. As shown in Fig. 9C, the growth of B. burgdorferi mutant B313 was significantly inhibited as compared with growth of B313/pBH in the presence of 25% NHS (p < 0.05). B313 containing the shuttle vector alone showed similar growth properties as the nontransformed B. burgdorferi mutant B313 (data not shown). This finding indicates that complement resistance can be increased when BhCRASP-1 is expressed in a heterologous B. burgdorferi strain. Heat inactivation of human serum before assaying the borrelial cells did not influence the growth of any strain (data not shown).
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Discussion |
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The previous findings that the relapsing fever spirochete B. hermsii expresses a receptor for FH, FhbA, and that surface-bound FH facilitates factor I-mediated cleavage of C3b suggest a suitable strategy of the pathogen to evade the first-line host defense via the complement system (18). FH binding has been reported for a number of bacterial species such as S. pyogenes (group A streptococcus) (50), Neisseria gonorrhoeae (10, 51), S. pneumoniae (11, 13, 52), B. burgdorferi (24), Borrelia afzelii (25), Borrelia recurrentis (53), Borrelia duttonii (53), Borrelia parkeri (17), and B. hermsii (18, 19). Moreover, for B. hermsii YOR it was found that it specifically binds both FH and FHL-1 via FhbA and that FH and FHL-1 interact with FhbA through the SCR domains 1–7 and SCR16–20 (19). In contrast, the presented plasma adsorption experiments and surface plasmon resonance analyses clearly showed that FH binding to BhCRASP-1 of B. hermsii HS1 is exclusively associated with SCR20. This is further substantiated by the fact that BhCRASP-1 also bound FHR-1, another member of the factor H family, exhibiting a C-terminal domain that is almost identical to the C terminus of FH but different to FHL-1, which consists of SCR1–7 (48). Thus, BhCRASP-1 and FhbA clearly express distinct biological activities in that both show similar binding potential for FH but different capacities to interact with FHL-1 and FHR-1. Although the actual function of FHR-1 is yet to be disclosed, it was suggested to be involved in the adhesion processes of the pathogen to neutrophils (54). The interaction of BhCRASP-1 with FH may be an important mechanism by which B. hermsii spirochetes control C3b deposition on their surface and escape opsonophagocytosis. In addition, FH may function in adherence due to its binding to surface glycosaminoglycans and host cell membrane receptors. In this context it is noteworthy that the C-terminal part of FH has previously been implicated in the binding to other bacterial surface structures, e.g., the sialylated lipooligosaccharide of N. gonorrhoeae and several lipoproteins of B. burgdorferi, including CRASP-3, -4, and -5 (10, 21, 22, 44).
Our results show that FH associated with BhCRASP-1 maintains its regulatory activity and controls C3b deposition and C3-convertase activity. Thus, the acquisition of FH molecules to surface-exposed BhCRASP-1 results in enhanced complement-regulatory activity, a process that would allow relapsing fever spirochetes to evade clearance by the innate immune system. The biological significance of BhCRASP-1 interaction with FH was further examined by using a B. burgdorferi strain as an amenable host to express a heterologous outer surface lipoprotein for studying complement resistance. In fact, B. burgdorferi was shown previously to successfully display surface-exposed lipoproteins, e.g., Vsp1 and Vsp2 of relapsing fever borreliae (55). In addition, the complementation of BbCRASP-1 or BbCRASP-2 expression in serum-sensitive borrelial cells imparts resistance to human serum (27, 29). Here we demonstrate that expression of the BhCRASP-1 of relapsing fever borreliae in the serum-sensitive Lyme disease spirochete B. burgdorferi B313 results in an increased resistance of the mutant strain to complement-mediated killing, suggesting the involvement of BhCRASP-1 in the immune evasion of B. hermsii.
To localize the peptide binding domain(s) of BhCRASP-1 for FH binding, BhCRASP-1 proteins with N- and C-terminal truncations were generated and used for functional analyses. Deletions of either the N terminus (fragment spanning residues 51–185) or the C terminus (fragment spanning residues 21–173) portion of BhCRASP-1 completely abrogated FH binding, suggesting that the FH binding site of BhCRASP-1 consists of a conformational rather than a contiguous linear peptide structure. Similar features have been previously reported for B. burgdorferi BbCRASP-1 and BbCRASP-3 (21, 22). However, Hovis and colleagues proposed that although determinants of the C-terminal domain of FhbA are important in FH/FHL-1 binding, the 10-aa C-terminal tail is dispensable (19). In this context the recent identification of two genetically distinct groups of B. hermsii is of interest (56). Together with the proposition of two clusters of FH binding proteins found in the B. hermsii genome, the combined studies thus suggest a differential association of FhbA and BhCRASP-1 with the two subgroups (57). However, further studies are required to settle this issue.
Several independent investigations have proposed that the binding of plasminogen to the surface of bacteria is of importance for the invasive capacity of a number of Borrelia species (32, 33, 35, 58, 59, 60). We have shown now that B. hermsii binds plasminogen to its outer surface and that bound plasminogen can, in turn, be converted to enzymatically active plasmin in the presence of exogenous human uPA as measured by the cleavage of the chromogenic plasmin substrate S-2251. The interaction of plasminogen with exponentially grown B. hermsii was first examined by the incubation of intact spirochetes with NHS, a natural source for plasminogen and FH, and both proteins, FH and plasminogen, were eluted from the spirochetal surface. To elucidate the putative plasminogen-binding capacity of the BhCRASP-1, ligand affinity blotting was used. As with FH, plasminogen binds to immobilized BhCRASP-1. Thus, plasmin-coated organisms could bind to the recently described plasminogen receptors on endothelium cells as a means of initial anchoring (61) and may use their enhanced proteolytic capacity to breach tight junctions of endothelium, cross basement membranes, and initiate pathophysiological processes in the affected organs (60). It is also known that, similar to Lyme disease, Borrelia relapsing fever species disseminate from the blood to many distant organs, including the brain (57). Our results show that plasminogen does not compete with FH for binding of immobilized BhCRASP-1 and vice versa. The efficient simultaneous binding of FH and plasminogen suggests that the two host proteins are bound to BhCRASP-1 of B. hermsii via distinct domains. In line with this observation it was found that BhCRASP-1 proteins with N- or C-terminal truncations exhibited a loss of binding to FH but not to plasminogen. Furthermore, the acquired proteolytic activity may also protect spirochetes against serum-derived nonspecific and specific antimicrobial compounds such as specific Abs. It has become evident that Staphylococcus aureus resists human innate immune defenses by activating human plasminogen into plasmin at the bacterial surface and that this in turn leads to degradation of surface-bound IgG and C3b (62). Similarly, BhCRASP-1-bound serine protease activity may act in concert with FH and factor I to strengthen the resistance of B. hermsii to human serum by promoting C3b inactivation. Therefore, BhCRASP-1 bound plasmin must be considered as another escape mechanism of B. hermsii during early infection.
To our knowledge, this is the first study showing the simultaneous and noncompetitive binding of FH and plasminogen to an outer surface protein of B. hermsii. This finding is of general importance and deserves further investigations to better understand the molecular interactions of FH and plasminogen with BhCRASP-1 as well as their roles in the virulence and pathogenesis of B. hermsii in humans. Our findings may have broad implications for the invasive potential of human pathogens and support the concept of their exploitation of host factors as a suitable survival strategy.
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1 We are indebted for the financial support of the Deutsche Forschungsgemeinschaft Grants Wa 533/7-1 (to R.W.) and Kr 3383/1-1 (to P.K.). This work forms part of the Ph.D. thesis of E.R. and P.H. 
2 The sequence presented in this article has been submitted to EMBL/GenBank under accession number AM408562.
3 E.R. and P.K. contributed equally to this work.
4 Address correspondence and reprint requests to Dr. Reinhard Wallich, Infectious Immunology Group, Institute for Immunology, University of Heidelberg, Im Neuenheimer Feld 305, Heidelberg, Germany. E-mail address: wallich{at}uni-hd.de
5 Abbreviations used in this paper: FH, factor H; FHL-1, FH-like protein 1; FHR, FH-related protein; CRASP-1, complement regulator-acquiring surface protein 1; BbCRASP-1, Borrelia burgdorferi CRASP-1; BhCRASP-1, Borrelia hermsii CRASP-1; NHS, normal human serum; Osp, outer surface protein; SCR, short consensus repeat; uPA, urokinase-type plasminogen activator.
Received for publication November 20, 2006. Accepted for publication March 13, 2007.
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1H for cleavage of C3b and C4b in solution. J. Exp. Med. 146: 257-270. 
). uPA-mediated plasminogen activation was inhibited by 50 mM tranexamic acid (
). When spirochetes were incubated in the absence of either uPA (
) or plasminogen (
), only weak plasmin activity was seen. This experiment was repeated three times with consistent results.
