Human Pathogenic Borrelia spielmanii sp. nov. Resists Complement-Mediated Killing by Direct Binding of Immune Regulators Factor H and Factor H-Like Protein 1

Abstract

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

Lyme disease is a multisystemic disorder caused by species of the Borrelia burgdorferi sensu lato complex (44). It is the most prevalent vector-borne zoonosis in Eurasia and North America, with about 23,000 newly reported clinical cases in 2005 occurring in the United States (8, 44). The B. burgdorferi sensu lato complex comprises at least 13 distinct species or genomic groups, including B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. japonica, B. valaisiana, B. lusitaniae, B. andersonii, B. bissettii, B. tanukii, B. turdi, B. sinica, B. californiensis, and B. spielmanii (39, 40). In Central Europe, B. burgdorferi sensu stricto, B. afzelii, and B. garinii are the most important causative agents of Lyme disease, while B. bissettii, B. lusitaniae, and B. valaisiana also appear to be associated with the disease (9, 11, 41, 46). More recently, B. spielmanii (formerly designated genospecies A14S) spirochetes have been isolated from patients with skin manifestations in The Netherlands, Germany, Denmark, Hungary, and Slovenia (12, 13, 31, 34, 39, 47, 48, 52, 54).

The ability of borreliae to perpetuate in their natural cycle in different reservoir hosts requires an array of strategies to survive in diverse environments and to overcome innate and adaptive immune responses. Certain Lyme disease genospecies are resistant to complement-mediated killing in vitro. Most B. afzelii isolates are serum resistant, B. burgdorferi isolates were classified as moderately serum resistant, and B. garinii isolates are sensitive to complement-mediated killing (21, 22, 29, 30, 49). The distinct patterns of complement susceptibility are consistent with the finding that serum-resistant B. afzelii isolates deposit small amounts of the late activation products C6 and C5b-9 membrane attack complex on their cell surfaces. In contrast, serum-sensitive B. garinii isolates show considerably higher amounts of activation products deposited on their surfaces (4, 5, 21). Recent studies have shown that resistance to complement-mediated killing correlates with the ability of serum-resistant B. burgdorferi and B. afzelii isolates to acquire the host immune regulators factor H and factor H-like protein 1 (FHL-1) (1, 17, 23, 51). Protection against complement attack by binding of complement regulators factor H and FHL-1 has also been demonstrated for a number of other important human pathogens, such as the relapsing fever spirochetes B. hermsii, B. recurrentis, and B. duttonii (32, 33, 42); Leptospira interrogans (50); Neisseria gonorrhoeae (37); Neisseria meningitidis (38); Streptococcus pyogenes (3, 20); and Streptococcus pneumoniae (14, 18, 19).

Factor H and FHL-1, the main immune regulators of the alternative pathway of complement activation, are structurally related proteins composed of several protein domains termed short consensus repeats (SCRs). Factor H is a 150-kDa glycoprotein composed of 20 SCR domains. In contrast, FHL-1 is a 42-kDa glycoprotein corresponding to a product of an alternatively spliced transcript of the factor H gene and consists of seven SCRs. The N-terminal seven SCRs of both complement regulators are identical with the exception of the C-terminal 4 amino acids of FHL-1 (26, 55, 56). Both plasma glycoproteins act as cofactors for factor I-mediated inactivation of C3b, accelerate the decay of the C3bBb convertase, and protect self surfaces from harmful attacks (26, 28, 35, 53).

In the present study, we investigated the abilities of B. spielmanii isolates obtained from Lyme disease patients, as well as the type strain, PC-Eq17 (a tick isolate), to resist complement-mediated killing. We demonstrate that serum resistance correlates with the ability to acquire the immune regulators factor H and FHL-1. Surface bound, both immune regulators retain their complement-regulatory activities for factor I-mediated C3b inactivation. Finally, we identified three surface-exposed proteins, designated BsCRASP-1 to -3, in B. spielmanii isolates.

(This work forms part of the M.D. thesis of P.H.)

MATERIALS AND METHODS

Bacterial isolates and culture conditions.

B. spielmanii isolates PC-Eq17 (DSM no. 16813^T = CIP 108855^T), A14S, PHap, PMai, and PMew, as well as B. burgdorferi isolate LW2, B. afzelii clonal isolate FEM1-D15, and B. garinii isolate G1, were grown at 33°C for 4 days to cell densities of 1 × 10⁷ ml⁻¹ in modified Barbour-Stoenner-Kelly (BSK) medium as described previously (23). B. spielmanii strain PC-Eq17 was isolated from Ixodes ricinus (40), and A14S, PHap, PMai, and PMew are skin isolates from erythema migrans patients (12, 52). The density of spirochetes was determined using dark-field microscopy and a Kova counting chamber (Hycor Biomedical, Garden Grove, CA).

Human sera and monoclonal and polyclonal antibodies.

Nonimmune human serum (NHS) obtained from 20 healthy human blood donors without known histories of spirochetal infections was used as source for factor H. Sera that proved negative for anti-Borrelia antibodies were pooled, stored as aliquots at −80°C, and thawed on ice before use.

Polyclonal rabbit αSCR1 to -4 antiserum, polyclonal goat anti-factor H antiserum (Calbiochem), or monoclonal antibody (MAb) B22 was used for detection of FHL-1 and factor H (26), and MAb VIG8 was applied to specifically detect factor H (36). MAb L41 1C11 was used for the detection of flagellin (16). Goat anti-human C3 (diluted 1/1,000 for immunofluorescence microscopy and 1/2,000 for Western blotting) and C6 (dilution, 1/50) antibodies were purchased from Calbiochem, and the anti-human C5b-9 MAb (dilution, 1/10) was from Quidel (San Diego, CA).

Expression of recombinant FHL-1.

Recombinant FHL-1 was expressed in insect cells infected with recombinant baculovirus (27). Briefly, Spodoptera frugiperda (Sf9) cells were grown at 28°C in monolayer cultures in protein-free expression medium for insect cells (BioWhittaker, Verviers, Belgium). Adherent Sf9 cells were infected with recombinant virus using a multiplicity of infection of 5. The culture supernatant was harvested after 9 days and subjected to affinity purification using Ni-nitrilotriacetic acid-agarose (QIAGEN, Hilden, Germany).

Serum susceptibility testing.

The serum susceptibilities of B. spielmanii isolates and B. garinii isolate G1 were assessed by applying a growth inhibition assay (21). Briefly, cells grown to mid-logarithmic phase were harvested, washed, and resuspended in fresh modified BSK medium. Spirochetes (1.25 × 10⁷) diluted in a final volume of 100 μl in BSK medium containing 240 μg ml⁻¹ phenol red were incubated with 50% normal human serum (NHS) or 50% heat-inactivated human serum in microtiter plates for 10 days at 33°C (Costar, Cambridge, MA). Modified BSK medium instead of human serum was included in all assays as a growth control. The growth of spirochetes was monitored by measuring the indicator color shift of the medium at 562/630 nm using an enzyme-linked immunosorbent assay reader (PowerWave 200; Bio-Tek Instruments, Winooski, VT). For calculation of the growth curves, Mikrowin version 3.0 software (Mikrotek, Overath, Germany) was used.

Serum adsorption experiments.

Spirochetes grown to mid-log phase and harvested by centrifugation (5,000 × g; 30 min; 4°C) were resuspended in 500 μl veronal-buffered saline (supplemented with 1 mM Mg²⁺, 0.15 mM Ca²⁺, 0.1% gelatin, pH 7.4), and after cell counting, a portion (2 × 10⁹) of the organisms were sedimented by centrifugation. The cell sediment was then resuspended in 750 μl NHS supplemented with 34 mM EDTA and incubated for 1 h at room temperature with gentle agitation. After three washes with 0.15 M NaCl, 0.03 M phosphate, 0.02% sodium azide, pH 7.2, containing 0.05% Tween 20, the proteins bound to the borreliae were eluted by incubation with 0.1 M glycine-HCl, pH 2.0, for 15 min. The bacterial cells were sedimented by centrifugation (14,000 × g; 20 min; 4°C), and the proteins in the supernatant were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.

SDS-PAGE, ligand affinity blotting, and Western blot analysis.

Borrelial cell lysates (15 μg) were subjected either to 10% Tricine-SDS-PAGE under reducing conditions or to 12.5% Laemmli SDS-PAGE under nonreducing conditions and transferred to nitrocellulose membranes (Protran BA83; Whatman, Dassel, Germany) as previously described (24). Briefly, after the transfer of the proteins onto nitrocellulose, nonspecific binding sites were blocked using 5% (wt/vol) dried milk in Tris-buffered saline (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Subsequently, the membranes were rinsed four times in Tris-buffered saline and incubated at 4°C overnight with NHS or culture supernatants containing recombinant FHL-1 protein. After four washings with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Tween 20, the membranes were incubated for 1 h with a 1/500 dilution of MAb B22 recognizing the N-terminal region SCR5 of factor H and FHL-1 or with MAb VIG8 (undiluted) directed against the C terminus of factor H. Following four washes with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Tween 20, the membranes were incubated with a secondary peroxidase-conjugated anti-mouse immunoglobulin G (IgG) antibody at a final dilution of 1/1,000 (DakoCytomation, Glostrup, Denmark) for 1 h at room temperature. Detection of bound antibodies was performed using 3,3′,5,5′-tetramethylbenzidine as a substrate.

Immunofluorescence assay for detection of complement proteins.

For indirect immunofluorescence assays, spirochetes were grown to mid-log phase, harvested by centrifugation at 5,000 × g for 30 min, washed, and resuspended in 300 μl phosphate-buffered saline (PBS). Spirochetes (6 × 10⁶) were incubated with either 25% NHS or 25% heat-inactivated NHS (hiNHS) for 30 min at 37°C with gentle agitation, washed three times with PBS containing 1% bovine serum albumin (PBS-BSA), and resuspended in 100 μl of the same buffer. Aliquots of 10 μl were then spotted on microscope slides and allowed to air dry overnight. After fixation with 100% acetone, the slides were dried for 1 h at room temperature and incubated for 1 h in a humidified chamber with antibodies against the complement components C3 (dilution, 1/1,000), C6 (dilution, 1/50), C5b-9 (dilution, 1/10), factor H, and FHL-1 (dilution, 1/20). Following three washes with PBS-BSA, the slides were incubated for 1 h at room temperature with 1:500 dilutions of appropriate Alexa 488-conjugated secondary antibodies (Molecular Probes, Leiden, The Netherlands). The slides were then washed three times with PBS-BSA and mounted in ProLong Gold antifade reagent containing the DNA-binding dye DAPI (4′,6′-diamidino-2-phenylindole) (Molecular Probes) before being sealed with coverslips. The slides were visualized at a magnification of ×1,000 using an Olympus CX40 fluorescence microscope.

Functional assay for cofactor assay of cell-bound factor H and FHL-1.

The cofactor activities of factor H and FHL-1 bound to borrelial cells were analyzed by measuring factor I-mediated conversion of C3b to inactivated C3b (iC3b). Spirochetes (5 × 10⁷) were incubated with either factor H (Calbiochem, Darmstadt, Germany) or recombinant FHL-1 protein (3 μg/ml each) for 1 h at room temperature with gentle agitation. After extensive washing with PBS, C3b (Calbiochem; 10 μg/ml) and factor I (Calbiochem; 50 μg/ml) were added to the cells, and the mixture was incubated for 30 min at 37°C. The cells were sedimented by centrifugation at 14,000 × g for 10 min, and the supernatants were mixed with sample buffer. The samples were then subjected to SDS-PAGE under reducing conditions and transferred onto a nitrocellulose membrane. C3b degradation products were evaluated by detection of α′-chain cleavage fragments of 68, 46, and 43 kDa by using polyclonal goat anti-C3 IgG at a final dilution of 1/2,000 (Calbiochem) and a secondary peroxidase-conjugated anti-goat IgG antibody (DakoCytomation, Glostrup, Denmark). For detection, 3,3′,5,5′-tetramethylbenzidine was used as a substrate.

In situ protease treatment of native spirochetes.

Whole cells of B. spielmanii isolate A14S were treated with proteases using a modification of a method described previously (7). Briefly, freshly harvested cells were washed twice with PBS-MgCl, and after centrifugation at 5,000 rpm for 10 min, the sedimented spirochetes were resuspended in 100 μl of this buffer. To 2 × 10⁸ intact borrelial cells (final volume, 0.5 ml), proteinase K in distilled water (Sigma-Aldrich, Deisenhofen, Germany) or trypsin in 0.001 N HCl (Sigma-Aldrich) was added to a final concentration of 12.5 to 100 μg/ml. Following incubation for 2 h at room temperature, proteinase K was terminated by adding 5 μl phenylmethylsulfonyl fluoride (Sigma-Aldrich; 50 mg/ml in isopropanol) and trypsin was inhibited by adding 5 μl phenylmethylsulfonyl fluoride (Sigma-Aldrich) and 5 μl pefabloc SC (Roche Diagnostic, Germany). The cells were then washed twice with PBS-Mg, resuspended in 20 μl of the same buffer, and lysed by sonication five times using a Branson B-12 sonifier (Heinemann, Schwäbisch Gmünd, Germany). Aliquots (10 μl) were separated using 10% Tricine-SDS-PAGE.

RESULTS

Serum resistance of B. spielmanii isolates.

To assess the serum sensitivity of B. spielmanii, human isolates A14S, PHap, PMai, and PMew, as well as tick isolate PC-Eq17, were incubated in 50% NHS or in 50% hiNHS for up to 10 days. Using a growth inhibition assay (21), different levels of serum susceptibility were observed among the five B. spielmanii isolates. Isolates A14S, PC-Eq17, PHap, and PMai were more resistant to complement-mediated lysis than isolate PMew, as demonstrated by a delay in growth in the presence of complement (Fig. 1). In contrast, growth of the serum-sensitive B. garinii isolate G1, which was used as a control, was strongly inhibited under the same conditions compared to the five B. spielmanii isolates. Using hiNHS instead of NHS, the growth of borrelial isolates was not affected and led to a continuous decrease of adsorbance.

FIG. 1.

Serum susceptibility among B. spielmanii isolates. A growth inhibition assay was used to investigate the susceptibility to human serum of B. spielmanii isolates A14S (A), PC-Eq17 (B), PMai (C), PHap (D), PMew (E), and the serum-sensitive B. garinii isolate G1 (F). Spirochetes were incubated in either 50% NHS or 50% hiNHS over a cultivation period of 10 days at 33°C. Color changes were monitored by measurement of the absorbance at 562/630 nm. All experiments were performed three times, and each test was done five times with very similar results. For clarity, only data from representative experiments are shown. The error bars represent standard errors of the mean.

Detection of deposited complement components C3, C6, and C5b-9 on the surface of B. spielmanii.

Since the B. spielmanii isolates exhibit differential serum susceptibilities, we analyzed the deposition of complement component C3 and late activated complement components C6 and C5b-9 (terminal complement complex) on the surfaces of isolates A14S, PC-Eq17, and PMew. After incubation of the spirochetes in NHS or heat-inactivated serum, the binding of complement components was analyzed by immunofluorescence microscopy. C3 bound strongly to all isolates tested (Fig. 2), while the intensities of C6 and C5b-9 binding varied markedly between the resistant isolates A14S and PC-Eq17 and the moderately serum-resistant isolate PMew. A mixed population containing a few strongly labeled cells and many weakly stained cells was observed for isolates A14S and PC-Eq17 (Fig. 2). In contrast, a higher number of cells of isolate PMew were positive for both C6 and C5b-9. Analysis of serum-sensitive B. garinii isolate G1 showed strong fluorescent staining for C3, C6, and C5b-9 for the majority of the cells. We noticed that spirochetes covered with complement components exhibited blebs of various sizes and showed signs of lysis and alterations in cell morphology (Fig. 2). To identify all spirochetes in a given field, counterstaining with DAPI was performed. Interestingly, the blebs exhibited a very strong fluorescent signal, whereas a number of complement-positive cells stained negative with DAPI, indicating that the borrelial DNA was highly concentrated in the blebs and that the DAPI-negative spirochetes might represent cell ghosts. As a control, spirochetes incubated with hiNHS showed no fluorescent staining. Taken together, B. spielmanii isolates differ in their abilities to activate complement, as previously demonstrated for B. burgdorferi sensu stricto, B. afzelii, and B. garinii (5, 21, 49).

FIG. 2.

Deposition of complement components C3, C6, and C5b-9 on the surface of B. spielmanii. Complement components deposited on B. spielmanii isolates A14S, PC-Eq17, and PMew, as well as the serum-sensitive B. garinii isolate G1, were detected by indirect immunofluorescence microscopy. Spirochetes were incubated with either 25% NHS or hiNHS for 30 min at 37°C with gentle agitation, and bound C3, C6, and C5b-9 were analyzed with specific antibodies against each component and appropriate Alexa 488-conjugated secondary antibodies. For visualization of the spirochetes in a given microscopic field, the DNA-binding dye DAPI was used. The spirochetes were observed at a magnification of ×1,000. The data were recorded with a DS-5Mc charge-coupled device camera (Nikon) mounted on an Olympus CX40 fluorescence microscope. The images shown are representative of at least 20 microscope fields.

Binding of complement regulators to B. spielmanii.

To assess the mechanism of complement resistance in B. spielmanii, we determined the binding of the human complement regulators factor H and FHL-1 to the surfaces of borrelial cells. To this end, B. spielmanii isolates A14S, PC-Eq17, PHap, PMai, and PMew were incubated with NHS as a natural source for factor H and FHL-1, which was supplemented with EDTA to prevent complement activation. After serum incubation, the wash and eluate fractions were separated by SDS-PAGE and subjected to Western blotting with anti-FHL-1 and anti-factor H antibodies. All tested strains of B. spielmanii bound factor H and FHL-1, although with distinct capacities (Fig. 3).

FIG. 3.

Binding of complement regulators factor H and FHL-1 by different B. spielmanii isolates. B. spielmanii isolates A14S, PC-Eq17, PMai, PHap, and PMew incubated in NHS-EDTA were extensively washed with 0.15 M NaCl, 0.03 M phosphate, 0.02% sodium azide, pH 7.2, containing 0.05% Tween 20, and bound proteins were eluted using 0.1 M glycine (pH 2.0). Both the last wash (w) and the eluate (e) fractions obtained from each strain were separated under nonreducing conditions in a 12.5% SDS-PAGE gel, transferred to nitrocellulose, and probed with either MAb VIG8 specific for SCR20 of factor H or MAb B22 for SCR5 of factor H and FHL-1.

The binding of complement regulators to isolates A14S and PC-Eq17 was further analyzed by immunofluorescence microscopy. Following incubation with NHS-EDTA, factor H was evenly distributed on the surfaces of isolates A14S and PC-Eq17, suggesting that the factor H-interacting proteins were homogeneously expressed and distributed on the borrelial surface (Fig. 4). As a negative control, serum-sensitive B. garinii isolate G1 was incubated with NHS-EDTA under identical conditions and stained for factor H detection. As expected, no fluorescent cells could be detected. For the detection of the spirochetes in a given microscopic field, the same slides were incubated with mounting medium containing DAPI.

FIG. 4.

Detection of factor H/FHL-1 on the surfaces of intact cells. Serum-resistant isolates A14S and PC-Eq17 and serum-sensitive B. garinii isolate G1 were incubated with NHS-EDTA. Bound proteins were detected by immunofluorescence microscopy after incubation with MAb B22 for factor H and FHL-1 (FH). For counterstaining, the DNA-binding dye DAPI was used to identify cells in a given microscopic field. The spirochetes were observed at a magnification of ×1,000. The data were recorded with a DS-5Mc charge-coupled device camera (Nikon) mounted on an Olympus CX40 fluorescence microscope. The images shown are representative of at least 20 microscope fields.

Cell-bound complement regulators display cofactor activity.

We next determined if factor H and FHL-1 bound to the surfaces of B. spielmanii cells act functionally as cofactors for serum protease factor I in cleaving C3b. Spirochetes were first incubated with factor H or FHL-1, and after washing of the spirochetes, factor I and C3b were added. After incubation, the cleavage products were detected by SDS-PAGE and Western blotting. As shown in Fig. 5, surface-bound factor H and FHL-1 retained cofactor activity, as indicated by the presence of representative C3b inactivation products (68-, 46-, and 43-kDa α′-chains). Borrelial cells preincubated in buffer alone with factor I did not promote cleavage of C3b, indicating that the B. spielmanii isolates studied lacked endogenous C3b degradation activity or cofactor activity for cleavage. Thus, binding of factor H and FHL-1 to the surface of B. spielmanii enhances complement control capacity.

FIG. 5.

Analysis of functional activities of factor H and FHL-1 bound to B. spielmanii. The cofactor activities of factor H and FHL-1 bound to spirochetes were analyzed by measuring factor I-mediated conversion of C3b to iC3b. B. spielmanii isolates PC-Eq17, A14S, PMai, PHap, and PMew were incubated with either factor H (A) or purified FHL-1 (3 μg/ml each) (B) for 60 min at room temperature. For control purposes, the cells were incubated without factor H. After extensive washing with PBS, C3b (Calbiochem, Darmstadt, Germany; 10 μg/ml) and factor I (FI) (Calbiochem, Germany; 50 μg/ml) were added, and the mixture was incubated for 30 min at 37°C. Subsequently, the probes were boiled for 5 min, subjected to 12.5% SDS-PAGE, and transferred onto a nitrocellulose membrane. The various C3b degradation products were visualized by Western blotting using a polyclonal goat anti-human C3 antiserum (Calbiochem). As a positive control, purified factor H or FHL-1 (50 ng each) was added to the reaction mixture, and as a negative control, C3b and factor I were incubated in the absence of complement regulators.

Identification of the borrelial protein(s) interacting with factor H and FHL-1.

To identify the bacterial protein(s) involved in factor H and FHL-1 binding, cell extracts from isolates A14S, PC-Eq17, PHap, PMai, and PMew were separated by a 10% Tris-Tricine gel, transferred to nitrocellulose, and incubated with either NHS as a source for factor H or recombinant FHL-1. Following incubation with factor H- or FHL-1-specific antibodies, a dominant factor H and FHL-1 binding protein of approximately 24.9 kDa, termed BsCRASP-1, and a second borrelial protein of 22.1 kDa, BsCRASP-2, were identified (Fig. 6). BsCRASP-1 was present in all B. spielmanii isolates studied, while expression of BsCRASP-2 was restricted to isolates A14S, PMai, and PMew. Stronger binding of BsCRASP-2 to factor H and FHL-1 than to BsCRASP-1 was detected in isolates A14S and PMai. Tick isolate PC-Eq17 expressed an additional factor H binding protein of approximately 15 kDa, termed BsCRASP-3 (Fig. 6A). Cell extracts from serum-resistant isolates B. burgdorferi LW2 and B. afzelii FEM1-D15, expressing up to five CRASP proteins, and a serum-sensitive, CRASP-negative isolate, B. garinii G1, served as controls.

FIG. 6.

Identification of factor H and FHL-1 binding proteins expressed within B. spielmanii isolates. Protein extracts (15 μg each) obtained from B. burgdorferi sensu stricto LW2, B. afzelii FEM1-D15, B. garinii G1, and B. spielmanii PC-Eq17, A14S, PMai, PHap, and PMew were separated by 10% Tris-Tricine SDS-PAGE and transferred to nitrocellulose. The membranes were incubated with either NHS as a source for factor H (A) or FHL-1 (B), and binding of the proteins was detected with MAb VIG8 specific for SCR20 of factor H or polyclonal serum specific for SCR1 to 4 of FHL-1. For detection of FlaB as a control, MAb L41 1C11 was applied. The identified CRASP proteins are indicated on the right, and the mobilities of the marker proteins are indicated on the left.

Surface exposure and protease sensitivity of BsCRASP-1.

To assess the surface exposure of BsCRASP-1 and BsCRASP-2 in situ, spirochetes were treated with proteinase K and trypsin to analyze the accessibility of proteins to proteolytic degradation. Treatment with proteinase K at concentrations up to 50 μg/ml resulted in the complete elimination of factor H binding by isolate A14S, indicating that BsCRASP-1 and, in particular, BsCRASP-2 were highly susceptible to proteolytic cleavage (Fig. 7). Lower concentrations of proteinase K led to partial inhibition of factor H binding. Similarly, treatment with trypsin resulted in decreased binding of factor H and FHL-1, indicating that BsCRASP-1 and BsCRASP-2 are more resistant to trypsin digestion (Fig. 7). The limited accessibility of OspA to proteinase K is reminiscent of previous reports using various B. burgdorferi strains (7). In contrast, OspB was highly sensitive to both proteases, even at low concentrations of ≤12.5 μg/ml. As a negative control, membranes were also screened with anti-FlaB antiserum. As expected, because of the periplasmic localization to the FlaB protein in Borrelia, FlaB was not degraded by either of the two proteases. These analyses demonstrated that BsCRASP-1 and BsCRASP-2 are exposed at the outer surface and thus are potentially available in vivo to interact with factor H and FHL-1.

FIG. 7.

Protease treatment affects surface expression of native BsCRASP-1 and BsCRASP-2 and binding to factor H and FHL-1. (A) B. spielmanii A14S cells were incubated with the indicated concentrations of proteinase K or trypsin. After 2 h of incubation, the cells were lysed by sonication, and each protein lysate was subjected to 10% Tris-Tricine SDS-PAGE. BsCRASP-1 and BsCRASP-2 were identified using recombinant FHL-1 and polyclonal antibody αSCR1 to 4 (dilution 1/1,000) specific for the N terminus of FHL-1/factor H by ligand affinity analysis. (B) Flagellin (FlaB) was detected with MAb L41 1C11 (dilution 1/1,000) by Western blotting. (C) Part of a Coomassie-stained 10% Tris-Tricine SDS-polyacrylamide gel is shown to demonstrate the susceptibility of OspA and OspB to proteolytic degradation.

DISCUSSION

Lyme disease spirochetes employ a broad range of strategies to survive and persist in the human host. It is far from being completely understood by what sophisticated means borreliae overcome the host's destructive immune defense, but immune escape has recently attracted particular attention. Several studies have demonstrated that serum-resistant B. burgdorferi sensu stricto and B. afzelii isolates acquire the host immune regulators factor H and FHL-1 (1, 23, 32, 45). The primary objective of the present study was to analyze the molecular mechanism(s) by which B. spielmanii sp. nov. evades the innate immune system of the human host. Here, we demonstrate for the first time, to our knowledge, that B. spielmanii strains isolated from Lyme disease patients resist complement-mediated killing. The complement-resistant phenotype appears to be accomplished by acquiring the immune regulators factor H and FHL-1.

B. spielmanii was recently determined to be a novel human-pathogenic genospecies of the B. burgdorferi sensu lato complex by multilocus sequence analysis (40, 43). In Central Europe, B. spielmanii is closely associated with garden and hazel dormice as the main reservoir hosts, but not with mice or voles. Furthermore, sequence analysis and polymorphic DNA fingerprinting distinguish these isolates from other Lyme disease genospecies (39). First reports on the prevalence of B. spielmanii in ticks and mammals point to a focal distribution of this genospecies at distinct areas in Central Europe, i.e., The Netherlands, France, Germany, Denmark, the Czech Republic, Slovenia, and Hungary (10, 13, 31, 39, 47, 52). Although B. spielmanii has frequently been detected in infected nymphal and adult ticks, a limited number of isolates were isolated from Lyme disease patients with erythema migrans (12, 13, 31, 52). Here, we present data on the serum susceptibilities of the largest collection of human B. spielmanii isolates. Previous studies on the complement resistance of B. burgdorferi sensu lato demonstrated that borrelial isolates differ substantially with regard to their sensitivities to human serum, as B. afzelii is mainly serum resistant, whereas the majority of B. burgdorferi sensu stricto isolates were classified as moderately serum resistant and isolates of the genospecies B. garinii were frequently classified as serum sensitive (5, 21, 49). Growth inhibition assays revealed that the majority of B. spielmanii strains displayed a serum-resistant phenotype similar to those of B. afzelii isolates. An earlier study of Lyme disease spirochetes provided evidence that differences in serum susceptibility correlate with differential depositions of the late complement components C6 and C5b-9 or the terminal complement complex (21). Isolates A14S, PC-Eq17, and PMew show deposition of various amounts of late complement activation products on their surfaces and represent a mixed population of positively and negatively stained cells. In contrast, larger amounts of surface-bound complement activation products were identified on isolate PMew, suggesting that complement deposition contributes to limited growth. It is important to note, however, that the deposition of late activated products is regulated at the level of C3, implying that factor H, the main immune regulator of the alternative pathway, plays an important role.

Recent studies have shown that the potential of B. burgdorferi sensu stricto and B. afzelii isolates to bind factor H and FHL-1 strictly correlates with serum resistance (1, 17, 23, 32, 51). All B. spielmanii isolates were able to acquire the immune regulators factor H and FHL-1 from human serum, and both complement regulators were uniformly distributed on the borrelial cell surface. This distribution suggests that factor H/FHL-1-interacting proteins on the spirochetal surface bind to the host complement regulators and thereby efficiently inhibit the formation of the C3 convertase. It is of interest that both immune regulators, when bound to the borrelial surface, maintain their cofactor activities for factor I-mediated C3b inactivation. Degradation of C3b was observed upon incubation with factor H and/or FHL-1, but not without complement regulators, indicating that B. spielmanii isolates lack endogenous C3b-cleaving activities.

Previous studies showed that B. burgdorferi sensu stricto and B. afzelii isolates express surface-exposed lipoproteins, collectively termed complement regulator-acquiring surface proteins (CRASPs), which specifically interact with serum factor H and/or FHL-1 (24). Expression of distinct CRASPs on the microbial surface has been implicated in the persistence and survival of spirochetes in the human host. Furthermore, complementation of serum-sensitive borrelial strains with BbCRASP-1, BbCRASP-2, or the factor H binding OspE protein increases or completely restores resistance to human serum (2, 6, 15), emphasizing a role for these lipoproteins in evading the innate immune system of the human host. In this study, B. spielmanii was shown to express most likely two surface-exposed factor H and FHL-1 binding proteins, designated BsCRASP-1 and BsCRASP-2. Assessed by ligand affinity blotting, BsCRASP-1 displayed a stronger binding intensity to FHL-1 than to factor H, which is reminiscent of BbCRASP-1, BaCRASP-1, and BbCRASP-2 (Fig. 6). Interestingly, BsCRASP-2 of A14S and PMai showed a stronger binding capacity to both immune regulators than the dominant BsCRASP-1 protein. Thus, it is tempting to speculate that differential expression levels of BsCRASP-1 and BsCRASP-2 or sequence differences that potentially account for their relative binding properties to factor H and FHL-1 are involved in the complement susceptibilities of individual B. spielmanii isolates. Moreover, tick isolate PC-Eq17 expressed an additional factor H binding protein, termed BbCRASP-3, comparable to the factor H binding BbCRASP-3 to BbCRASP-5 proteins of B. burgdorferi and BaCRASP-4 and -5 of B. afzelii (23). Therefore, we hypothesize that BsCRASP-3 belongs to the factor H binding Erp protein family (25, 45). Investigations are under way to isolate and functionally characterize BsCRASP-1 from distinct B. spielmanii isolates to provide further insight into the molecular interaction of factor H and FHL-1 with BsCRASP-1, as well as their roles in the virulence and pathogenesis of B. spielmanii in humans.

Due to the limited number of isolated B. spielmanii strains and the fragmentary information available, one can only speculate on their prevalence in humans (12, 34). It has been shown by Richter et al. (39) that the garden and hazel dormice appear to be the main reservoir hosts for B. spielmanii. Therefore, the geographical distribution of this genospecies is more restricted than those of the other human-pathogenic Lyme disease spirochetes. As the garden dormice have adapted to distinct ecotonal habitats, their distribution is somewhat restricted to particular landscapes. Due to the exclusive host-pathogen relationship of the dormouse-associated B. spielmanii spirochetes and the specific adaptation of their reservoir host(s), it is to be expected that the genospecies would rarely be detected in human biopsies.

The association of B. spielmanii with garden dormice might reflect an adaptation to the individual host's complement system, as previously shown for certain Lyme disease spirochetes, especially avian-associated B. garinii spirochetes (30). The fact that most B. spielmanii isolates exhibit resistance to human complement might argue for their competence to infect and survive in the human host. However, it has also been shown that B. spielmanii is transmitted more efficiently to dormice than B. afzelii spirochetes, indicating that humans are not the preferred host for B. spielmanii (39). Studies on the prevalence of B. spielmanii in patients with Lyme disease who reside in the same geographical area where infected dormice are abundant will help to elucidate the potential of this genospecies to cause clinical manifestations other than erythema migrans.

In summary, this study demonstrates that B. spielmanii acquires immune regulators, factor H and FHL-1, on the borrelial surface, which contribute to resistance against complement-mediated lysis. The characterization of BsCRASP-1 represents an important step forward and will expand our understanding of the molecular basis of the pathogenesis of this novel Lyme disease spirochete.