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* Department of Bacteriology and Immunology, Haartman Institute and Helsinki University Central Hospital, and
Peptide and Protein Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland; and
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20 years ago (1). A wealth of knowledge on the spirochete has accumulated during the last decades (2, 3). There are three major human-pathogenic genospecies of B. burgdorferi, which differ in their geographic distribution as well as in their most common clinical manifestations (4). Of these, only B. burgdorferi sensu stricto has been found in North America, while in Europe the most common genospecies are Borrelia garinii and Borrelia afzelii. Lyme borreliosis can be treated with antibiotics, but in chronic conditions the treatment may be ineffective. Despite initial attempts (5), no vaccine is currently in use in humans against B. burgdorferi. Borrelia spirochetes can persist in the human body for prolonged periods, even for decades, as shown in patients with acrodermatitis chronica atrophicans (ACA)3 lesions (6). In some patients with chronic disease, immune or other less well-characterized mechanisms are the cause of the symptoms. For example, in PCR-positive and culture-negative ACA lesions, live bacteria are scanty or not present, but nevertheless the symptoms prevail (7). In other long-term disease manifestations, immune mechanisms are common even without detectable spirochetes. For example, both PCR-positive and -negative Lyme arthritis have been described (8). In borreliosis, there is a clear predisposition toward typical clinical manifestations with a particular genospecies. Thus, B. afzelii is the most common finding in ACA, Borrelia garinii in neuroborreliosis, and B. burgdorferi in arthritis. Considering the ability of the pathogen to persist in the human body, it is obvious that B. burgdorferi spirochetes must have developed efficient mechanisms for evading the innate as well as the adaptive immune responses. As one key mechanism, we have described the binding of the complement system inhibitors factor H (FH) and FH-like protein 1 (FHL-1) by Borrelia strains that are resistant to serum killing (9, 10). The importance of FH binding to Borrelia has been documented also in studies by Kraiczy et al. (11), Stevenson et al. (12), and McDowell et al. (13, 14).
FH binding is a complement and phagocytosis evasion mechanism used by several different bacteria, e.g., by pathogenic streptococci (15, 16), pneumococci (17, 18), and group B streptococci (19). According to the current understanding, B. burgdorferi spirochetes use several immune evasion mechanisms. Serum resistance in Borrelia has been shown to be mediated by FH binding to the outer surface protein E (OspE) and its orthologs, such as P21, which is a surface lipoprotein encoded by the cp32 plasmid in the B. burgdorferi sensu stricto 297 strain (10, 13, 14, 20, 21). In addition, another group of FH-binding proteins with apparent molecular mass ranging from 27.5 kDa (13, 14, 21, 22) to 35 kDa (9) has been described.
In its enzootic cycle, the B. burgdorferi spirochete infects various mammals (such as mice, voles, and deer), and ticks are the vectors that transmit the bacteria from one host to another. Stevenson et al. (12) observed FH binding to borrelial surface molecules from the sera of different mammals. We originally observed that both human and rabbit FH bound to Borrelia (9). FH binding to B. burgdorferi inhibits the alternative pathway of complement activation and promotes C3b inactivation on the surface of the bacteria (9, 10, 14, 22). The expression of the binding target OspE protein on Borreliae is induced following a blood meal by the ticks. There are numerous plasmids in the B. burgdorferi strains 297 and B31, both of which encode sets of different, but often homologous Osps. In strain 297, genes in two distinct circular cp32 plasmids encode OspE-related proteins, OspE and P21 (23). Thus, multiple Osps on a single Borrelia strain could interact with FH (9, 11, 22). OspE has been shown to become up-regulated in the mammalian host when the temperature changes from 23°C to 37°C or when the spirochetes get into contact with host-associated factors (24, 25, 26, 27, 28). This could be particularly relevant in rodents, which are a major reservoir of Borreliae in nature. The consequent serum resistance seems to be an important factor in the ecology of Borreliae (29).
Because of the importance of the FH binding to Borreliae, we have, in this study, examined in more detail the interactions between FH and OspE by deletion mutagenesis, peptide mapping, and alanine scanning. We have located the OspE binding site on both human and mouse FH. OspE was found to bind via multiple sites to the C terminus (short consensus repeats (SCRs) 18–20) of both human and mouse FH. The binding required lysine residues on OspE. In addition, binding was found to another FH-related plasma protein, FH-related protein 1 (FHR-1), which shows homology in its C terminus to FH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The GST fusion proteins of OspE from the B. burgdorferi sensu stricto strain N40 were generated by cloning PCR-amplified products into the p-GEX-2T vector, as previously described (10). Subsequently, a GST-OspE pulldown technique (30) was used to identify potential Borrelia OspE-interacting proteins in human serum. For this, both normal human serum and serum from a volunteer deficient in FHR-1 (FHR-1DS) were used. One-milliliter serum samples were incubated with 50 µl of 50% slurry of glutathione-Sepharose 4B (Amersham Biosciences, Freiburg, Germany) beads for 2 h at 4°C with gentle mixing and then centrifuged (20,800 x g, 2 min) at 4°C to collect the supernatants. One-milliliter portions of the precleared sera (at dilutions 1/2 and 1/10) were mixed with 50 µl of glutathione-Sepharose. A total of 10 µg of GST was added to the control tube, and 16 µg of GST-OspE was added to the other tubes, followed by gentle mixing for 2 h at 4°C. The beads were washed three times with PBS and the GST-OspE protein, and any proteins bound to it were eluted by adding 50 µl of 20 mM reduced glutathione. After centrifugation, the supernatants containing the eluted proteins were collected for further analysis.
SDS-PAGE and Western blot analysis
The various eluted fractions from the GST-OspE pulldown experiment were subjected to SDS-PAGE through 4% stacking and 10% separating gels. For Western blot analysis, proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% (w/v) dried milk in PBS for 30 min and incubated at room temperature for 1 h with polyclonal goat anti-human FH Ab (Calbiochem, La Jolla, CA; diluted 1/5000). This Ab also recognizes human FHL-1 and the family of FHRs (FHR-1 to FHR-5). A donkey anti-goat IgG Ab (Jackson ImmunoResearch Laboratories, West Grove, PA; diluted 1/5000) was used as the secondary Ab.
Generation and purification of P21 deletion mutants and synthesis of C-terminal peptides
P21 is an OspE paralog from the B. burgdorferi sensu stricto 297 strain. The truncated versions of P21 lacking the N terminus (P21
1–35) or C-terminal regions of varying lengths (P21105–166, P21143–166, and P21152–166) were made initially as GST fusion proteins (Fig. 1). The mutant P21 constructs were generated by directionally cloning PCR-amplified products into the BamHI and SalI cloning sites of pGEX-4T-2 vector using primers P211–35, 5'-GCG GAT CCA AAT TTA CTG TAA AAA TTA AAA-3'; P21–3', 5'-GCC GTC GAC TTT TAA ATT TTT TTT AAG ATC-3'; P21–5', 5'-GCG GAT CCT GCA AAA TTC ATA CTT CAT ATG-3'; P21105–166-3', 5'-GCC GTC GAC CTC ATC AGT TTT AAA CGA ACC-3'; P21143–166-3', 5'-GCC GTC GAC ATT TCC TGA AAA TGT AAT ATA-3'; and P21152–166-3', 5'-GCC GTC GAC AAC TTT ATC CCC GGA ATC C-3' (sites added for cloning are underlined). All resulting fusion constructs were subjected to sequence analysis to verify that no mutations were introduced by the PCR amplification step. The full-length GST fusion proteins were produced, purified, and cleaved free of the GST moiety, as previously described (31, 32). C-terminal peptides from OspE-297, OspEF-related protein A (ErpA)-B31, and ErpP-B31, as well as a scrambled version of ErpP-B31 were synthesized at the Oklahoma University Health Sciences Center core facility. The N termini of peptides were modified with 
-aminobutyric acid (GABA) by using a derivatized fMOC for the final addition during peptide synthesis.
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The C-terminal parts of mouse and human FH containing SCR domains 18–20 were generated as follows. DNA coding for SCR18–20 of murine and human FH was amplified by PCR using the clone containing full-length cDNA of mouse FH (Z. Cheng, J. Hellwage, H. Seeberger, P. Zipfel, S. Men, and S. Jokiranta, manuscript in preparation) and human liver cDNA library (Stratagene, La Jolla, CA), respectively. The primers for the mouse FH fragment were 5'-GGA TTG GAC CCT GCA GAA GCA AAG ATA A-3' (forward, restriction site underlined) and 5'-GTC GAC ATG ATT TTG TAC ACA AGT GGG-3' (reverse), and the primers for human FH fragment were 5'-GAA TTC AAA GAG ACA CCT CCT GTG TG-3' (forward) and 5'-CCG CGG CTA TCT TTT TGC ACA AGT TGG-3' (reverse). The purified PCR products were cloned into expression vectors pPICZ
A and pPICZB (Invitrogen, Carlsbad, CA) for mouse and human FH, respectively, to generate a sequence encoding SCR18–20 of mouse FH, followed by a polyhistidine (His-6) tag and a sequence encoding SCR18–20 of human FH. The resulting plasmid constructs were linearized and transformed into Pichia pastoris strain X33 by electroporation (Bio-Rad Gene Pulser II; Bio-Rad, Hercules, CA). The transformed cells were grown on yeast extract peptone dextrose medium plates containing Zeocin (100 µg/ml; Cayla, Toulouse, France) at 30°C for 3 days, and the clones were selected according to the manufacturer’s protocol. The recombinant proteins were expressed in buffered methanol-complex medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 4 x 10–5% biotin, 1% methanol) with a daily addition of methanol (1% v/v) before harvesting. Human FH SCR19–20 fragment was generated similarly as the SCR18–20 fragment using the following forward primer: 5'-GA ATT CAA AAA GAT TCT ACA GGA AAA TGT G-3'.
The expressed mouse FH SCR18–20 recombinant protein was purified using Ni2+ NTA agarose (Invitrogen), according to the manufacturer's protocol. Further purification was performed by heparin affinity chromatography (Pharmacia, Uppsala, Sweden) using Hitrap heparin column and elution with a salt gradient from 50 to 200 mM. Purity of the recombinant proteins was examined by SDS-PAGE and found to be >95% (mouse factor H (moH) 18–20 and human factor H (huH) 18–20 fragments were expressed as glycosylated forms). The antigenicity of the fragments was verified by immunoblotting analysis using goat anti-human FH antiserum (Calbiochem). The protein concentrations were determined by bicinchoninic acid protein assay (Pierce, Rockford, IL).
Surface plasmon resonance
Surface plasmon resonance-binding analyses were conducted using the Biacore 2000 biosensor instrument (Biacore, Uppsala, Sweden). Purified mutant P21–297 proteins (5.6 µM) were amine coupled to carboxyl groups of carboxymethylated dextran (CM5) sensor chips, according to the manufacturer’s instructions. The buffer was chosen according to the pI of the analyte: maleate and sodium acetate buffers were used for proteins whose calculated pI values were over or below 6, respectively. The coupling levels of proteins were: 2468, 9284, 2293, 3403, 1990, and 7439 resonance units for P21 (Figs. 2 and 3A); P21152–166, P21143–166, and P21 (Figs. 3B and 4); and P211–35 and P21105–166, respectively. The binding of complement FH (Calbiochem; purity >95% by SDS-PAGE) to immobilized proteins was assayed at 25°C in 50 mM veronal-buffered saline. Similar binding tests were performed with the biotin-GABA-linked C-terminal peptides of OspE proteins. The peptides were coupled to a streptavidin chip and tested for FH-binding ability. The binding of recombinant human FH fragments huH18–20 (0.044 mM) and huH19–20 (0.10 mM) and the recombinant mouse FH fragment moH18–20 (0.061 mM) to P21–297 was analyzed similarly using veronal-buffered saline as a buffer. The bound proteins were removed with a regeneration buffer containing 3.0 M NaCl (pH 4.7). To see whether binding of FH could be inhibited by heparin, FH (300 nM) binding to P21 was assayed in the presence of 0.3 or 3.0 µM heparin 17/19 kDa (Sigma-Aldrich, St. Louis, MO). Similar inhibition analyses were performed with the deletion mutants of P21 and soluble C-terminal P21 peptides (see below).
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The OspE-related amino acid sequences chosen for peptide-scanning analysis from the National Center for Biotechnology Information protein sequence databank (http://www.ncbi.nlm.nih.gov) were OspE-N40 (AAA22959) (33), OspE-297 (AAC34953) (31), P21–297 (AAC34957) (31), and ErpA-B31 (AAF07400) (34). Amino acid sequences of the tested proteins were aligned using the Megalign and ClustalX programs. Sequences from the five putative FH binding regions (20) were chosen from ErpA and from the proteins giving the strongest FH-binding signal strength for each region. In reanalysis of region II, however, the binding of FH was weak or absent. Each amino acid in the selected sequences was changed to alanine, one at a time, and the synthesized peptides were 15–17 aa long. The peptides were synthesized as spots onto polyethylene glycol-derivatized cellulose membranes (AIMS Scientific Products, Braunschweig, Germany) using the peptide-scanning instrument AutoSpot Robot ASP222 (Abimed Analysen-Technik, Langenfeld, Germany). Subsequently, a protein overlay assay with radiolabeled FH (9, 35) was conducted. The membranes were washed and exposed on an x-ray film (Fuji Photo Film, Tokyo, Japan).
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Results |
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FH from multiple animal species binds to the borrelial OspE (9, 12, 29). Thus, we wanted to localize the borrelial OspE binding sites on both human and mouse FH. As a representative of the OspE family of proteins, we used the OspE paralog protein P21 that is encoded by the B. burgdorferi sensu stricto strain 297. Because our earlier studies suggested a binding site in the SCR15–20 region (10), we first tested binding of human FH constructs containing SCRs 18–20 or 19–20 to surface-coupled P21 using the surface plasmon resonance technique. A human FH construct containing SCRs 18–20 bound to the P21 protein with a high affinity (Fig. 2A). For the shorter construct huH19–20, both P21 association and dissociation occurred more slowly (Fig. 2A), indicating that the FH19–20 fragment remained strongly bound to P21. Additional experiments showed that the mouse C-terminal FH construct moH18–20 also binds to P21–297 (Fig. 2B).
Binding of FHR-1 to OspE
Because human blood plasma contains several FHR proteins (FHR-1, -2, -3, -4, and -5) whose C-terminal SCRs resemble those of FH, we analyzed whether OspE could bind to serum proteins other than FH. To identify potential OspE-interacting proteins, normal human serum (1/2 or 1/10 dilution) and FHR-1DS were first precleared from any GST-binding proteins and thereafter incubated with the GST-OspE fusion protein and glutathione-Sepharose beads. After extensive washing of the beads, the GST-OspE-bound proteins were eluted, separated by SDS-PAGE, and analyzed by silver staining and Western blotting. The eluted proteins were detected as bands 40 and 155 kDa by silver staining. Western blotting with a polyclonal goat anti-human FH Ab was used to further characterize the eluted proteins. As a control, purified FH (Fig. 5, lane 1) was separated in the same gel. As shown in Fig. 5 (lanes 2 and 3), FHR-1 (37 kDa), FHR-1
(43 kDa), and FH (155 kDa) were detected among the GST-OspE-bound proteins based on their typical mobility and because they were not present in the FHR-1DS sample (Fig. 5, lane 4). The binding interactions were specific because FH, FHR-1, and FHR-1 were not present among GST-bound proteins (data not shown). The binding of FHR-1 to OspE is probably due to the three most C-terminal domains (SCRs 3, 4, and 5) of FHR-1, which are very homologous (100, 100, and 97%, respectively) to FH SCR domains 18, 19, and 20. The finding of interaction between FHR-1 and OspE suggests that Borreliae also use FHR-1 for their protection.
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We wanted to define the FH sites necessary for OspE binding among the five putative sites previously characterized by peptide mapping (20). We therefore constructed N- or C-terminal deletions of varying lengths that are shown in Fig. 1 and tested FH binding of the mutant proteins in Biacore analysis. The P21–297 mutants were coupled to the sensor chip, and human FH was used as the fluid-phase ligand. Of the mutants, P211–35 showed a virtually identical FH binding to the full-length P21 (Kd 8.4 nM; Fig. 3A), indicating that the N-terminal amino acid residues 1–35 of P21 are not required for FH binding. In contrast, the mutant P21105–166 showed no binding of FH (Fig. 3A), while the mutant P21143–166 as well as the deletion mutant P21152–166 that lacked only 15 C-terminal residues had only a minimal ability to bind FH (Fig. 3B).
Next, we tested whether the shortest FH constructs with OspE-binding activity would bind to the C-terminally truncated P21 proteins. As shown in Fig. 4, some binding of the FH constructs SCR18–20 and SCR19–20 occurred to the C-terminally truncated mutant P21 proteins P21143–166 and P21152–166, although the binding was found to be significantly reduced when compared with full-length P21. This indicated that region V, consisting of the C-terminal 15 aa residues, is critical for FH binding. Importantly, however, this does not exclude the possibility that additional sites contribute to the binding. This is because weak residual binding of FH and of the huH18–20 and huH19–20 fragments was observed to the C-terminally truncated mutants of P21 (Figs. 3 and 4, A and B).
FH binding by C-terminal peptides
Because analysis of the deletion mutants suggested that the C termini of OspE proteins are important for FH binding, we analyzed whether surface-associated peptides representing the C terminus of OspE are sufficient for the binding. However, biotin-GABA-coupled peptides synthesized from the C termini of OspE paralogs OspE-297, P21–297, and ErpP-B31 did not bind FH in Biacore analysis. Also, soluble forms of the individual peptides did not inhibit the binding of FH to OspE (data not shown). This suggested that either the peptides did not adopt the correct conformation in solution or that the binding requires multiple binding sites on P21 for FH.
Inhibition of FH binding to P21 by truncated mutants of P21 and by heparin
In the next experiment, fluid-phase P21 and mutants thereof were tested for their ability to inhibit FH binding to surface-coupled P21. For comparison, heparin was tested as a control. As shown in Fig. 6A, inhibition of FH binding was seen with P21 and its N-terminal mutant, but not with the mutants with deletions in the C terminus. This further confirmed that the C-terminal binding site in P21 was the most essential one for FH binding. Heparin (Fig. 6B) inhibited binding to FH already at an equimolar concentration (300 nM), which is in agreement with the ionic nature of the interaction between P21 and FH. On FH, a heparin binding site is located in SCR20 (36). Thus, binding of heparin to SCR20 could hinder the interaction between SCR18–20 and FH. In contrast, heparin could also bind to positively charged residues in P21. To test whether basic (or other) residues in the C terminus of P21 could be involved in binding FH, we next synthesized peptides with changes in individual residues.
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Because the C-terminal OspE peptides immobilized on cellulose membranes bound FH, we analyzed the characteristics of FH binding to OspE by performing an alanine-scanning analysis on peptides from all the five putative binding domains of OspE. To scan for potentially important amino acids of OspE in its interaction with FH, Ala was substituted for each amino acid of each of the regions I-V separately in peptides synthesized on a cellulose membrane. Radiolabeled FH was applied on the membrane, and binding was detected using autoradiography. Fig. 7 shows binding of 125I-labeled FH to the peptides and the effects of the amino acid replacements. In this assay, FH bound to peptides I, III, IV, and V. The replacement of any of the three lysines (Lys162, Lys163, and Lys166) in peptide V completely abolished FH binding (Fig. 7), indicating that these residues were crucial for FH binding (Fig. 7). Similarly, the replacement of two (of three) lysine residues in peptide IV (Lys143 and Lys145) inhibited FH binding. Lysines appeared to contribute to binding also in peptide III and weakly in peptide I, while other amino acid replacements had no appreciable effects on FH binding. In conclusion, in this assay, lysine residues, particularly those close to the C terminus of OspE, constituted the FH-binding determinants.
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Discussion |
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Borreliae are able to infect a number of mammalian species in nature. In addition to human FH, mouse and rabbit FH also have been shown to bind to borrelial OspE. We observed binding of human and mouse FH constructs containing only the most C-terminal SCRs to P21–297. Thus, it is likely that human and rodent FH bind to OspE proteins in an analogous manner. Human FH SCRs 18–20 bound to P21–297 with kinetics similar to whole FH, suggesting that most, if not all, binding activity of FH is contained within the SCR18–20 region.
In addition to FH, another plasma protein of the FH family, FHR-1 was found to bind to OspE. FHR-1 was identified by the typical two-polypeptide band pattern (FHR-1 and FHR-1), m.w., and reactivity with a polyclonal anti-FH Ab. It is likely that FHR-1 interacts with OspE with its SCR domains 3–5, which are homologous to FH SCRs 18–20. The fact that no other FHR proteins could be detected in the OspE-Sepharose absorption test could be due to their low concentrations in plasma or to a more limited homology of their C termini with FH. The functions of FHR-1 are still not certain, but it has been shown to bind to C3b and to plasma lipoproteins. One possibility is that by binding to C3b, the FHR-1 protein (37) can inhibit the binding of factor B to C3b and thus formation of the alternative pathway C3-convertase C3bBb. The conservation of homologous C termini in FH and FHR-1, and possibly in other FHR proteins as well, suggests an important function for this region.
Mapping of the FH binding site on OspE should be important, e.g., for any future attempts to interfere with the FH-OspE interaction by immunization. Deletion mutants of the representative OspE protein P21 showed varying degrees of FH binding, with the binding strength generally decreasing with longer deletions at the C terminus. In contrast, an N-terminal deletion did not decrease the binding affinity, indicating that: 1) not just any deletion in rOspE protein abolishes FH-binding activity, and 2) the most N-terminal part (residues 1–35) in P21 does not affect the overall conformation of OspE to an extent that would influence FH binding. Interestingly, Metts et al. (21) made a longer N-terminal deletion (45 aa with P21 as the reference sequence in an alignment with P21, ErpA, and ErpP) to the OspE proteins ErpA and ErpP from the B31M1 strain of B. burgdorferi and found loss of FH binding. This suggests that an FH binding site or an OspE conformation-determining region is located roughly in the area between aa 35 and 46 (according to sequence numbering of P21).
The 15 most C-terminal residues containing the region V were found to be essential for the binding activity of the whole OspE protein. However, C-terminal peptides containing the region V coupled to a Biacore chip with a biotin-GABA linker did not show FH binding, and soluble peptides representing this region did not inhibit FH binding to OspE. These results suggest that region V is not sufficient for binding. In the Biacore experiments, weak binding of deletion constructs containing sites I-III and I-IV, respectively, to FH and its fragments SCR18–20 and 19–20 was observed, suggesting that domains III and IV are involved in the binding. Binding of OspE to the SCRs 18–20 of FH may involve multiple interactions in the III-V region to account for the high affinity between OspE and FH. Our data thus agree with the proposal by Metts et al. (21) that the interaction of OspE with FH depends on conformational or discontinuous epitopes.
The alanine scanning identified a key role for lysines in interactions of the binding regions of OspE-related proteins with FH. This is consistent with the ionic nature of the binding revealed by the Biacore experiments and by heparin inhibition of the interaction. The positive charges of the lysines apparently contribute to the binding interaction. These could involve not only lysine residues in regions III-V, but the Lys41 and Lys43 residues in the N-terminal part close to region I. This part contains the sequence KIKNK that was deleted from the nonfunctional N-terminal mutants of Metts et al. (21), but not from our P211–35 mutant that was functionally active. Because lysines are relatively common in the Borrelia proteome, constituting 10% of the whole amino acid content, it is not surprising that they are used for important functions. Although lysines turned out to contribute to FH binding in our peptide analyses, the results do not exclude a role for other Borrelia peptides that could have an equal or even higher affinity for FH.
In contrast to the OspE family proteins, the 35-kDa FH-binding protein on B. burgdorferi was found to bind preferentially the FHL-1 protein (9), which is the product of an alternatively spliced FH gene. FHL-1 contains only the seven N-terminal SCR domains of FH, indicating that the main binding site of the 35-kDa protein is in the N-terminal part of FH. This would be analogous to the interaction of a 27- to 27.5-kDa complement regulator acquiring surface protein-1 (Bba68) with SCR domains 5–7 (22, 38). McDowell et al. (13, 14) reported various FH-binding proteins in B. burgdorferi strains (16–31 kDa, classes I and II), B. afzelii strains (18–33 kDa), and selected strains of B. garinii (20–28 kDa), as well as in other genospecies of B. burgdorferi sensu lato (19–28 kDa). Thus, in addition to OspE, another family of FH-binding proteins exists, and further work is needed to define their relative importance. It is interesting to note that the cp32 plasmid carrying genes for OspE paralogs contains a set of tightly packed genes that together constitute a late operon typical for bacteriophages (34, 39, 40, 41, 42). Horizontal transfer of OspE genes in phages would allow transfer of C resistance genes between different Borreliae strains. This may also be related to the widespread nature of Borreliae in nature, e.g., in rodents.
In this study, we were able to show that both human and mouse FH interact with OspE-related proteins, especially P21, with their C-terminal SCR domains 18–20. Binding requires multiple sites and possibly a distinct conformation, and lysine residues on P21. The C-terminal region V is important for the binding interaction: when it is removed from P21, binding to FH is nearly abolished. The advantages of knowing the FH binding regions in OspE proteins include the potential possibility to immunize model animals with peptides covering the FH binding sites of OspE. This is especially important, because Abs in mice immunized with full-length OspE are directed against the V region of OspE, which is not required for FH binding (21). The strong binding of FH to OspE could thus have prevented Ab formation against the binding sites. Alternatively, conformationally altered forms of OspE could perhaps be used for immunization. We hope that such strategies could lead to the generation of Abs that block the FH-OspE interaction and thereby promote killing of Borreliae.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Seppo Meri, Department of Bacteriology and Immunology, P.O. Box 21, Haartmaninkatu 3, FIN-00014 University of Helsinki, Helsinki, Finland. E-mail address: seppo.meri{at}helsinki.fi
3 Abbreviations used in this paper: ACA, acrodermatitis chronica atrophicans; CM5, carboxymethylated dextran; Erp, OspEF-related protein; FH, factor H; FHL-1, FH-like protein 1; FHR-1, FH-related protein 1; FHR-1DS, FHR-1-deficient serum; GABA, -aminobutyric acid; Osp, outer surface protein; SCR, short consensus repeat; moH, mouse factor H; huH, human factor H.
Received for publication January 5, 2004. Accepted for publication March 3, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein has separate binding sites for human factor H and IgA-Fc. J. Biol. Chem. 277:12642.This article has been cited by other articles:
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  A. Seling, C. Siegel, V. Fingerle, B. L. Jutras, C. A. Brissette, C. Skerka, R. Wallich, P. F. Zipfel, B. Stevenson, and P. Kraiczy Functional Characterization of Borrelia spielmanii Outer Surface Proteins That Interact with Distinct Members of the Human Factor H Protein Family and with Plasminogen Infect. Immun., January 1, 2010; 78(1): 39 - 48. [Abstract] [Full Text] [PDF]
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M. R. Kenedy, S. R. Vuppala, C. Siegel, P. Kraiczy, and D. R. Akins CspA-Mediated Binding of Human Factor H Inhibits Complement Deposition and Confers Serum Resistance in Borrelia burgdorferi Infect. Immun., July 1, 2009; 77(7): 2773 - 2782. [Abstract] [Full Text] [PDF]
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J. V. McDowell, B. Huang, J. C. Fenno, and R. T. Marconi Analysis of a Unique Interaction between the Complement Regulatory Protein Factor H and the Periodontal Pathogen Treponema denticola Infect. Immun., April 1, 2009; 77(4): 1417 - 1425. [Abstract] [Full Text] [PDF]
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 C. Siegel, J. Schreiber, K. Haupt, C. Skerka, V. Brade, M. M. Simon, B. Stevenson, R. Wallich, P. F. Zipfel, and P. Kraiczy Deciphering the Ligand-binding Sites in the Borrelia burgdorferi Complement Regulator-acquiring Surface Protein 2 Required for Interactions with the Human Immune Regulators Factor H and Factor H-like Protein 1 J. Biol. Chem., December 12, 2008; 283(50): 34855 - 34863. [Abstract] [Full Text] [PDF]
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K. M. Hovis, J. C. Freedman, H. Zhang, J. L. Forbes, and R. T. Marconi Identification of an Antiparallel Coiled-Coil/Loop Domain Required for Ligand Binding by the Borrelia hermsii FhbA Protein: Additional Evidence for the Role of FhbA in the Host-Pathogen Interaction Infect. Immun., May 1, 2008; 76(5): 2113 - 2122. [Abstract] [Full Text] [PDF]
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 A. K. Zaiss, M. J. Cotter, L. R. White, S. A. Clark, N. C. W. Wong, V. M. Holers, J. S. Bartlett, and D. A. Muruve Complement Is an Essential Component of the Immune Response to Adeno-Associated Virus Vectors J. Virol., March 15, 2008; 82(6): 2727 - 2740. [Abstract] [Full Text] [PDF]
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E. A. Rogers and R. T. Marconi Delineation of Species-Specific Binding Properties of the CspZ Protein (BBH06) of Lyme Disease Spirochetes: Evidence for New Contributions to the Pathogenesis of Borrelia spp. Infect. Immun., November 1, 2007; 75(11): 5272 - 5281. [Abstract] [Full Text] [PDF]
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T. Bykowski, M. E. Woodman, A. E. Cooley, C. A. Brissette, V. Brade, R. Wallich, P. Kraiczy, and B. Stevenson Coordinated Expression of Borrelia burgdorferi Complement Regulator-Acquiring Surface Proteins during the Lyme Disease Spirochete's Mammal-Tick Infection Cycle Infect. Immun., September 1, 2007; 75(9): 4227 - 4236. [Abstract] [Full Text] [PDF]
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M. E. Woodman, A. E. Cooley, J. C. Miller, J. J. Lazarus, K. Tucker, T. Bykowski, M. Botto, J. Hellwage, R. M. Wooten, and B. Stevenson Borrelia burgdorferi Binding of Host Complement Regulator Factor H Is Not Required for Efficient Mammalian Infection Infect. Immun., June 1, 2007; 75(6): 3131 - 3139. [Abstract] [Full Text] [PDF]
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 K. M. Chung, M. K. Liszewski, G. Nybakken, A. E. Davis, R. R. Townsend, D. H. Fremont, J. P. Atkinson, and M. S. Diamond From the Cover: West Nile virus nonstructural protein NS1 inhibits complement activation by binding the regulatory protein factor H PNAS, December 12, 2006; 103(50): 19111 - 19116. [Abstract] [Full Text] [PDF]
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L. Lu, Y. Ma, and J.-R. Zhang Streptococcus pneumoniae Recruits Complement Factor H through the Amino Terminus of CbpA J. Biol. Chem., June 2, 2006; 281(22): 15464 - 15474. [Abstract] [Full Text] [PDF]
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K. M. Hovis, J. P. Jones, T. Sadlon, G. Raval, D. L. Gordon, and R. T. Marconi Molecular Analyses of the Interaction of Borrelia hermsii FhbA with the Complement Regulatory Proteins Factor H and Factor H-Like Protein 1 Infect. Immun., April 1, 2006; 74(4): 2007 - 2014. [Abstract] [Full Text] [PDF]
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K. M. Hovis, E. Tran, C. M. Sundy, E. Buckles, J. V. McDowell, and R. T. Marconi Selective Binding of Borrelia burgdorferi OspE Paralogs to Factor H and Serum Proteins from Diverse Animals: Possible Expansion of the Role of OspE in Lyme Disease Pathogenesis Infect. Immun., March 1, 2006; 74(3): 1967 - 1972. [Abstract] [Full Text] [PDF]
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J. V. McDowell, J. Lankford, L. Stamm, T. Sadlon, D. L. Gordon, and R. T. Marconi Demonstration of Factor H-Like Protein 1 Binding to Treponema denticola, a Pathogen Associated with Periodontal Disease in Humans Infect. Immun., November 1, 2005; 73(11): 7126 - 7132. [Abstract] [Full Text] [PDF]
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 C. S. Brooks, S. R. Vuppala, A. M. Jett, A. Alitalo, S. Meri, and D. R. Akins Complement Regulator-Acquiring Surface Protein 1 Imparts Resistance to Human Serum in Borrelia burgdorferi J. Immunol., September 1, 2005; 175(5): 3299 - 3308. [Abstract] [Full Text] [PDF]
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M. Biedzka-Sarek, R. Venho, and M. Skurnik Role of YadA, Ail, and Lipopolysaccharide in Serum Resistance of Yersinia enterocolitica Serotype O:3 Infect. Immun., April 1, 2005; 73(4): 2232 - 2244. [Abstract] [Full Text] [PDF]
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 J. V. McDowell, M. E. Harlin, E. A. Rogers, and R. T. Marconi Putative Coiled-Coil Structural Elements of the BBA68 Protein of Lyme Disease Spirochetes Are Required for Formation of Its Factor H Binding Site J. Bacteriol., February 15, 2005; 187(4): 1317 - 1323. [Abstract] [Full Text] [PDF]
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J. V. McDowell, J. Wolfgang, L. Senty, C. M. Sundy, M. J. Noto, and R. T. Marconi Demonstration of the Involvement of Outer Surface Protein E Coiled Coil Structural Domains and Higher Order Structural Elements in the Binding of Infection-Induced Antibody and the Complement-Regulatory Protein, Factor H J. Immunol., December 15, 2004; 173(12): 7471 - 7480. [Abstract] [Full Text] [PDF]
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