Inadequate Binding of Immune Regulator Factor H Is Associated with Sensitivity of Borrelia lusitaniae to Human Complement

Bacterial strains and culture conditions.Borrelial strains listed in Table 1 were cultured until mid-exponential phase (5 × 10⁷ cells per ml) at 33°C in Barbour-Stoenner-Kelly (BSKII) medium as described previously (28). Cultures of Escherichia coli JM109 and TOP10 were propagated routinely in 2× YT medium (Becton Dickinson GmbH, Heidelberg, Germany) supplemented with ampicillin (100 μg/ml).

Human sera, monoclonal and polyclonal antibodies, and human serum proteins.Nonimmune human serum (NHS) was tested for the presence of anti-Borrelia IgM and IgG antibodies by use of commercially available enzyme-linked immunosorbent assays (ELISAs) (Enzygnost borreliosis/IgM and Enzygnost Lyme link VlsE/IgG; Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). Only sera proven to be negative for anti-Borrelia IgM or IgG antibodies were pooled and used as a source of CFH for ligand affinity blotting. Purified CFH, polyclonal goat anti-CFH antiserum, human factor I, and human complement C3b were purchased from Calbiochem, Bad Soden, Germany. The cloning, expression, and purification of CFH, FHL1, and CFHR1 have been described previously (16, 30, 31). Polyclonal rabbit anti-SCR1-4 antiserum and monoclonal antibody (MAb) JHD 7.10 were used for detection of FHL1 and CFHR1, respectively (17, 30). For the detection of CFH, MAb IXF9 was applied (43), and a polyclonal anti-glutathione S-transferase (GST) antibody from GE Healthcare, Freiburg, Germany, was used. Goat anti-human C3 (dilutions of 1/1,000 for immunofluorescence microscopy and 1/2,000 for Western blotting) and anti-human C6 (dilution of 1/50) antibodies were purchased from Calbiochem, and monoclonal anti-human C5b-9 antibody (dilution of 1/10) was from Quidel (San Diego, CA). If not stated otherwise, anti-SCR1-4 was used at a final dilution of 1/1,000; anti-GST was used at a final dilution of 1/2,000; and MAbs IXF9, JHD 7.10, and B22 were used undiluted.

Serum sensitivity testing.Serum sensitivity of borrelial strains was assessed employing a growth inhibition assay as previously described (6, 27, 36). Briefly, highly motile spirochetes (1.25 × 10⁷) diluted in a final volume of 100 μl in BSKII medium containing 240 μg ml⁻¹ phenol red were incubated with 50% nonimmune human serum (NHS) or 50% heat-inactivated NHS (hiNHS) in microtiter plates for 10 days at 33°C (Costar, Cambridge, MA). Growth of spirochetes was monitored by daily measuring of the indicator color shift of the medium at 562/630 nm using an ELISA reader (PowerWave 200; Bio-Tek Instruments, Winooski, VT). For calculation of the growth curves, Mikrowin version 3.0 software (Mikrotek, Overath, Germany) was used.

Immunofluorescence assay for detection of deposited complement components.For detection of activated complement components deposited on the borrelial surface, an immunofluorescence assay was performed as previously described (19). In brief, spirochetes (6 × 10⁶) were incubated in 25% NHS or, as a control, in 25% hiNHS for 30 min at 37°C with gentle agitation. Ten microliters of cell suspension was spotted on glass slides, allowed to air dry overnight, and fixed in methanol. After 1 h of incubation at 37°C with polyclonal antibodies directed against the complement component C3 (Calbiochem) or C6 (Calbiochem) or a MAb directed against C5b-9 (Quidel), slides were washed and subsequently incubated with Alexa 488-conjugated antibodies directed against either goat or mouse antibodies (Molecular Probes). After being washed, the slides were mounted with ProLong Gold antifade reagent (Molecular Probes) containing DAPI (4′,6-diamidino-2-phenylindole).

Binding of complement proteins to spirochetes in EDTA-treated human serum.Borreliae (1 × 10⁹ cells) grown to mid-log phase were washed and resuspended in 750 μl NHS supplemented with 34 mM EDTA (pH 8.0) to avoid complement activation. After 1 h of incubation at room temperature and four washes with PBSA (0.15 M NaCl, 0.03 M phosphate, 0.02% sodium azide, pH 7.2) containing 0.05% Tween 20, proteins bound to the cell surface were eluted with 100 mM glycine-HCl (pH 2.0) for 15 min. Cells were removed by centrifugation at 14,000 × g for 10 min at 4°C, and both the supernatant and the last wash were separated by Laemmli SDS-PAGE under nonreducing conditions and analyzed by Western blotting as previously described (19).

Opsonization of borrelial cells and analysis of covalently bound C3 fragment.Spirochetes from logarithmic-phase cultures were harvested, washed three times, and resuspended in veronal-buffered saline (VBS). Opsonization was carried out by incubation of borrelial cells (2 × 10⁸) in 10% NHS, 10% NHS-EGTA, or 10% NHS-EDTA for 30 min at 37°C. To differentiate between the classical and alternative pathways of complement activation, NHS had been preincubated for 30 min at 37°C either with 10 mM EGTA, 4 mM MgCl₂ in VBS to specifically inactivate the classical pathway or with 10 mM EDTA to abolish activation of both the classical and alternative pathways. Non-covalently bound C3 was removed by washing the spirochetes with phosphate-buffered saline (PBS) containing 500 mM NaCl. Activated and covalently bound C3 was subsequently eluted from the borrelial surface by incubation of the cells in 1 M hydroxylamine, 0.2 M Na₂CO₃ (pH 11) for 60 min at 37°C. After centrifugation, the supernatants were adjusted to pH 7.0 by addition of 2 M HCl, separated by Laemmli SDS-PAGE under reducing conditions, and analyzed by Western blotting as previously described (19).

Cofactor assays with whole borrelial cells.Cofactor activity of CFH bound to borrelial cells was analyzed by measuring factor I-mediated conversion of C3b to iC3b, as previously described extensively (16, 19). In brief, 4 × 10⁷ cells immobilized onto microtiter plates were incubated with purified CFH (50 ng) for 60 min at room temperature. After the cells were washed, human C3b (Calbiochem) and human factor I (Calbiochem) were added to the cells and the reaction mixtures were incubated for 60 min at 37°C. The cells were sedimented by centrifugation, and the supernatants were mixed with sample buffer, subjected to SDS-PAGE under reducing conditions, and transferred onto a nitrocellulose membrane. C3b degradation products were visualized by Western blotting using polyclonal goat anti-C3 IgG (Calbiochem) (dilution of 1/2,000) and 3,3′,5,5′-tetramethylbenzidine as the substrate.

Enzyme-linked immunosorbent assay.Binding of CFH, CFHR1, and FHL1 to recombinant borrelial proteins was analyzed by ELISA as described previously (52). Briefly, purified GST fusion proteins were immobilized onto microtiter plates overnight at 4°C, and unspecific binding sites were blocked with 0.2% gelatin in PBS for 6 h at 4°C. CFH (Calbiochem), CFHR1, or FHL1 (5 μg/ml each) was added to the wells and incubated overnight at 4°C. After the wells were washed with PBS, protein complexes were identified using a polyclonal goat anti-CFH antibody followed by a secondary peroxidase-conjugated anti-goat IgG antibody. The reaction was developed with 1,2-phenylenediamine dihydrochloride (Sigma-Aldrich).

SDS-PAGE, ligand affinity blot analysis, and Western blot analysis.Whole-cell lysates obtained from each borrelial isolate or from purified recombinant proteins (500 ng per lane) were subjected to 10% Tris-Tricine SDS-PAGE under reducing conditions and transferred to nitrocellulose as previously described (28).

PCR cloning and purification of recombinant proteins.Various oligonucleotides listed in Table 2 were selected for amplification of the orthologous ospE genes of B. lusitaniae. Amplicons purified were cloned into the pCR2.1 TOPO vector (Invitrogen, Carlsbad, CA) and subsequently sequenced. The ospE-like gene of B. lusitaniae isolate MT-M8 was then subcloned into the pGEX 6P-1 expression vector by PCR using primers OspE 55(+) BamHI and OspE 3nc(−) XhoI, resulting in plasmid pGEX P38_MT-M8. The recombinant protein contained an amino-terminal GST tag, with the OspE segment beginning with that protein's first amino acid following the cysteine lipidation site. Expression of the GST-OspE fusion protein in E. coli JM109 and affinity purification on a glutathione-Sepharose column were performed as recommended by the manufacturer (GE Healthcare, Freiburg, Germany). Generation of recombinantly expressed CspA, CspZ, and ErpP proteins has been described elsewhere (15, 24, 25).

Analysis of B. lusitaniae mRNA levels.Total RNA was extracted from cultured spirochetes (1 × 10⁹ cells) grown to mid-log phase by using TRIzol reagent and Max bacterial enhancement reagent (Invitrogen, Carlsbad, CA) as described previously (8). Briefly, isolated RNA was resuspended in water and treated with DNase I (Ambion, Austin, TX). Following inactivation of DNase I using DNase inactivation reagent (Ambion), a 1-μg aliquot of each DNA-free RNA preparation was reverse transcribed using a first-strand cDNA synthesis kit (Roche Diagnostic GmbH, Mannheim, Germany) with random hexamers and avian myeloblastosis virus (AMV) reverse transcriptase. As controls, reaction mixtures containing all components except AMV reverse transcriptase were prepared and treated similarly. Templates and primers were annealed for 10 min at room temperature, followed by cDNA synthesis at 42°C for 1 h. Reverse transcriptase was inactivated by incubating the reaction mixtures at 95°C for 5 min, followed by 10 min at 4°C. All cDNAs and appropriate controls were diluted 10-fold before being used as templates for reverse transcriptase PCR (RT-PCR) or quantitative real-time PCR.

RT-PCR was performed using Taq polymerase (Invitrogen) and oligonucleotides FlaB lusF and FlaB lusR or OspE F2 and OspE R2 for amplification of the flaB or ospE genes of B. lusitaniae, respectively (Table 2). All amplicons were 198 bp in size. RT-PCR conditions consisted of a 1-min initial 94°C denaturation, followed by 45 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 30 s. To verify amplicon sizes and purity, all RT-PCR products were separated by agarose gel electrophoresis, and the amplified DNA fragments were visualized by ethidium bromide. In addition, amplicons were sequenced to confirm their identities.

Quantitative real-time PCR was performed using a LightCycler thermal cycler (Roche Applied Science) as recommended by the manufacturer. In brief, each reaction mixture contained cDNA (50 ng), LightCycler FastStart DNA Master SYBR green I (Roche Applied Science), and oligonucleotide primers FlaB lusF and FlaB lusR or OspE F2 and OspE R2. All cDNA samples were analyzed in triplicate. The conditions for quantitative real time PCR consisted of a 2-min initial 94°C denaturation, followed by 45 cycles of 94°C for 5 s, 50°C for 5 s, and 72°C for 30 s. To generate standard curves, serial dilutions of purified amplicons of the flaB and ospE genes, respectively, were included in every assay. To calculate the copy number of the respective transcript present in each cDNA sample and for melting-curve analysis, Light Cycler software version 3.5 (Roche Applied Science) was utilized.

Nucleotide sequence analysis.The deduced amino acid sequence of the OspE protein of B. lusitaniae MT-M8 was aligned using the DNAstar Lasergene 99 software package. The secondary-structure prediction was obtained using GOR4 (J. Garnier et al., 1996), available at http://www.expasy.org .

Nucleotide sequence accession number.The OspE-encoding ospE gene sequence reported in this paper has been deposited in the EMBL/GenBank databases under accession no. FN822242.

Serum sensitivity of B. lusitaniae isolates.To assess the sensitivity of B. lusitaniae to complement-mediated killing, 16 isolates of different biological and geographical origins (Table 1) were grown in the presence of 50% nonimmune human serum (NHS) or in 50% heat-inactivated NHS (hiNHS) for up to 10 days. In the presence of NHS, growth of B. lusitaniae isolates, including the human isolate PoHL, was strongly inhibited, as evidenced by minor changes in absorbance values (NHS/hiNHS ratios of 2.8 to 4.0) (Table 1). When identical experimental conditions were employed, serum-resistant strains B. burgdorferi B31-e2, B. afzelii FEM1-D15, and B. spielmanii A14S showed growth in NHS, as indicated by a significantly lower NHS/hiNHS ratio of <1.4 at day 10. In the presence of hiNHS, the growth of borrelial isolates was unaffected. Taken together, these results indicate that all B. lusitaniae isolates were highly susceptible to human complement and were classified as serum-sensitive strains.

Detection of deposited complement components C3, C6, and TCC on the surface of B. lusitaniae.Next, we analyzed deposition of the complement components C3, C6, and TCC on the surface of B. lusitaniae isolates following incubation in NHS by immunofluorescence microscopy. Large amounts of C3, C6, and TCC were deposited on the surface of the serum-sensitive B. lusitaniae isolates, in particular, MT-M8, RBU Pm2-N6, PoHL, and IP1-N1, as well as the serum-sensitive control strain B. garinii G1 (Fig. 1). The remaining B. lusitaniae isolates showed the same prominent labeling (data not shown). In contrast, serum-resistant strains B. burgdorferi B31-e2, B. spielmanii A14S, and B. afzelii FEM1-D15 showed no or marginal fluorescent staining for C3, C6, and TCC. Counterstaining with DAPI was performed to identify all spirochetes in a given field. Cells depositing complement components showed extensive bleb formation and cell fragmentation. Although the cells themselves were DAPI negative, the blebs appeared to accumulate DNA, suggesting that the DAPI-negative cells observed represent “cell ghosts.” When spirochetes were incubated with hiNHS, cell morphology remained intact and fluorescent staining was undetectable (data not shown). Thus, incubation in NHS leads to complement-mediated lysis of B. lusitaniae isolates.

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FIG. 1.

Deposition of complement components C3, C6, and TCC on the surface of B. lusitaniae. Complement components deposited on the surfaces of four representative B. lusitaniae isolates (MT-M8, RBU Pm2-N6, PoHL, and IP1-N1), B. garinii G1, B. burgdorferi B31-e2, B. spielmanii A14S, and B. afzelii FEM1-D15 were visualized by indirect immunofluorescence microscopy. Spirochetes were incubated with 25% NHS for 30 min at 37°C with gentle agitation, and bound C3, C6, and TCC were analyzed with specific antibodies against each component (αCompl.) 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 ×100. The data were recorded with a DS-5Mc charge-coupled-device (CCD) camera (Nikon) mounted on an Olympus CX40 fluorescence microscope. Each panel shown is representative of at least 20 microscope fields.

Complement activation and C3 deposition and degradation after opsonization.Borreliae can activate complement by either the classical or the alternative pathway (4, 6, 57). To compare the contributions of the alternative and classical pathways to complement activation, each B. lusitaniae isolate was incubated in 10% NHS, 10% EGTA-chelated NHS (for specific inhibition of the classical pathway), or 10% EDTA-chelated NHS (for inhibition of both complement pathways). After incubation, covalently bound C3 was released from the bacterial surface by using hydroxylamine, and C3 degradation products were analyzed by Western blotting. B. lusitaniae isolates showed no significant differences in complement activation and C3b deposition (Fig. 2). All isolates bound the 75-kDa β-chain common to C3b and iC3b, the 68-kDa α′-chain of iC3b, and the 43-kDa α′-chain of C3c after incubation with NHS (Fig. 2A). The presence of the 105-kDa α′-chain indicates deposition of intact C3b on the cell surface. All B. lusitaniae isolates, B. garinii strain G1, and B. burgdorferi strain B31-e2 activated C3, whereas no covalently bound C3 was released from the surface of B. spielmanii A14S or B. afzelii FEM1-D15. In addition, similar C3 fragmentation patterns were obtained from B. lusitaniae isolates, B. garinii G1, and B. burgdorferi B31-e2 following incubation in NHS-EGTA, indicating that these particular Borrelia isolates activate complement predominately via the alternative pathway (Fig. 2B). Opsonization of C3 was undetectable when B. spielmanii A14S and B. afzelii FEM1-D15 were incubated with EGTA-chelated NHS. The restricted complement activation of these particular isolates could be explained either by efficient release of covalently bound C3 due to binding of complement regulators or by inhibition of C3 deposition through the production of a slime layer as described previously (27). As expected, when spirochetes were incubated with EDTA-chelated serum, C3 was not activated (data not shown). Thus, all B. lusitaniae isolates activated complement mainly by the alternative pathway.

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FIG. 2.

Complement activation and C3 deposition. B. lusitaniae isolates and control strains B. garinii G1, B. burgdorferi B31-e2, B. spielmanii A14S, and B. afzelii FEM1-D15 were incubated in 10% NHS (A) or 10% EGTA-chelated NHS (B) for 30 min. After opsonization, covalently bound C3 fragments were released from the bacterial surface by using hydroxylamine, and the resulting degradation products were analyzed by Western blotting. The 105-kDa band represents the α′-chain of C3b, and the 75-kDa β-chain is common to all C3 fragments. The degradation fragment of 68 kDa indicates the α′-chain of iC3b (α′68-chain), and the 43-kDa band represents C3c. Purified C3b was used as a control and was identified by the uncleaved 115-kDa α-chain. The mobility of the marker proteins (Precision Plus protein standard) is indicated on the left.

Identification of serum protein binding to B. lusitaniae.To further examine whether B. lusitaniae can bind to human serum proteins and in particular to members of the CFH family, borrelial cells were incubated in NHS-EDTA to avoid complement activation, followed by extensive washings and elution of bound proteins from the spirochetal surface using 0.1 M glycine-HCl (pH 2.0). The last wash and the eluted fraction were then separated by SDS-PAGE and subjected to Western blotting with a polyclonal anti-CFH antiserum. A faint band of 150 kDa corresponding to CFH was detected in the eluted fractions of all B. lusitaniae isolates (Fig. 3). Isolates SDA1-N1, MT-M8, MT-W4, MT-W16, RBU Pm2-N6, RBU La5-L3, HHS La1-L3, and PoHL in addition bound CFHR1α (43 kDa) and CFHR1β (37 kDa), the two glycosylated forms of CFHR1. Moreover, isolate MT-M8 acquired CFHR2 (24 kDa) and its glycosylated form CFHR2α (29 kDa) on the surface. As expected, serum-sensitive B. garinii isolate G1 did not bind CFH or any other members of the CFH protein family. In contrast, serum-resistant isolates B. burgdorferi B31-e2, B. spielmanii A14S, and B. afzelii FEM1-D15 bound CFH and FHL1 to their surfaces, as previously described (19).

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FIG. 3.

Binding of serum proteins by B. lusitaniae isolates. B. lusitaniae isolates as well as control strains B. garinii G1, B. burgdorferi B31-e2, B. spielmanii A14S, and B. afzelii FEM1-D15 incubated in NHS-EDTA were extensively washed with PBSA containing 0.05% Tween 20, and bound proteins were eluted using 0.1 M glycine (pH 2.0). The last wash (w) and the eluate fraction (e) obtained from each isolate were separated using nonreducing 12.5% SDS-PAGE, transferred to nitrocellulose, and probed with a polyclonal anti-CFH antiserum (Calbiochem, Darmstadt, Germany). Purified CFH was used as a positive control. The mobility of the marker proteins (Precision Plus protein standard) is indicated on the left. The band corresponding to FHL1 is indicated by an asterisk.

Cofactor activity of CFH bound to B. lusitaniae.To investigate the conflicting observation that serum-sensitive B. lusitaniae isolates could acquire CFH on their surfaces, a cofactor assay was employed. Intact borrelial cells with CFH attached to the surface were analyzed for the capacity to inactive C3b. To this end, we incubated spirochetes with purified CFH, factor I, and C3b for 60 min and analyzed the C3b inactivation products by Western blotting. CFH, when bound to 13 of the 16 isolates, failed to produce the characteristic C3b cleavage pattern (68-, 46-, and 43-kDa α′-chain) (Fig. 4). When incubated with isolates RBU Pm2-N6, HHS La1-L3, BBWS2-W2, and IP1-N1, however, CFH retained some of its complement regulatory activity, based on the appearance of the 68-, 46-, and 43-kDa bands. When serum-sensitive B. garinii G1 was used, C3b degradation could not be observed. In contrast, CFH bound to serum-resistant B. burgdorferi B31-e2, B. spielmanii A14S, and B. afzelii FEM1-D15 retained its cofactor activity, as indicated by the presence of representative C3b inactivation products. Taken together, these experiments indicate that CFH bound to the majority of the tested B. lusitaniae isolates failed to inactivate C3b.

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FIG. 4.

Analysis of the functional activities of CFH bound to B. lusitaniae. Spirochetes immobilized to microtiter plates were used to capture CFH. After sequential addition of C3b and factor I, bound CFH retained cofactor activity by enabling factor I-mediated cleavage of C3b to iC3b. Following incubation, the mixture was separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose, and C3b and its degradation products were analyzed using a C3 antiserum (Calbiochem, Darmstadt, Germany). As a positive control, purified CFH (50 ng) was incubated with C3b and factor I, and as a negative control, complement proteins were incubated in the absence of CFH. The mobilities of the α′-chain and the β-chain of C3b and the cleavage products of the α′-chain (α′-68, α′-46, and α′-43) are indicated. +, incubation with all complement proteins; −, incubation in the absence of CFH.

Identification of CFH-binding proteins in B. lusitaniae.To extend our analysis on the identification of potential CFH-binding proteins produced by diverse borrelia species, ligand affinity blotting (28) was performed with cell lysates obtained from the 16 B. lusitaniae isolates. Following incubation with NHS and a polyclonal anti-CFH antiserum, a CFH-binding protein of approximately 16 kDa was identified solely for B. lusitaniae isolate MT-M8 (Fig. 5). Concerning isolates RBU Pm2-N6 and ZWU3-N4, very weak signals could also be detected. For comparison, cell lysate obtained from serum-resistant B. burgdorferi sensu stricto isolate LW2 showed three CFH-binding CRASP proteins (CspA, ErpP, and ErpA). In agreement with our previous results, CFH-binding proteins were undetectable in the serum-sensitive B. garinii isolate G1 (19, 28, 52). Taken together, these results indicate that only 1 of the 16 B. lusitaniae isolates tested expresses a 16-kDa protein that possesses CFH-binding capability.

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FIG. 5.

Identification of CFH-binding proteins of B. lusitaniae isolates. Cell lysates (30 μg each) obtained from diverse B. lusitaniae isolates and control strains B. burgdorferi sensu stricto LW2 and B. garinii G1 were separated by 10% Tris-Tricine SDS-PAGE and transferred to nitrocellulose. The membranes were incubated with NHS as a source for CFH, and binding of the proteins was detected by a polyclonal anti-CFH serum. The identified CRASP proteins, CspA, CspZ, and ErpP of B. burgdorferi LW2 and OspE of B. lusitaniae MT-M8, are indicated on the right, and the mobility of the marker proteins is indicated on the left.

Cloning and characterization of the CFH-binding protein of B. lusitaniae MT-M8.According to the binding properties and the molecular mass, we speculated that the 16-kDa protein of isolate MT-M8 could represent a member of the polymorphic CFH/CFHR1-binding Erp family. Consequently, a set of oligonucleotide primers suitable to amplify various Erp-encoding genes was employed (25, 51) (Table 2). Sequence analysis of the achieved 766-bp amplicon revealed an open reading frame that exhibited 61.2% sequence identity to the erpA, erpC, and erpP genes of B. burgdorferi sensu stricto B31. This erp-like gene encodes a unique protein consisting of 192 amino acid residues, with a calculated molecular mass of 21.5 kDa. The N terminus of this protein is homologous 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 (14, 50), suggesting that the putative protein is lipidated and located at the outer surface of the spirochetes. To confirm the surface localization of the 16-kDa protein, we treated intact borrelial cells with proteolytic enzymes. Treatment with proteinase K at the lowest concentration (2.5 μg/ml) led to a complete elimination of the 16-kDa band, whereas trypsin treatment had no effect (data not shown). Because the CFH-binding protein of 16 kDa is readily accessible to proteinase K, it appears to be located on the outer surface of B. lusitaniae.

Binding properties of the recombinant CFH-binding protein of B. lusitaniae MT-M8.We analyzed the binding activity of the CFH-binding protein of B. lusitaniae MT-M8 to diverse members of the CFH protein family, in particular, CFH, FHL1, and CFHR1. A GST-tagged fusion protein using oligonucleotides provisionally termed OspE 55(+) BamHI and OspE 3nc(−) XhoI was expressed (Table 2). After cloning and overproduction in E. coli, ligand affinity blotting and ELISA were performed to investigate binding of CFH, CFHR1, and FHL1 (Fig. 6). The recombinant OspE derived from B. lusitaniae MT-M8 failed to bind FHL1 and CFHR1 and bound to CFH less efficiently than did CspA, CspZ, and ErpP of B. burgdorferi sensu stricto LW2 (20, 21, 29, 30) (Fig. 6A). Each of the three B. burgdorferi sensu stricto CRASP proteins used as controls bound to CFH and FHL1 (CspA and CspZ) or CFH and CFHR1 (ErpP). As expected, purified GST protein did not bind to any of the three complement regulators. Subsequently, we quantified the binding activity of each recombinant protein to CFH by ELISA (Fig. 6B). Consistent with our ligand affinity blot analyses, binding of CspA, CspZ, and ErpP to CFH was up to 6-fold more efficient than that of OspE of B. lusitaniae MT-M8.

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FIG. 6.

Binding capability of OspE of B. lusitaniae MT-M8 to human serum proteins. (A) Binding capabilities of CFH, CFHR1, and FHL1 to purified GST fusion proteins were analyzed by ligand affinity blotting. Recombinant proteins (500 ng/lane) were subjected to 10% Tris-Tricine SDS-PAGE and blotted to nitrocellulose membranes. GST fusion proteins were detected by using anti-goat GST antibody. For detection of CFH and CFHR1 bound to CRASP proteins, membranes were incubated with NHS as a source of CFH or with purified CFHR1. Protein complexes were then visualized using MAb IXF9 or JHD 7.10, respectively. Binding of FHL1 was detected using MAb B22 or polyclonal anti-SCR1-4 antiserum specific for the N-terminal region of CFH and FHL1. The CFH/FHL1-binding CspA and CspZ proteins, the CFH/CFHR1-binding ErpP protein, and purified GST served as controls. (B) Binding of CFH to recombinant CRASP proteins was also quantified by ELISA. Proteins (500 ng each) were immobilized onto a microtiter plate and incubated with purified CFH. For detection of protein complexes, a polyclonal anti-CFH antiserum was used. Reaction mixtures were run in duplicate or triplicate, and all experiments were repeated at least twice, with very similar results; the figure displays a representative experiment. Error bars represent standard deviations (SDs).

OspE expression by B. lusitaniae isolates.In order to examine expression levels of the ospE gene of B. lusitaniae in vitro, quantitative real-time PCR was performed. Total RNA isolated from each of the B. lusitaniae isolates grown to mid-logarithmic phase was reverse transcribed, and cDNAs were subjected to quantitative real-time PCR to measure the ospE transcript levels. As depicted in Fig. 7, ospE was expressed in all isolates, albeit at different levels. As expected, ospE expression levels were lower than flaB expression levels (Fig. 7). Overall, these results demonstrated that ospE transcripts were present in all B. lusitaniae isolates cultivated in vitro.

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FIG. 7.

ospE expression levels among B. lusitaniae isolates. Illustrated are quantitative real-time PCR results from spirochetes grown at 33°C and harvested at mid-logarithmic growth phase. The relative expression levels of the ospE gene are presented as copies of ospE transcript per 10² copies of the constitutively expressed B. lusitaniae flaB gene. Each experiment was performed at least two times in triplicate, and error bars represent standard deviations (SDs).

Taken together, these experiments indicate that spirochetes of the genospecies B. lusitaniae are highly susceptible to human complement-mediated killing. All isolates activate complement mainly via the alternative pathway, resulting in deposition of complement components C3, C6, and TCC on the surface. Surprisingly, B. lusitaniae binds human complement regulators CFH and CFHR1. Based on its reduced activity, we hypothesize that CFH, when attached to the surface of B. lusitaniae, is sterically hindered and unable to retain full complement regulatory activity, thereby conferring a serum-sensitive phenotype.