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

2.1 Binding of complement regulators to different B. burgdorferi isolates

To determine how Borreliae control complement activity on their surface, we inverstigated whether serum-resistant and serum-sensitive isolates differ in their binding capacities for human complement regulators. Serum-resistant and serum-sensitive isolates were incubated in NHS (derived from non-immune, non-vaccinated individuals). After extensive washing bound proteins were eluted, separated by SDS-PAGE and analyzed by Western blotting for binding of the two central human complement regulators FHL-1/reconectin and Factor H. The results demonstrate that all analyzed serum-resistant isolates (EB1, EB2, FEM1-C1, FEM1-D15, PKo, and FEM2) bound both complement regulators, while none of the analyzed serum-sensitive isolates (PBr, PSth, G1, G2 and A87S_B) bound these plasma proteins at a comparable intensity (Fig. 1). FHL-1/reconectin was detected as a 42-kDa and Factor H as a 150-kDa protein. Binding was considered specific as the majority of the bound protein was detected in the elute fraction and only a minor fraction was present in the wash fraction. Binding of these regulators was concentration dependent; for FHL-1/reconectin, binding was detected when 10⁸ or 10⁹ cells were incubated in 750 μ l NHS, although with the serum-sensitive isolate PSth no binding was observed using the same number of cell (data not shown).

2.2 Adsorption from human serum

To determine the adsorption of the human complement regulators from serum in more detail, we compared the concentration of the two regulators in human plasma to that in the wash and elute fraction (Fig. 2). Using the serum-resistant isolate FEM1-D15 and antisera specific for FHL-1/reconectin and Factor H, a large accumulation of the FHL-1/reconectin protein was detected on the surface of Borreliae (Fig. 2A, compare lanes 1 and 3). In addition, an antiserum that detects several members of the Factor H protein family, i.e. FHR-1α, FHR-1β, FHR-2α and FHR-2 31, revealed binding of these four proteins to isolate FEM1-D15 and higher levels of the two FHR-2 isoforms on the Borreliae surfaces as compared to human serum (Fig. 2A; lanes 5 and 6). In contrast, the serum-sensitive isolate PSth did not bind any of the regulators or additional members of the Factor H protein family (Fig. 2B).

2.3 Identification of other cell bound proteins

Having demonstrated specific binding and enrichment of complement regulators from NHS, we wanted to determine whether plasma proteins in general were absorbed from the Borreliae isolates, or whether borrelial outer surface proteins were removed from the bacterial surface under these conditions. The wash and elute fractions of serum-resistant and serum-sensitive isolates were obtained after incubation in PBS and NHS, and the samples were separated by SDS-PAGE and analyzed by silver staining. More proteins were detected in the elute fraction of the serum-sensitive isolate PSth compared to the serum-resistant isolate FEM1-D15 (Fig. 3A; compare lanes 4 and 8). For a direct comparison of the mobility, purified Factor H (lane 9) and recombinant FHL-1/reconectin (lane 10) were separated in the same gel. This analysis indicates that adsorption of human plasma proteins is specific, but also that several proteins are released from the bacterial surface during the conditions used for elution. In particular, the small number and low amounts of protein detected in the serum-resistant isolate FEM1-D15, which strongly absorbs both FHL-1/reconectin and Factor H from human serum (Fig. 3B), highlights the specificity of the adsorption. The slower mobility of the plasma form of FHL-1/reconectin to the recombinant protein (Fig. 3A and 4B) is explained by the histidine tag of 3.15 kDa that is attached to the recombinant form.

2.4 Localization of the binding domain of FHL-1/reconectin to B. burgdorferi

Recombinantly expressed FHL-1/reconectin protein was assayed for binding to serum-resistant isolate FEM1-D15 and compared to serum-sensitive isolate PSth. As found with the serum form, the recombinant FHL-1/reconectin also bound specifically to the serum-resistant isolate FEM1-D15, but not to the serum-sensitive PSth (Fig. 4A, lane 6 and Fig. 4B lane 6).

FHL-1/reconectin is composed of seven individually folding SCR domains 15. Binding of selected deletion mutants of FHL-1/reconectin, which have individual SCR domains sequentially deleted from the C terminus, to serum-resistant Borreliae allowed the interacting domain(s) to be localized. Again binding to serum-resistant isolate FEM1-D15 was analyzed and compared to that of the serum-sensitive isolate PSth. In addition to recombinant FHL-1/reconectin deletion mutants consisting of SCR 1–6, SCR 1–5 bound to serum-resistant isolate FEM1-D15, but not to serum-sensitive isolate PSth (Fig. 4A, B). No binding was observed for deletion mutant, which include SCR 1–4, or even further deletions. These data indicate the existence of two interacting sites on the host FHL-1/reconectin protein. Thus, the human immune regulator may either bind to two different borrelial proteins or has two distinct sites which interact with a single borrelial protein.

2.5 Binding of FHL-1/reconectin to intact Borreliae

Immunofluorescence staining of intact, unfixed cells revealed binding of FHL-1/reconectin to serum-resistant FEM1-D15. After incubation with recombinant FHL-1/ reconectin, the human immune regulator was distributed over the entire cells, suggesting that the binding borrelial proteins were equally expressed on the surface (Fig. 5A). This staining pattern is considered specific as it was not detected with serum-sensitive PSth cells (Fig. 5B). To demonstrate intactness of the bacterial cells and to exclude that staining resulted from damaged membranes, Borreliae were incubated with an antibody specific for the surface-expressed outer membrane protein C (OspC), which resulted in uniformly labeled cells (Fig. 5C, D). Intactness of the bacteria was further confirmed by demonstrating lack of reactivity for the cytosolic HSP70 protein (data not shown). The staining pattern revealed a different distribution of the FHL-1/Factor H binding protein(s), which shows a spotted/granular pattern and a uniform distribution of OspC.

2.6 Cell bound FHL-1/reconectin displays cofactor activity

To determine whether FHL-1/reconectin bound to the surface of Borreliae displays complement regulatory activity, we analyzed the C3b inactivating capacity of surface-attached and of eluted proteins following incubation of serum-resistant isolate FEM1-D15 and serum-sensitive isolate PSth in NHS or with the recombinant FHL-1/reconectin protein. Upon incubation with human serum or recombinant FHL-1/reconectin cofactor activity for Factor I-mediated cleavage of C3b was observed in serum-resistant isolate (FEM1-D15), whereas no or only a negligible conversion occurred in serum-sensitive isolate (PSth). Significant functional activity of absorbed FHL-1/reconectin and Factor H could be demonstrated on the microbial surface (Fig. 6A) or after elution of the proteins from the bacterial surfaces (Fig. 6B). Borrelia treated in veronal-buffered saline (VBS) and their elutes revealed no cofactor activity. No activity was observed when Borreliae were incubated in a medium containing an unrelated recombinant protein (rprotein) expressed in the same system. These results demonstrate that acquisition of FHL-1/reconectin to the surface of Borreliae enhances their complement control capacity. That a protease activity is responsible for the C3b-inactivating activity can be excluded, as inactivation was observed after incubation in NHS or recombinant FHL-1/reconectin protein, but not after treatment of the same genospecies in VBS or upon incubation with an unrelated protein synthesized in the same expression system (Fig. 6).

2.7 Identification of the borrelial protein(s) binding FHL-1/reconectin or Factor H

Having demonstrated binding of two human complement regulators to B. burgdorferi, we wanted to identify the bacterial protein(s) involved in this interaction. Cell extracts were generated from various serum-resistant B. afzelii isolates (EB1, EB2, FEM1-C1, FEM1-D15, PKo, and FEM2) and serum-sensitive B. garinii isolates (PBr, PSth, G1, G2, and A87S_B) and used for ligand blotting. These extracts were separated on a 10% tricine gel, transferred to nitrocellulose membrane and incubated with recombinant FHL-1/reconectin. The blot was developed using a rabbit antiserum specific for SCR 1–4 of FHL-1/reconectin and Factor H (Fig. 7). All six serum-resistant B. afzelii isolates showed a dominant binding protein of approximately 27.5 kDa. The different mobilities of these binding proteins of the six serum-resistant B. afzelii extracts reveal the heterogeneity of the proteins. In addition, a second binding protein of lower mass was identified for EB2, FEM1-D15 and PKo. This protein of approximately 20 or 21 kDa also shows heterogeneity in size, and is either expressed at lower concentration on the borrelial surface or is bound by the immune regulators with apparently lower intensity. Expression of this smaller protein appears more restricted as it is only detected in three of the six serum-resistant isolates.

2.8 Localization of FHL-1/reconectin binding domain(s)

To differentiate the two borrelial binding proteins and to characterize the domain of FHL-1/reconectin which binds to these proteins, the various recombinant deletion mutants were used for ligand blotting. This experiment showed different binding characteristics of the 27.5- and 20-kDa borrelial proteins of serum-resistant isolate FEM1-D15 for FHL-1/reconectin. The 27.5-kDa protein bound the intact protein SCR 1–7 and the deletion mutants containing SCR 1–6 and SCR 1–5 with weaker intensity, but did not bind deletion mutants SCR 1–4, SCR 1–3 and SCR 1–2 (Fig. 8). The 20-kDa borrelial protein bound the intact FHL-1/reconectin protein and the mutant SCR 1–6 weakly, but did not bind SCR 1–5. In summary, these experiments reveal the existence of two B. afzelii proteins of 27.5 and 20/21 kDa, which both bind the human complement regulator FHL-1/reconectin. However, these two proteins bind this human immune regulator with different affinities and interact with different domains.

2.9 Further characterization and localization of the binding proteins to the outer membrane

The mapping experiments describe above showed binding of both FHL-1/reconectin and Factor H to intact Borreliae and the existence of two borrelial binding proteins. To identify the binding preference of the two borrelial proteins for the two human immune regulators, due to the lack of a specific FHL-1/reconectin antibody, we used a differential staining approach. The reactivity of an antiserum specific for FHL-1/reconectin and Factor H was compared to that obtained with mAb VIG8, which is specific for the Factor H. mAb VIG8 revealed specific binding of Factor H to the 20-kDa protein (Fig. 9A, lane 2), while the polyclonal antiserum, which detects both immune regulators, identified both borrelial proteins as ligands (Fig. 9A, lane 1).

To confirm the surface localization of the two borrelial FHL-1/Factor H binding proteins, an outer membrane protein fraction was prepared from wild-type serum-resistant FEM1, separated on tricine gel and, after transfer to nitrocellulose, was used for ligand blotting. The strong intensity of the detection signal showed enrichment of the two binding proteins in this membrane extract (Fig. 9B).

4.1 Borreliae isolates and culture conditions

All Borreliae isolates used in this study were obtained from human biopsy material. Clonal FEM1-D15 and the OspC-lacking variant FEM1-C1 25 were derived from wild-type B. afzelii isolate FEM1 by twofold single-colony plating on solid medium as described 26. Serum-resistant B. afzelii EB1, EB2, FEM1-C1, FEM1-D15, PKo, and FEM2, serum-sensitive B. garinii PBr, PSth, G1, G2, and A87S_B 24 were cultured until mid-log phase (5×10⁷ cells/ml) at 33°C in modified Barbour-Stoenner-Kelly (BSK) medium 27. Samples of 1.8 ml were dispensed into screw-cap tubes (Nunc, Wiesbaden, Germany), frozen at –70°C, and used as stock cultures. Prior to use, afrozen suspension of spirochetes was thawed and inoculated into fresh BSK medium. The density of spirochetes was determined using dark-field microscopy and a Kova counting chamber (Hycor Biomedical, Garden Grove, CA).

4.2 Non-immune human serum

Sera (NHS) from twenty healthy human blood donors without a known history of a spirochetal infection were tested for the presence of IgG and IgM antibodies against B. burgdorferi by acommercial whole cell ELISA (Dade Behring, Marburg, Germany) and a immunoblot with recombinant proteins (Mikrogen, Martinsried, Germany). Only sera that proved negative in all assays were combined to form the NHS pool.

4.3 Expression of recombinant proteins

Recombinant proteins were expressed in insect cells infected with recombinant baculovirus. The cloning of various recombinant deletion constructs, expression and purification was performed as described 17, 28. Spodoptera frugiperda cells (Sf9) were grown at 28°C in monolayer cultures in protein-free expression medium (BioWhittaker, Verviers, Belgium) in the presence of streptomycin (100 μg/ml), penicillin (100 U/ml) and amphotericin B (250 ng/ml) (Life Technologies, Eggenstein, Germany). Adherent Sf9 cells were infected with recombinant virus using a multiplicity of infection of five. The culture supernatant was harvested after 9 days and used directly for adsorption experiments.

4.4 Serum adsorption experiments

Borreliae (5×10⁸ cells) were grown to mid-log phase, harvested by centrifugation (5,000×g, 30 min, 4°C) and resuspended in 100 μl VBS (supplemented with 1 mM Mg²⁺, 0.15 mM Ca²⁺, 0.1% gelatin, pH 7.4). The cell suspension was then incubated in 750 μl NHS supplemented with 0.34 mM EDTA or in 1.4 ml supernatant of Sf9 insect cells infected with recombinant FHL-1/reconectin or the various recombinant deletion mutants (consisting of SCR 1–6, SCR 1–5, SCR 1–4, SCR 1–3, SCR 1–2) 17 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 (PBSA) 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 SDS-PAGE and Western blot experiments.

4.5 Isolation of borrelial outer membrane fractions

Fractions of the outer membranes of wild-type B. afzelii FEM1 were isolated as described previously 30.

4.6 SDS-PAGE and Western Blot analysis for detection of absorbed FHL-1/reconectin and Factor H

Wash and elute fractions obtained from the various Borreliae isolates were subjected to SDS-PAGE using 10% or 12% gels and, after transfer onto nitrocellulose membranes, proteins werevisualized by semidry blotting 17. The membranes were blocked with 5% (w/v) dried milk in PBS for 30 min and incubated overnight with polyclonal rabbit anti-Factor H SCR1–4 antibody (raised against the N-terminal SCR 1–4 of the protein) as described 19.

For the detection of binding proteins for FHL-1/reconectin and Factor H whole cell extracts of the indicated Borreliae isolates were generated by sonication using a Branson B-12 sonifier (Heinemann, Schwäbisch Gmünd, Germany) and separated by tricine-SDS-PAGE through 4% stacking and 10% separating gels as described previously 25. For Western blot analysis, proteins were transferred to nitrocellulose membranes as described above.

4.7 Immunofluorescence assay for detection of bound FHL-1/reconectin on intact borrelial cells

For indirect immunofluorescence assays with unfixed cells, Borreliae (1×10⁸ cells) were grown to mid-log phase, harvested by centrifugation (5000×g, 30 min, 4°C), washed and resuspended in 200 μ l of culture supernatant of Sf9 insect cells containing recombinant FHL-1/reconectin. After incubation for 1 h at room temperature with gentle agitation the cell suspension was washed three times with PBS containing 1% BSA and incubated for 60 min with polyclonal rabbit anti-Factor H SCR1–4 antibody or mouse mAb 93–84/07 directed against OspC which was kindly provided by H. Peters (Dade Behring). Following three washes with PBS, the Borreliae were incubated for 60 min with a fluorescein-conjugated swine anti-rabbit IgG antibody or fluorescein-conjugated goat anti-mouse IgG antibody (Dako, Glostrup, Denmark) at a dilution of 1:50 in PBS containing 1% BSA, washed, and mounted for microscopy. Microscopy was performed with an Olympus CX40 fluorescence microscope at a magnification of ×1,000.

4.8 Functional assay for cofactor activity of FHL-1/reconectin and Factor H

Cofactor activity of and FHL-1/reconectin and Factor H was tested on borrelial surface as well as in elutes from the bacteria after adsorption of serum or recombinant FHL-1/reconectin by measuring Factor I-mediated conversion of C3b to iC3b. In brief, washed pellets of serum or FHL-1/reconectin treated Borreliae or elutes from pretreated Borreliae were incubated with optimal concentrations of purified C3b (Calbiochem, Bad Soden, Germany) and purified Factor I (Sigma, Deisenhofen, Germany) for 15 min at 37°C. Supernatants obtained by centrifugation of the bacteria as well as the elutes were tested for iC3b by ELISA. iC3b-ELISA was performed using neoepitope-specific mouse monoclonal anti-iC3b IgG as capture antibody and biotinylated rabbit ant-C3c IgG (Dako, Hamburg, Germany) as detector antibody. The reaction was visualized by the addition of streptavidin-peroxidase, followed by o-phenylenediamine/H₂O₂ as substrate. Purified iC3b (Calbiochem) was taken as standard. Control experiments included Borreliae preincubated with buffer instead of serum and with an unrelated recombinant protein expressed in the identical system. Elutes from serum-treated Borreliae were also tested directly for iC3b.