INFECTION AND IMMUNITY, Apr. 2005, p. 2351–2359 0019-9567/05/$08.00⫹0 doi:10.1128/IAI.73.4.2351–2359.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 4
Identification and Functional Characterization of Complement Regulator-Acquiring Surface Protein 1 of the Lyme Disease Spirochetes Borrelia afzelii and Borrelia garinii Reinhard Wallich,1* Joseph Pattathu,1 Veronique Kitiratschky,1 Christiane Brenner,1 Peter F. Zipfel,2 Volker Brade,3 Markus M. Simon,4 and Peter Kraiczy3 Infectious Immunology Group, Institute for Immunology, University of Heidelberg, Heidelberg,1 Department of Infection Biology, Leibniz Institute for Natural Products Research and Infection Biology, Jena,2 Institute of Medical Microbiology, University Hospital of Frankfurt, Frankfurt,3 and Max Planck Institute for Immunobiology, Freiburg,4 Germany Received 7 August 2004/Returned for modification 23 September 2004/Accepted 28 October 2004
Complement regulator-acquiring surface protein 1 (CRASP-1) is the dominant factor-H-like protein 1 (FHL-1)- and factor-H-binding protein of Borrelia burgdorferi and is suggested to contribute to persistence of the pathogen. The prototype CRASP-1 of B. burgdorferi sensu stricto (CRASP-1Bb) has been formerly characterized. As shown recently, serum-resistant Borrelia afzelii strains express a unique FHL-1 and factor Hbinding protein, designated CRASP-1Ba. Here, we describe for the first time the isolation and functional characterization of the gene encoding the full-length CRASP-1Ba of 28 kDa, which, upon processing, is predicted to be 26.4 kDa. CPASP-1Ba of B. afzelii spirochetes is associated with a genetic locus encoding the orthologous gbb54 gene family that maps to the linear plasmid of approximately 54 kb. Ligand affinity blotting techniques demonstrate that both native and recombinant CRASP-1Ba molecules strongly bind to FHL-1 and much more weakly to factor H. The FHL-1 and factor-H-binding site in CRASP-1Ba is shown to be localized to a 12-amino-acid residue domain at the C terminus of the protein. For comparison, the corresponding cspA-like gene(s) of a serum-sensitive Borrelia garinii strain has also been cloned and characterized. Most notably, two CRASP-1-related B. garinii proteins were identified; however, both molecules bind only weakly to FHL-1 and not at all to factor H. The present identification of the binding site of CRASP-1Ba represents an important step forward in our understanding of the pathogenesis of Lyme disease and may be helpful to design therapeutic regimens to interfere with complement evasion strategies of human pathogenic Borrelia strains. The human pathogenic genospecies Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii are the causative agents of Lyme borreliosis, the most commonly reported vector-borne infectious disease in Eurasia and the United States (37). Spirochetes of the B. burgdorferi complex are transmitted to humans and vertebrate hosts primarily by Ixodes sp. ticks. Adaptation to the diverse environmental conditions, including sophisticated means of evading the vertebrate hosts’ immune system, in particular complement occurs at the first line of defense following infection (20). One strategy of B. burgdorferi and B. afzelii (2, 16) and many human pathogenic microorganisms such as Streptococcus pyogenes (9, 10, 28), Streptococcus pneumoniae (26), Neisseria meningitidis (30), Neisseria gonorrhoeae (31), Echinococcus granulosus (6), and Yersinia enterocolitica (5) to resist complement-mediated killing is the expression of receptors for host-derived fluid-phase complement regulatory proteins, such as factor H protein and/or factor-Hlike protein 1 (FHL-1), on the surface (17). FHL-1 and factor H, the main immune regulators of the alternative pathway of complement activation, are structurally related proteins and organized into individually folding protein domains termed
short consensus repeats (SCRs). FHL-1, an alternatively spliced variant of the factor H gene, consists of seven SCRs, and the factor H protein includes 20 SCR domains. The first seven SCRs at the N terminus of both complement regulators are identical, but FHL-1 includes an extension of 4 amino acids at the C terminus. When bound to the outer surface of a pathogen, factor H and FHL-1 inhibit complement activation by rapidly inactivating at the microbial surface newly formed C3b and by accelerating the decay of the C3 convertase of the alternative pathway (2, 18, 27). Previous studies showed that serum-resistant B. afzelii as well as moderately serum-resistant B. burgdorferi strains express a number of lipoproteins, termed complement regulatoracquiring surface proteins (CRASPs), which bind FHL-1 and/or factor H (17). According to their binding properties, CRASPs are divided into three groups: group I expresses proteins that bind both FHL-1 and factor H, e.g., CRASP-1Bb, CRASP-1Ba, CRASP-2Bb, CRASP-2Ba; groups II and III express proteins that selectively bind either FHL-1 or factor H, e.g., CRASP-3Ba and CRASP-3Bb to CRASP-5Bb and CRASP4Ba to CRASP-5Ba, respectively (15, 17). In addition, OspE paralogs (OspE, p21, ErpA, ErpC, ErpP) of independent B. burgdorferi strains were shown to bind factor H (1, 8, 11, 13, 24, 32). Notably, earlier analysis showed that all serum-resistant B. burgdorferi sensu stricto strains express CRASP-1Bb, the prominent outer surface protein for binding of FHL-1 and factor H (12). Furthermore, serum resistance of Borrelia strains was
* Corresponding author. Mailing address: Infectious Immunology Group, Institute for Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg, Germany. Phone: 496221-564090. Fax: 49-6221-565611. E-mail:
[email protected] -heidelberg.de. 2351
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found to directly correlate with the expression of CRASP-1Bb and complement-regulating activities of FHL-1 and factor H are maintained upon binding to the surfaces of spirochetes (16). The cspA gene encoding CRASP-1Bb of B. burgdorferi strain ZS7 (35) is localized to the 54-kb linear plasmid (lp54) and represents a member of the orthologous gene family gbb54 (7). To date, respective molecular data on CRASP-1 orthologous proteins of B. afzelii and B. garinii strains are lacking. The aim of this study was to identify and functionally characterize the corresponding CRASP-1 molecules CRASP-1Ba and CRASP1Bg of B. afzelii (strain MMS) and B. garinii (strain ZQ1), respectively. MATERIALS AND METHODS Borrelial strains and culture conditions. B. burgdorferi ZS7 and LW2, B. afzelii MMS and FEM1-D15, and B. garinii ZQ1 were investigated. Unless otherwise stated, all strains were cultured until mid-log phase (5 ⫻ 107 cells per ml) at 33°C in modified Barbour-Stoenner-Kelly (BSKII) medium. Samples of 1.8 ml were then dispensed into screw-cap tubes (Nunc, Wiesbaden, Germany), frozen at ⫺70°C, and used as stock cultures. Prior to use, a frozen suspension of spirochetes was thawed and inoculated into fresh BSKII medium. The density of spirochetes was determined using a Kova counting chamber (Hycor Biomedical, Garden Grove, Calif.) and dark-field microscopy. Genomic library screening and PCR cloning. A B. afzelii strain MMS genomic library constructed in pUEX1 was screened using a cspA gene probe of B. burgdorferi strain ZS7. A 5-kb clone (#3.3) containing cspA and adjacent downstream genes including mmsa66 was identified and selected for further analysis. A 1.4-kb fragment (#66A) containing the complete mmsa66 gene and upstream DNA was amplified by PCR with an SAWADY high-fidelity PCR system (peqlab, Erlangen, Germany) from the genomic DNA of B. afzelii strain MMS by using primers BBA66R9 and 66F7. By using primers R5⬘1 and A74R1, a 1.5-kb fragment (#73A) containing the complete mmsa72 and mmsa73 genes was amplified (Fig. 1; Table 1). The fragments were cloned into pGEM-Teasy (Promega, Mannheim, Germany). The following reaction conditions were used: 94°C for 30 s, 45°C for 30 s, and 68°C for 30 s for 30 cycles. The gbb54 locus on lp54 of B. garinii ZQ1 was identified and cloned employing a PCR strategy. A 1.4-kb fragment (#66G) containing the zqa66 gene was amplified by PCR as described above from the genomic DNA of B. garinii strain ZQ1 by using primers BBA66R10 and ZQA66F7 and ligated in the vector pGEM-Teasy. Similarly, a 2.1-kb fragment (#67G) containing the zqa67 and zqa68 genes, a 3.2-kb fragment (#70G) containing zqa69 and zqa70 and the C-terminal part of zqa71, and a 2.5-kb fragment (#72G) harboring partial zqa71, zqa72, and zqa73 genes was amplified by using primers ZQA66R4 and 140/152F2, 26-R-V and 22-F-V, and 22-R-V and BBA73F2, respectively (Fig. 1; Table 1). Plasmid DNA was prepared from the presumptive clones with the QIAprep kit (QIAGEN, Hilden, Germany), and the Borrelia DNA inserts were sequenced using the BigDye Terminator cycle sequencing kit (PE Applied Biosystems, Foster City, Calif.) in accordance with the manufacturers’ recommendations. SDS-PAGE, ligand affinity blot analysis, and Western blotting. Borrelial cell lysates (15 g) or purified recombinant proteins (400 ng) were subjected to 10% Tris-Tricine–sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) under reducing conditions and transferred to nitrocellulose as previously described (13, 15). Briefly, after transfer of proteins onto nitrocellulose, nonspecific binding sides were blocked using 5% (wt/vol) dried milk in TBS (50 mM Tris-HCl [pH 7.4], 200 mM NaCl, 0.1% Tween 20) for 6 h at room temperature. Subsequently, membranes were rinsed four times in TBS and incubated at 4°C overnight with nonimmune human serum (NHS) or culture supernatants containing either recombinant FHL-1 or a variety of deletion mutants of factor H. After four washings with 50 mM Tris-HCl (pH 7.5)–150 mM NaCl–0.2% Tween 20 (TBST), membranes were incubated for 3 h with either a polyclonal rabbit antiserum recognizing the N-terminal region of factor H (␣SCR1-4) or a monoclonal antibody (MAb), VIG8, directed against the C terminus of factor H. Following four washes with TBST, blot strips were incubated with a secondary peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody or with a secondary peroxidase-conjugated anti-mouse IgG antibody (Dako, Glostrup, Denmark) for 60 min at room temperature. Detection of bound antibodies was performed by using 3,3⬘,5,5⬘-tetramethylbenzidine as substrate. Expression and purification of recombinant CRASP-1 orthologous proteins.
FIG. 1. cspA containing gene clusters in different European Borrelia strains. Linear representation of the genomic organization of the gbb54 gene cluster (bba66 to bba73) on lp54 plasmids from three European strains: B. burgdorferi ZS7, B. afzelii MMS, and B. garinii ZQ1 is given. Strain names are shown above each gene cluster. Grey arrows indicate open reading frames with homologous genes in other lp54 plasmids. Black arrows represent unique cspA genes encoding the FHL-1- and factor-H-binding proteins CRASP-1Bb and CRASP-1Ba. White arrows indicate B. garinii-encoded CRASP-1-like proteins that could weakly bind, if at all, FHL-1. Plasmids harboring genomic Borrelia DNA isolated from hybridizing clones (dotted line) or by using a PCR strategy (solid lines) are shown below each schematic.
Recombinant CRASP-1 and orthologous proteins were expressed as fusion proteins with glutathione S-transferase (GST). The genes lacking their leader sequences were cloned into the pGEX-2T vector (Amersham Bioscience, Freiburg, Germany). The ligation mixtures were used to transform Escherichia coli DH5-␣ or JM109 as described previously (34). Expression of the respective recombinant B. burgdorferi GST fusion proteins, affinity purification on glutathione-Sepharose columns, and endoproteinase thrombin cleavage of the GST fusion proteins were performed as recommended by the manufacturer (Amersham Bioscience). Cterminal deletion mutant CRASP-1Ba26-231 was constructed by PCR using pGEX sequencing primer #13 in combination with primer #14 and the CRASP-1Ba gene without the leader sequence cloned in pGEX-2T as template (Table 1). The amplified DNA fragments were digested with BamHI and ligated into pGEX-2T (Amersham Bioscience). Expression and purity of generated GST fusion proteins were confirmed by employing Tris-Tricine–SDS-PAGE, and protein concentrations were determined by a Bradford assay (Bio-Rad, Munich, Germany). Expression of recombinant proteins of FHL-1 and factor H. Recombinant FHL-1 protein (FH1-7) and deletion constructs of factor H (FH1-2, FH1-3, FH1-4, FH1-5, FH1-6, FH8-20, FH15-20, and FH19-20) were expressed in Spodoptera frugiperda Sf9 insect cells infected with recombinant baculovirus (19). The cloning of various deletion constructs, expression, and purification have been described previously (19). Serum adsorption experiments using intact borrelial cells. Borreliae (2 ⫻ 109 cells) were grown to mid-log phase, harvested by centrifugation (5,000 ⫻ g, 30 min, 4°C), and resuspended in 100 l of veronal-buffered saline (supplemented with 1 mM Mg2⫹–0.15 mM Ca2⫹–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 of NHS-EDTA for 1 h at room temperature with gentle agitation. After three washes with PBSA (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 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 ⫻ g for 20 min at 4°C, and the supernatant was analyzed. RT-PCR analysis. For reverse transcription (RT)-PCR, total RNA was isolated from spirochetes cultured at 33°C in BSKII medium by the acid guanidinium thiocyanate phenol-chloroform method using TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s recommendations. RNA was treated with DNase and reverse transcribed, and PCR was performed
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TABLE 1. Oligonucleotides used in this study Oligonucleotide
Sequence (5⬘-3⬘)
Designation
Localization
1 2 3 4 5 6 7 8 9 10 11 12 13 14
TGTTTCCCGTAAAGCTTGCTAC TAGGTTTAATTGTATTCAGGTTGG AATTCATTTCTAGGTCTGAGG CAGGAATAATATTTGAAAATTAAATC AATGTCGTTATCCCCAATG TGCTGAGCTTGTTGGGCTTACTAC GGATGAAGTTTCGAGCTGTAG TCCCTGTGTTGAGGCTGGTCG ATACACCTTAGCAAACATTGCC CTAAGCCTTTAAATAAAAAAAATC CCGATAAGGATCAATTAGC TCGAGGAATAGTATTTTG GGGCTGGCAAGCCACGTTTGGTG GATTACTAAATTAATGGGATTAAAAGGATCCAGGT CTTAGTAATTTTC
BBA66R9 R5⬘I A74R1 66F7 BBA66R10 ZQA66R4 26-R-V 22-R-V BBA73F2 22-F-V 140/152F2 ZQA66F7 pGEX(⫹) CRASP-1Ba 687(⫺)
Upstream of mmsa66 cspA Downstream of mmsa73 mmsa67 Upstream of zqa66 zqa66 zqa68 zqa71 Downstream of zqa73 zqa71 Downstream of zqa68 zqa66 GST cspA (C terminus)
as described above. PCR-amplified gene fragments were loaded onto agarose gels and visualized by staining. In situ protease treatment of spirochetes. Borrelial cells of B. burgdorferi ZS7, B. afzelii MMS, and B. garinii ZQ1 were treated with proteases as described previously (12). Briefly, spirochetes were grown to mid-exponential phase, sedimented by centrifugation at 5,000 ⫻ g for 30 min, washed twice with cold phosphatebuffered saline (PBS) containing 5 mM MgCl2 (PBS-Mg), and resuspended in 100 l of the same buffer. To 108 borrelial cells (final volume of 0.5 ml), proteinase K or trypsin was added to a final concentration of 12.5 to 100 g/ml. Following incubation for 1 h at room temperature, digestion with proteinase K or trypsin was terminated by the addition of 5 l of phenylmethylsulfonyl fluoride or by the addition of 5 l of phenylmethylsulfonyl fluoride and 5 l of AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride], respectively. Borrelial cells were then washed twice with PBS-Mg, resuspended in 20 l, and lysed by sonication five times using a Branson B-12 sonifier (Heinemann, Schwa¨bisch Gmu ¨nd, Germany). Equal volumes of borrelial lysates were subjected to Tris-Tricine–SDS-PAGE, and proteins were transferred to nitrocellulose membranes as described previously (15). Susceptibility of proteins to protease digestion was assessed by Western or ligand affinity blotting with the appropriate monoclonal or polyclonal antibodies, following by incubation with a secondary horseradish peroxidase-conjugated IgG antibody, and visualized by the addition of 3,3⬘,5,5⬘-tetramethylbenzidine. Human sera, polyclonal antibodies, and MAbs. NHS obtained from 20 healthy human blood donors without a known history of spirochetal infections was used as a source for factor H. Sera that proved negative for anti-Borrelia antibodies were combined to form the NHS pool. Polyclonal rabbit anti-SCR1-4 antiserum (␣SCR1-4) was used for the detection of FHL-1 (18), and MAb VIG8 against factor H, LA3 against Hsp70, and LA22.1 against flagellin were described elsewhere (29, 36). Nucleotide sequence accession numbers. The nucleotide sequences of the gbb54 gene cluster of B. afzelii MMS and B. garinii ZQ1 have been deposited in the EMBL and GenBank databases under accession no. AJ786368 and AJ786369, respectively.
RESULTS Identification and genomic organization of the gbb54 orthologous gene family in B. afzelii MMS and B. garinii ZQ1 harboring CRASP-1. In order to identify the key FHL-1 and factor-H-binding CRASP-1 of B. afzelii, screening of a genomic library with a 700-bp fragment of the cspA (formerly named zsa68) gene from B. burgdorferi strain ZS7 as probe led to the identification of a cspA encoding a 5-kb DNA fragment from B. afzelii strain MMS, designated #3.3 (35). In addition, a PCR-based approach was used to clone upstream (#66A) and downstream (#73A) paralogs of cspA (Fig. 1). Similarly, the linear plasmid (lp54)-located gbb54 gene cluster in B. garinii ZQ1 was cloned employing PCR to allow a comparison of
CRASP-1 among the three human pathogenic Borrelia species. Prefixes were used according to the specific strain designations as follows: for B. burgdorferi ZS7-specific genes, zsa66 through zsa73 (unpublished data); for B. afzelii MMS, mmsa66 through mmsa73; and for B. garinii ZQ1, zqa66 through zqa73. The gbb54 gene cluster of each Borrelia genospecies harbored homologous alleles in similar locations (Fig. 1). According to The Institute for Genomic Research designation, a66 and a73 genes encode for putative p35 antigens. As depicted in Fig. 1, with the exception of mmsa72 and zsa67, all other genes of the gbb54 orthologous gene family in both B. afzelii and B. burgdorferi are arranged in the same orientation. In contrast to B. afzelii and B. burgdorferi, all gbb54 orthologous genes of B. garinii are arranged in the same orientation and, in addition, exhibit similar sizes (705 to 768 bp). The analysis of gene expression of the gbb54 gene family members of strains MMS and ZQ1 by RT-PCR showed specific mRNA for all five genes of B. afzelii analyzed (a67, a68, a69, a70, and a71) (Fig. 2A). In contrast, two of the six genes (a67 through a72) of B. garinii ZQ1 investigated, a71 and a72, were not expressed in spirochetes propagated in vitro (Fig. 2B). Binding of complement regulators FHL-1 and factor H to native and recombinant CRASP-1 orthologs. Upon incubation with spirochetal cell lysates, FHL-1 was found to bind strongly to a 25.9-kDa protein of ZS7 (CRASP-1Bb) and a 27.4-kDa protein of MMS (CRASP-1Ba) but only weakly to a protein of 27.7 kDa of ZQ1 (CRASP-1Bg) (Fig. 3A). When binding of factor H to proteins derived from strains ZS7, MMS, and ZQ1 was analyzed by immunoblot using a factor-H-specific antibody, it was shown that CRASP-1Bb and CRASP-1Ba showed weak binding whereas no binding was detected for CRASP1Bg. The finding that similar amounts of flagellin were detectable in all three samples suggests either that the various CRASP-1 proteins are differentially expressed in the three strains or that they exhibit different binding characteristics for the two complement regulators (Fig. 3A). Recombinant proteins encoded by members of the gbb54 orthologous gene family of B. afzelii were produced in E. coli and analyzed for binding of host complement regulators FHL-1 and factor H (Table 2). From these recombinant B. afzelii proteins tested, only the cspA-encoded recombinant pro-
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FIG. 2. RT-PCR analyses of gene expression of gbb54 family members. Total genomic DNA and cellular RNA samples isolated from in-vitro-propagated B. afzelii MMS (A) and B. garinii ZQ1 (B) were tested for mmsa67-to-cspA and zqa67-to-zqa72 expression, respectively. Specific mRNA transcripts were amplified by RT-PCR (lane 1) plus DNase (lane 2) plus RNase (lane 3) or in the absence of reverse transcriptase (lane 4). Control samples containing total genomic DNA (lane 5) or no DNA (lane 6) were amplified by PCR.
tein, named CRASP-1Ba, showed strong binding for FHL-1 (Table 2). When recombinant proteins of B. garinii were tested, weak interaction of FHL-1 with gene products of zqa68 and zqa71, termed CRASP-1Bg and CRASP-1Bg, respectively, but not those of zqa67, zqa69, zqa70, and zqa72, were detectable (Table 2; Fig. 3B). Note that expression of zqa71 in cultivated spirochetes was not detected (Fig. 2). Binding of factor H to recombinant B. afzelii proteins tested was observed exclusively for CRASP-1Ba. None of the B. garinii proteins were able to bind to this complement regulator (Fig. 3B). For comparison and as a control, binding of FHL-1 and factor H to CRASP-3Bb was included (Fig. 3B) (13). Characteristics of CRASP family members are summarized in Table 3. Comparative amino acid sequence analysis of CRASP-1 orthologs. The CRASP-1 amino acid sequences of the three human pathogenic Borrelia genospecies ranged in length from 238 to 256 amino acid residues, and all four CRASP-1 orthologs exhibited molecular masses of approximately 25 to 27 kDa. CRASP-1Ba appeared to be most closely related to CRASP-1Bb (48% identity). The leader sequences (amino acids 1 to 25) of all four proteins are rather conserved (⬇70% identity) among the three genospecies. Comparison of CRASP-1Ba, CRASP-1Bg, CRASP-1Bg, and CRASP-1Bb showed a high degree of variability, with sequence identities ranging from 45 to 54% (Fig. 4). Elsewhere in the sequence, there were marked differences in the positions of charged amino acids. Searching the CRASP-1 sequences for putative factor-H-binding motifs similar to those previously identified
in CRASP-1Bb (12) by using the DNAstar Lasergene 99 software package revealed three potential regions within the middle and near the C terminus of the CRASP-1 molecules (Fig. 4). Mapping of the complement regulator-binding site on CRASP-1Ba. To localize the FHL-1–factor H-binding domain (12) of CRASP-1Ba, a C-terminal deletion mutant encompassing 11 amino acid residues was generated and the binding properties of wild-type CRASP-1Ba and the mutant CRASP-1Ba26-231 to FHL-1 and factor H were analyzed (Table 2). Only the full-length form of CRASP-1Ba strongly bound FHL-1, whereas the potential of the truncated mutant CRASP-1Ba26-231 was greatly reduced. In contrast, binding to factor H was completely abolished by using mutant CRASP-1Ba26-231, suggesting that the critical binding sites for both complement regulators do overlap. Furthermore, these findings indicate that the C terminus of CRASP-1Ba is relevant for the binding of complement regulators FHL-1 and factor H, similar to CRASP-1Bb. Mapping of the CRASP-1Ba-binding site of FHL-1 and factor H. Previously, we have reported that binding of both FHL-1 and factor H to native CRASP-1Ba is predominantly mediated via SCR5 to SCR7 (12). In this study, we aimed to precisely map the binding sites of FHL-1 and factor H to recombinant CRASP-1Ba of B. afzelii strain MMS by employing the ligand affinity blotting technique. As shown in Fig. 5 CRASP-1Ba strongly binds FHL-1 (lane 6) and, in addition, deletion constructs FH1-6 (lane 5) and FH1-5 (lane 4), but not constructs FH1-4 (lane 3), FH1-3 (lane 2), or FH1-2 (lane 1). Applying
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FIG. 3. Analysis of the complement regulator-binding capabilities of native and recombinant CRASP-1 molecules of B. burgdorferi sensu lato strains. Binding capabilities of FHL-1 and/or factor H to cell lysates or to recombinant CRASP-1 molecules were analyzed by the ligand affinity blot technique. Whole-cell lysates of strains ZS7, MMS, and ZQ1 (A) or recombinant CRASP-1Bb, CRASP-1Ba, CRASP-1Bg, CRASP-1Bg, or CRASP-3Bb (400 ng/lane) (B) were subjected to 10% Tris-Tricine–SDSPAGE and blotted to nitrocellulose membranes. These membranes were then incubated with either recombinant FHL-1 or with NHS for detection of factor H binding. Bound proteins were visualized using antiserum (␣SCR1-4) specific for the N-terminal region of FHL-1 or MAb VIG8 specific for the C-terminal region of factor H. Flagellin and Hsp70 were detected using MAbs LA22.1 or LA3, respectively. The mobilities of the marker proteins are indicated to the right.
deletion constructs representing the C-terminal SCRs of factor H, i.e., FH8-20, FH15-20, and FH19-20, no binding to CRASP1Ba was observed (Fig. 5, lanes 8 through 10). In comparison to FHL-1 (lane 6), native factor H (lane 7) showed a weak binding to CRASP-1Ba. These data indicate that SCR5 to SCR7 of FHL-1 and factor H are critical for interaction with CRASP1Ba.
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Determination of surface exposure of CRASP-1 orthologous proteins. In order to determine if CRASP-1Bb orthologs are surface exposed, their susceptibility to proteolysis was tested. To this end, cells of B. burgdorferi ZS7, B. afzelii MMS, and B. garinii ZQ1 were treated with proteinase K or trypsin. Cell lysates were fractionated by SDS-PAGE and immunoblotted, and the respective proteins were detected either by MAbs or by binding of FHL-1. As shown in Fig. 6, treatment with proteinase K resulted in the complete loss of FHL-1 binding to both CRASP-1Bb and CRASP-1Bg. In contrast, degradation of CRASP-1Ba was incomplete following incubation with proteinase K even at high concentrations. The limited susceptibility of CRASP-1Ba to proteinase K is reminiscent of a previous report on the differential susceptibilities of OspA/OspB from independent B. burgdorferi strains to proteolysis (22). Upon treatment with trypsin, a more site-specific protease, all CRASP-1 orthologs were completely degraded after 1 h of incubation at a concentration of either 50 g/ml (CRASP-1Bb and CRASP1Bg) or 100 g/ml (CRASP-1Ba) (Fig. 6). As expected, flagellin and Hsp70 were resistant to trypsin and proteinase K even at the highest concentration applied. These data demonstrate that all three CRASP-1 orthologs are surface-exposed proteins and suggest that they are capable of interacting with FHL-1 and/or factor H in vivo. Moreover, expression of exposed CRASP-1 molecules seems to be variable for distinct Borrelia genospecies. Demonstration of cell-bound FHL-1 and factor H. To assess the binding of both complement regulators to the surfaces of borrelial cells in a more physiologic assay, intact spirochetes were incubated with NHS, a natural source for FHL-1 and factor H, that was supplemented with EDTA to prevent complement activation. Complement regulators were adsorbed to the borrelial surface and subsequently eluted by employing a pH shift assay. Cell supernatants were analyzed for the presence of FHL-1 and factor H. With the exception of B. garinii ZQ1, FHL-1 could be detected in the eluted fractions of B. burgdorferi ZS7 and LW2 and B. afzelii MMS and FEM1-D15, indicating that the interaction of FHL-1 with B. burgdorferi and B. afzelii strains is more efficient compared to that with B. garinii ZQ1. In addition, it was found that
TABLE 2. Alignment of the C-terminal region of CRASP-1Bb orthologous proteins and binding properties to FHL-1 and/or factor H Protein or deletion mutant
ZSA68/CRASP-1Bb MMSA67 MMSA68 MMSA69 MMSA70 MMSA71/CRASP-1Ba CRASP-1Ba26–231 ZQA67 ZQA68/CRASP-1Bg ZQA69 ZQA70 ZQA71/CRASP-1Bg ZQA72 a b
C-terminal sequence of CRASP-1Bb orthologous proteinsa
QNTQDNEKILAEHFNKYYKDFDTLKPAFY TTLQTNSEELEAYLKTTYEDILKPDSNEIYDLMIEIGRELQENL YDATAEITKIKEILNPNYNLPTLTIPGNQN ESIKTNEDKLAAHMNENYEEFTSLKPI ANIKTNKEALVKHMDENYTNLNSFKPTN QNIQYNDEELAKHMDENYKDLDSFNPIN QNIQYNDEELAKHMDENY ENIKTNAEQLAKHINENYKEFNSLKPIH DNIKSNEDQLAKHMDENYKEFDPLNLDY ENIKSDKDQLAKHMDENYKNLHSFKTVYY NNIKSDAEALANHMNENYKDHEYLKPID YNIKNNIEELAWHIDANYKTLDSFKSIN HNITNDMEELAWKIHEVCEELGTLKPIK
Binding tob: FHL-1
Factor H
⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫾ ⫺ (⫾) ⫺ ⫺ (⫾) ND
⫹ ⫺ ⫺ ⫺ ⫺ ⫾ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ND
Conserved amino acids are indicated in bold. The putative factor H-binding motif is underlined. ⫹⫹, strong binding intensity; ⫹, moderate binding intensity; ⫾, weak binding intensity; (⫾), extremely weak binding intensity; ⫺, no binding; ND, not done.
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INFECT. IMMUN. TABLE 3. Features of CRASP family membersa
CRASP designation
Gene name
CRASP-1Bb CRASP-3Bb CRASP-4Bb CRASP-5Bb CRASP-1Ba CRASP-1Bg CRASP-1Bg
cspA erpP erpC erpA cspA NA NA
Strain
B. B. B. B. B. B. B.
burgdorferi ZS7 burgdorferi ZS7 burgdorferi LW2 burgdorferi LW2 afzelii MMS garinii ZQ1 garinii ZQ1
Systematic designation
TIGRb name
Gene familyc
Binding activity tod:
Reference
ZSA68 ZSN38 NA LWP38 MMSA71 ZQA68 ZQA71
BBA68 BBN38 NA BBP38 BBA71 BBA68 BBA71
gbb54 gbb162 gbb162 gbb162 gbb54 gbb54 gbb54
FHL-1, FH FH FH FH FHL-1, FH ⫹/⫺ ⫹/⫺
12 13 11 11 This study This study This study
a
NA, not available. TIGR, The Institute for Genomic Research. c Gene localization and description of the orthologous gene family were used according to the nomenclature of type strain B31 (7). d FH, factor H; ⫹/⫺, extremely weak binding to FHL-1. b
factor H was present in the eluate fractions of B. burgdorferi ZS7 but not in those of B. afzelii MMS and B. garinii ZQ1 (Fig. 7). Furthermore, factor H was found at high levels in the eluate of B. afzelii FEM1-D15 and B. burgdorferi LW2, indicating that both strains are capable of adsorbing factor H more efficiently. The lack of factor H in the eluate fractions of B. afzelii MMS and B. garinii ZQ1 could be explained by the absence of specific factor-H-binding CRASPs which are found to be expressed in FEM1-D15 (CRASP5Ba) and LW2 (CRASP-3Bb to CRASP-5Bb) (15, 16). DISCUSSION This study is the first account on the identification and functional characterization of the receptor(s) for complement regulators FHL-1 and factor H, CRASP-1Ba and CRASP-1Bg, of
members of the Lyme disease spirochetes B. afzelii and B. garinii, respectively. The data presented here demonstrate that CRASP-1Ba of B. afzelii MMS binds strongly to FHL-1 and weakly to factor H via its C terminus, similar to CRASP-1Bb of B. burgdorferi ZS7 (15, 16). In contrast, two corresponding CRASP-1 molecules from B. garinii ZQ1, CRASP-1Bg and CRASP-1Bg, were found to bind only weakly to FHL-1 and not at all factor H. Molecular analysis of the CRASP-1Ba-encoding gene cspA showed considerable sequence homology to other members of the gbb54 orthologous gene family, e.g., the CRASP-1Bb-encoding gene. Interspecies homology of the CRASP-1Bb and CRASP-1Ba sequences was about 48%. Due to the observed heterogeneity among charged and hydrophobic amino acids of CRASP-1Ba and CRASP-1Bb, folding of the mature proteins
FIG. 4. Alignment of deduced amino acid sequences of CRASP-1 orthologous proteins. The sequences of CRASP-1 from B. burgdorferi ZS7 (CRASP-1Bb), B. afzelii MMS (CRASP-1Ba), and B. garinii ZQ1 (CRASP-1Bg and CRASP-1Bg) strains were aligned with CLUSTAL. Gaps introduced to maximize alignments are indicated by dots. Identical amino acid residues in at least three of four sequences are marked by black shaded boxes. Putative factor-H-binding regions are indicated by grey boxes.
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FIG. 5. Mapping of the binding domains of factor H and FHL-1 for CRASP-1Ba. Purified recombinant CRASP-1Ba (lanes 1 to 10) was separated by 10% Tris-Tricine–SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with either recombinant FHL-1 (FH1-7) or several deletion constructs of factor H (FH1-2, FH1-3, FH1-4, FH1-5, FH1-6, FH8-20, FH15-20, or FH19-20) or with human serum (FH). Bound proteins were visualized using antisera specific for SCR1 to SCR4 (␣SCR1-4) and SCR20 of factor H (␣SCR20-V1G8). The size of the indicated binding molecule was derived from the mobility of marker proteins.
FIG. 7. Binding of complement regulator FHL-1 and factor H by different borrelial strains. Strains ZS7, MMS, ZQ1, FEM1-D15, and LW2 incubated in NHS-EDTA were extensively washed with PBSATween 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 by 10% Tris-Tricine–SDS-PAGE under nonreduced conditions, transferred to nitrocellulose, and probed with rabbit antiserum (␣SCR1-4) recognizing both FHL-1 and factor H.
may vary considerably and thus may cause conformational and, consequently, functional changes. Current investigations are aimed at the crystallographic analysis of the CRASP-1 orthologs of B. afzelii and B. burgdorferi to resolve this topic. As shown before for mature CRASP-1Ba (15), the recombinant form binds strongly to FHL-1 and less efficiently to factor
FIG. 6. Accessibility of proteases to native CRASP-1 proteins. Borrelial cells of B. burgdorferi ZS7, B. afzelii MMS, and B. garinii ZQ1 were incubated with either proteinase K or trypsin at concentrations of 12.5 to 100 g/ml or in buffer without any protease (0). After 1 h of incubation, cells were lysed by sonication as described in Materials and Methods. Each protein lysate was then subjected to 10% Tris-Tricine– SDS-PAGE, blotted to membranes, and analyzed by Western or ligand affinity blotting. CRASP-1Bb, CRASP-1Ba, and CRASP-1Bg were detected after incubation with recombinant FHL-1, and binding of the protein was visualized with antiserum (␣SCR1-4) specific for the Nterminal region of FHL-1. Intracellular Hsp70 and periplasmatic flagellin were detected with MAbs LA3 and LA22.1.
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H. In contrast, all other recombinant spirochetal proteins of the gbb54 orthologous gene family of B. afzelii MMS tested to this end, i.e., MMSA67, MMSA68, MMSA69, and MMSA70, showed no binding to FHL-1 and factor H at all. Ligand blot experiments revealed that the binding affinity of CRASP-1Ba for FHL-1 was higher than that of CRASP-1Bb, a finding supporting the observed differential susceptibility of B. afzelii and B. burgdorferi strains to human-complement-mediated lysis (15). The demonstration that removal of an 11-amino-acid domain from the C terminus of CRASP-1Ba inhibits the binding of FHL-1 and drastically reduces the binding of factor H is in line with previous experiments employing CRASP-1Bb (12) and indicates that the C terminus of CRASP-1Ba is critical for interaction with both complement regulators. Therefore, the C-terminal regions of CRASP-1 molecules derived from B. afzelii and B. burgdorferi may be particularly rewarding for further analysis of CRASP-1 and FHL-1–factor H interactions and for studies on amino acid sequence constraints in antigenic variants (unpublished data). Earlier studies on the binding of complement regulators to human pathogenic microorganisms (e.g., S. pyogenes, N. gonorrhoeae) provided evidence that the C-terminal regions of FHL-1 (SCR5 through SCR7) and factor H (SCR19 and SCR20) are predominantly involved in these interactions (8, 10, 28, 38). Using the deletion constructs of FHL-1 and factor H, we previously localized the binding domain for the native CRASP-1Ba to SCR5 through SCR7 (15, 23). FHL-1 and factor H, bound to either recombinant CRASP-1Bb or intact B. burgdorferi and B. afzelii strains mainly via SCR7, retain their cofactor activity for factor-I-mediated C3b inactivation (12) such that their potential to control the alternative pathway of complement activation is maintained. In addition, recombinant CRASP-1Ba, like its native counterpart, similarly binds FHL-1 and factor H via SCR7, thereby preventing complement activation. In contrast to the observed FHL-1–factor H interaction with the C termini of spirochetal CRASP-1 molecules, the two FHL-1-binding proteins M5 and M6 of S. pyogenes interact with their N-terminal hypervariable regions with FHL-1 (9).
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WALLICH ET AL.
Unfortunately, to this end, no further attempts have been made to characterize the sequence motif of the respective binding sites. However, the fact that the hypervariable regions of M5 and M6 proteins and possibly other M proteins are involved in the interaction with FHL-1 indicates that similar structures rather than similar sequence motifs are responsible for ligand binding (9). Complement-resistant B. afzelii strains, which readily express CRASP-1Ba, efficiently bind FHL-1 and factor H on their outer surface, whereas complement-sensitive B. garinii strains bind, if at all, only marginally to the two complement regulators (16). The potential of B. afzelii to bind FHL-1 and factor H directly correlates with their complement-resistant phenotype. Comparison of the FHL-1- and factor-H-binding efficacies of two B. burgdorferi strains, ZS7 and LW2, and two B. afzelii strains, MMS and FEM1-D15, revealed that individual strains acquire various amounts of FHL-1 and factor H on the cell surface, suggesting differential expression of CRASPs. Moreover, the fact that B. burgdorferi LW2 expresses up to five different CRASPs (CRASP-1Bb to CRASP-5Bb), B. afzelii FEM1-D15 and B. burgdorferi ZS7 express two CRASPs each (CRASP-1Ba, CRASP-5Ba and CRASP-1Bb, CRASP-3Bb, respectively), and B. afzelii MMS expresses only one CRASP (CRASP-1Ba) (15) suggests that the complement susceptibility of individual borrelial strains is regulated by the differential expression of various CRASPs with distinct binding properties. It has been demonstrated that CRASPs comprise three different groups according to their binding capability to complement regulators factor H and FHL-1 (11, 17). Group I proteins (e.g., CRASP-1Bb) are related to the gbb54 orthologous family and bind to both factor H and FHL-1, whereas group III proteins (e.g., CRASP-3Bb to CRASP-5Bb) are members of the polymorphic Erp protein family and bind to only factor H. Previous work indicated that serum-sensitive B. garinii strains lack CRASP molecules and are unable to bind FHL-1 or factor H for survival in the infected host (3, 4, 14, 15, 21, 33). However, the present study shows that B. garinii ZQ1 also expresses a protein homologous to CRASP-1Bb and CRASP-1Ba, termed CRASP-1Bg. However, when employing recombinant protein, binding of FHL-1 was rather low compared to that for CRASP-1Bb and CRASP-1Ba. Furthermore, interaction of recombinant B. garinii CRASP-1Bg and CRASP-1Bg with FHL-1 could only be observed in an enzyme-linked immunosorbent assay test system but not when the ligand affinity blot technique was employed. The finding that binding of FHL-1 or factor H could not be detected on the cell surface of B. garinii suggests that CRASP-1 either displays low-affinity binding to complement regulators and/or is only marginally exposed to the cell surface. It remains to be determined whether CRASP-1Bg is expressed and functionally active during mammalian infection. Previous studies using B. afzelii FEM1 showed the up-regulation of CRASP-1Ba expression when low-passage spirochetes were cultured at 37°C, suggesting that increased expression of CRASP-1 molecules may be particularly relevant in maintaining bacterial integrity during infection and adaptation to the human host (15). Borrelia spirochetes are maintained in nature by tick-vertebrate transmission cycles, and their resistance to complement seems to match the known reservoir status of a wide variety of potential hosts. Thus, persistence of spirochetes seems to be
INFECT. IMMUN.
restricted by the genetic background of spirochetes, including the expression of CRASP-1, as well as the quality and source of complement (20, 21). One could speculate that during the coevolution of B. burgdorferi and vector-mammalian hosts, the gbb54 orthologous gene family is generated by gene duplication to cope with the innate immunity, including complement, of the various reservoir hosts. Consequently, multiple CRASPs expressed on the spirochetal surface may ensure the survival of a distinct Borrelia strain in different animal hosts. In line with this assumption, it was shown that the Erp proteins display different binding affinities for factor H derived from various animal hosts, suggesting that the temporal expression of multiple Erp proteins conveys resistance to complement-mediated killing in a wide range of vertebrate hosts (25, 32). In conclusion, the characterization of CRASP-1Ba as a novel candidate virulence factor of Borrelia critical for the evasion of innate immunity increases our knowledge of the persistence and pathogenesis of Lyme borreliosis and may help to develop further strategies to prevent and treat B. burgdorferi infections. ACKNOWLEDGMENTS We gratefully acknowledge the expert technical assistance of Juri Habicht, Christa Hanssen-Hu ¨bner, and Andrea Ho ¨nes. This work was funded by the Thu ¨ringer Ministerium fu ¨r Wissenschaft, Forschung und Kunst, and the Deutsche Forschungsgemeinschaft, project Zi 432/5 and Br 446/11-3. REFERENCES 1. Alitalo, A., T. Meri, H. Lankinen, I. Seppa ¨la ¨, P. Lahdenne, P. S. Hefty, D. Akins, and S. Meri. 2002. Complement inhibitor factor H binding to Lyme disease spirochetes is mediated by inducible expression of multiple plasmidencoded outer surface protein E paralogs. J. Immunol. 169:3847–3853. 2. Alitalo, A., T. Meri, L. Ra ¨mo ¨, T. S. Jokiranta, T. Heikkila ¨, I. J. T. Seppa ¨la ¨, J. Oksi, M. Viljanen, and S. Meri. 2001. Complement evasion by Borrelia burgdorferi: serum-resistant strains promote C3b inactivation. Infect. Immun. 69:3685–3691. 3. Brade, V., I. Kleber, and G. Acker. 1992. Differences of two Borrelia burgdorferi strains in complement activation and serum resistance. Immunobiology 185:453–465. 4. Breitner-Ruddock, S., R. Wurzner, J. Schulze, and V. Brade. 1997. Heterogeneity in the complement-dependent bacteriolysis within the species of Borrelia burgdorferi. Med. Microbiol. Immunol. 185:253–260. 5. China, B., M. P. Sory, B. T. NⴕGuyen, M. De Bruyere, and G. R. Cornelis. 1993. Role of the YadA protein in prevention of opsonization of Yersinia enterocolitica by C3b molecules. Infect. Immun. 61:3129–3136. 6. Diaz, A., A. Ferreira, and R. B. Sim. 1997. Complement evasion by Echinococcus granulosus: sequestration of host factor H in the hydatid cyst wall. J. Immunol. 158:3779–3786. 7. Fraser, C. M., S. Casjens, W. M. Huang, G. G. Sutton, R. Clayton, R. Lathigra, O. White, K. A. Ketchum, R. Dodson, E. K. Hickey, M. Gwinn, B. Dougherty, J. F. Tomb, R. D. Fleischmann, D. Richardson, J. Peterson, A. R. Kerlavage, J. Quackenbush, S. Salzberg, M. Hanson, R. van Vugt, N. Palmer, M. D. Adams, J. Gocayne, J. Weidman, T. Utterback, L. Watthey, L. McDonald, P. Artiach, C. Bowman, S. Garland, C. Fujii, M. D. Cotton, K. Horst, K. Roberts, B. Hatch, H. O. Smith, and J. C. Venter. 1997. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390: 580–586. 8. Hellwage, J., T. Meri, T. Heikkila, A. Alitalo, J. Panelius, P. Lahdenne, I. J. Seppa ¨la ¨, and S. Meri. 2001. The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi. J. Biol. Chem. 276:8427–8435. 9. Johnsson, E., K. Berggard, H. Kotarsky, J. Hellwage, P. F. Zipfel, U. Sjobring, and G. Lindahl. 1998. Role of the hypervariable region in streptococcal M proteins: binding of a human complement inhibitor. J. Immunol. 161:4894–4901. 10. Kotarsky, H., J. Hellwage, E. Johnsson, C. Skerka, H. G. Svensson, G. Lindahl, U. Sjobring, and P. F. Zipfel. 1998. Identification of a domain in human factor H and factor H-like protein-1 required for the interaction with streptococcal M proteins. J. Immunol. 160:3349–3354. 11. Kraiczy, P., K. Hartmann, J. Hellwage, C. Skerka, M. Kirschfink, V. Brade, P. F. Zipfel, R. Wallich, and B. Stevenson. 2004. Immunological characterization of the complement regulator factor H-binding CRASP and Erp proteins of Borrelia burgdorferi. Int. J. Med. Microbiol. 293(Suppl. 37):152– 157.
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