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Infection and Immunity, May 2008, p. 2113-2122, Vol. 76, No. 5
0019-9567/08/$08.00+0     doi:10.1128/IAI.01266-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Kelley M. Hovis,1 John C. Freedman,1 Hongming Zhang,1 Jonathan L. Forbes,1 and Richard T. Marconi1,2*

Department of Microbiology and Immunology,1 Center for the Study of Biological Complexity, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298-06782

Received 14 September 2007/ Returned for modification 16 January 2008/ Accepted 13 February 2008


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ABSTRACT
 
Borrelia hermsii, an etiological agent of tick-borne relapsing fever in North America, binds host-derived serum proteins including factor H (FH), plasminogen, and an unidentified 60-kDa protein via its FhbA protein. Two distinct phylogenetic types of FhbA have been delineated (FhbA1 and FhbA2). These orthologs share a conserved C-terminal domain that contains two alpha helices with a high predictive probability of coiled-coil formation that are separated by a 14-amino-acid loop domain. Through site-directed mutagenesis, we have identified residues within these domains that influence the binding of both mouse and human FH, plasminogen, and/or the 60-kDa protein. To further investigate the involvement of FhbA in the host-pathogen interaction, strains that are either FhbA+ (isolate YOR) or FhbA– (isolate REN) were tested for serum sensitivity. Significant differences were observed, with YOR and REN being serum resistant and serum sensitive (intermediate), respectively. To test the abilities of these strains to infect and persist in mice, mice were needle inoculated, and infectivity and persistence were then assessed. While both strains REN and YOR infected mice, only the FhbA+ YOR strain persisted beyond day 4. Survival of the YOR isolate in blood correlated with the upregulation of the fhbA gene, as demonstrated by real-time reverse transcriptase PCR. These data advance our understanding of the unique interactions of FhbA with individual serum proteins and provide support for the hypothesis that FhbA is an important contributor to the pathogenesis of the relapsing fever spirochete B. hermsii.


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INTRODUCTION
 
Over 17 species of Borrelia have been identified as being causative agents of relapsing fever. In North America, serious outbreaks of tick-borne relapsing fever (TBRF) caused by Borrelia hermsii and Borrelia turicatae have been described (5, 9, 10, 19, 22, 54, 55, 57, 58). The impact of TBRF is most strongly felt in Africa. In regions of Tanzania, Sudan, and Ethiopia, TBRF is among the top 10 killers of children under the age of 5 years, and nearly 40% of children under the age of 1 year will develop the disease (13, 14, 56).

TBRF spirochetes are transmitted to animals through the bite of soft-bodied Ornithodoros ticks. Within days of transmission, the spirochetes achieve remarkably high levels in the blood (spirochetemia) that may reach 106 to 108 spirochetes ml–1 of blood. Characteristic overt symptoms of TBRF include recurring fever, which coincides with high-level spirochetemia (20). The antibody response to the spirochetes targets primarily surface antigens of the Vlp or Vsp protein families (6-9). These proteins mediate a well-defined antigenic variation system. The ability of the spirochetes to persist indicates that they can also evade the innate arm of the immune system. It has recently been established that B. hermsii is able to bind factor H (FH) and FH-like protein 1 (FHL-1) (28, 41), which are host-produced negative regulators of the alternative complement system (53). This immune evasion mechanism has been demonstrated for several important human pathogens (4, 15, 18, 21, 25, 26, 40, 41, 43, 44, 47, 49). The binding of FH and/or FHL-1 to the pathogen surface locally downregulates the production of C3b and increases the efficiency of factor I-mediated C3b cleavage (62, 63). This is thought to result in the inhibition of C3b-mediated opsonization and phagocytosis of the pathogen. It has also been proposed that the binding of cell-anchored forms of complement regulators may also facilitate adherence and invasion (37, 47).

The FH/FHL-1-binding protein of B. hermsii has been designated FhbA (27). This protein has also been referred to as BhCRASP-1 (52). However, since BhCRASP-1 displays high amino acid identity with FhbA, we continue to employ the original FhbA nomenclature (27). FhbA is a 20-kDa surface protein that is encoded by a linear plasmid of 200 kb (27-29). It has been postulated that fhbA may be carried by a bacteriophage that has integrated into a 170-kb linear plasmid to form a 200-kb linear plasmid (29). While FhbA has been demonstrated to bind FH in a biologically competent manner that allows for it to serve as a cofactor in the factor I-mediated cleavage of C3b, little is known regarding its functional roles in vivo (41). Recent data demonstrating that FhbA can also bind plasminogen suggest that FhbA may contribute to the host-pathogen interaction in several ways. Experimentally infected mice and naturally infected humans elicit an early and strong immunoglobulin G (IgG) response to FhbA, demonstrating that FhbA expression is initiated during the earliest stages of infection (29). Analyses of FhbA sequences from a panel of isolates recovered from human TBRF patients revealed that there are two distinct phyletic types of FhbA that we have designated FhbA1 and FhbA2 (29). Individual strains carry only a single fhbA gene that is either fhbA1 or fhbA2. Divergence within fhbA is consistent with and reflective of the two major phlyetic lineages of B. hermsii (48).

The biological significance and molecular basis of the interaction of FH protein family members with microbially produced binding proteins have been the focus of several recent studies (1, 3, 16, 18, 21, 23, 24, 28, 29, 32, 34, 38, 42, 45, 49, 50). We have demonstrated that the binding interaction between FH and the spirochetal FH-binding proteins OspE, CspA (BbCRASP-1), and CspZ (BbCRASP-2) involves conformational determinants (38, 42, 45, 51). Independent N- or C-terminal truncations of these proteins or site-directed mutagenesis of specific alpha helices abolished ligand binding ability. Specifically, it was demonstrated that alpha helices harboring the heptad a to g repeat domain that is associated with coiled-coil formation probability are required for the presentation of the FH or infection-induced antibody binding sites. FhbA1 and FhbA2 also possess helices that have a high predicted probability of coiled-coil formation. We previously postulated that an antiparallel coiled-coil interaction in FhbA presents a unique serine-rich loop domain that is directly or indirectly involved in FH binding (28). The goals of this study were to assess the contribution of the coiled-coil elements and the loop domain of FhbA to the formation of the FH-binding site. In addition, we investigate the ability of FhbA1, FhbA2, and site-directed or deletion variants of FhbA to bind FH and serum proteins from both mice and humans. Lastly, we demonstrate that the FhbA phenotype is associated with serum resistance and with the ability of spirochetes to persist in the blood of infected mice. The data presented within enhance our understanding of the molecular basis of the interaction of complement regulatory proteins with Borrelia, explore additional virulence functions for FhbA, and provide insight into the biological function of FhbA in the pathogenesis of the TBRF spirochete B. hermsii.


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MATERIALS AND METHODS
 
Bacterial cultivation. The B. hermsii isolates YOR, REN, and FRE employed in this study were originally recovered from human relapsing fever patients in the United States. YOR and FRE produce FhbA2- and FhbA1-type proteins, respectively. REN is a high-passage isolate that does not produce FhbA. It lacks a 30-kb segment of the linear plasmid (lp200) that carries the fhbA gene. Additional strains that served as controls included B. garinii G25 (serum sensitive) and B. burgdorferi B31MI 5A4 (serum resistant). All strains were cultivated at 33°C in BSK-H complete medium (Sigma-Aldrich) supplemented with heat-inactivated rabbit serum (6% for Lyme disease spirochetes and 12% for relapsing fever spirochetes) (Sigma-Aldrich). Growth was monitored using dark-field microscopy.

Generation of infection serum and analysis of the infective potential of FhbA+ and FhbA– B. hermsii strains in mice. C3H-HeJ mice were infected with B. hermsii YOR (FhbA+) or REN (FhbA–) by intradermal inoculation of 103 spirochetes (in phosphate-buffered saline) between the shoulder blades as previously described (29). Infection was assessed by microscopic analysis of blood smears (obtained by tail snip) at days 2, 4, and 7 postinoculation. Terminal bleeds were conducted after 8 weeks, and serum was recovered. In this study, serum collected from infected animals is referred to as infection serum.

Production of recombinant proteins using LIC. All recombinant proteins were generated using a PCR-based strategy and ligase-independent cloning (LIC) methodologies. To generate a template for PCR, bacteria from 100-µl aliquots of actively growing cultures were recovered by centrifugation. The cells were washed, suspended in 50 µl water, and lysed by boiling. One-microliter aliquots of the supernatant were used as a template for PCR. All primers are described in Table 1. PCR was performed using Taq polymerase and standard conditions. Primers were constructed with 5' extensions that allow for annealing of the amplicon with the pET32-Ek/LIC vector (Novagen). All LIC methodologies were used according to instructions provided by the supplier (Novagen), and the sequences of all final constructs were confirmed through automated DNA sequencing (MWG Biotech). All recombinant proteins were generated with an N-terminal fusion of 17 kDa that contains a Trx tag, a His tag, and an S tag. The recombinant proteins were purified using the His-Bind purification kit as directed by the manufacturer (Novagen).


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  		TABLE 1. Primers used in this study

Site-directed mutagenesis of FhbA. Site-directed mutagenesis of the B. hermsii YOR FhbA2 gene was conducted using a two-step PCR-based approach and mutagenic primers as previously described (38). In brief, the desired regions of the gene were amplified as two separate amplicons. For most constructs, the 5' portion of the gene was PCR amplified using primer FhbA(+)LIC paired with an antisense mutagenic primer. The 3' portion of the gene was amplified using a forward mutagenic primer (that complements the antisense mutagenic primer) and primer FhbA(–)LIC. The two amplicons were then combined, and the primer set composed of FhbA(+)LIC and FhbA(–)LIC was added. The complementarity between the 3' end of the upstream gene fragment and the 5' end of the downstream gene fragment allowed the amplicons to self-prime. Additional site-directed mutations were introduced into some constructs using the approach described above. For those constructs that required more than one round of mutagenesis to introduce all of the desired site-directed substitutions, the primers used in each round of mutagenesis are indicated by subscripts (Table 1). To generate constructs that had site-directed mutations in the 5' end of the gene (i.e., the FhbA2 cc1 construct), primer FhbA(+)LIC was replaced with a mutagenic LIC primer. The mutated amplicons were then used to generate recombinant protein using LIC methodologies and purification procedures described above. The mutations were confirmed through automated DNA sequencing (MWG Biotech).

SDS-PAGE and immunoblot analyses. Purified recombinant proteins were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) in 15% precast Criterion gels (Bio-Rad) and transferred onto a polyvinylidene difluoride membrane (Millipore) by electroblotting as previously described (36). All polyvinylidene difluoride membranes were blocked for 1 h in blocking buffer (phosphate-buffered saline containing 0.2% Tween 20 and 5% nonfat dry milk). Expression of the recombinant proteins was confirmed using horseradish peroxidase (HRP)-conjugated S protein (1:40,000 dilution; Novagen). For immunoblot analyses, serum collected from infected mice was used at a dilution of 1:1,000. HRP-conjugated goat anti-mouse IgG (1:40,000 dilution; Pierce) served as the secondary antibody. Bound HRP-conjugated antibody or S protein was detected by chemiluminescence using the SuperSignal West Pico substrate (Pierce). All dilutions were prepared in blocking buffer, and mixtures for all steps were incubated for 1 h at room temperature.

ALBI analyses. To assess the ability of recombinant proteins to bind human FH, FH affinity ligand binding immunoblot (ALBI) assays were performed as previously described (45). In brief, membrane-immobilized proteins were incubated with purified human FH (5 ng µl–1; Calbiochem), and bound FH was detected using goat anti-human FH antiserum (1:1,000; Calbiochem). Rabbit anti-goat IgG (1:40,000; Pierce) served as the secondary antibody, and detection was done by chemiluminescence as described above.

Reverse ALBI analyses. To compare the abilities of FhbA1 and FhbA2 and a subset of the mutated FhbA proteins to bind to serum proteins from humans and mice, the reverse ALBI approach was employed as previously described (30). Briefly, serum samples were fractionated by SDS-PAGE in 7.5% Criterion gels (Bio-Rad) under nonreducing conditions and transferred onto membranes by electroblotting. Nonreducing conditions maintain disulfide bonds that have been demonstrated to be required for the binding of FH-binding proteins to FH and other serum proteins (30). The membrane-immobilized serum proteins were incubated with purified recombinant FhbA proteins (75 ng µl–1 in blocking buffer at room temperature for 2 h). Unbound protein was removed by washing, and bound protein was detected through its N-terminal S tag using HRP-conjugated S protein (1:40,000 dilution; Novagen). The migration position of plasminogen was determined by screening a membrane with goat anti-human plasminogen antiserum (1:1,000 dilution; Rockland Immunochemicals) and rabbit anti-goat IgG (1:40,000 dilution; Pierce) as the secondary antibody.

Serum sensitivity assay. Borrelia cells grown in BSK complete medium containing either 6% or 12% rabbit serum were added to either normal human serum (NHS) or heat-inactivated NHS (hiNHS) at a ratio of 1:1 to a final volume of 300 µl. The reaction mixtures were incubated at 37°C, and at 0, 2, and 6 h, a 100-µl aliquot was removed for live/dead staining using the Live/Dead BacLight bacterial viability kit (Molecular Probes) as directed by the manufacturer. At each time point, 15 40x fields were visualized, cells were counted using an Olympus BX51 fluorescence microscope with a fluorescein or rhodamine filter, and the percent survival was calculated.

Determination of fhbA mRNA level and spirochete numbers in mice. To assess spirochete numbers in the blood of infected animals and to measure the mRNA levels of fhbA during infection, blood was collected 4 and 7 days after inoculation of mice infected with strains YOR and REN. EDTA (3 mM) and NaCl (7 mM) were added to each blood sample to prevent clotting. DNA and RNA were then extracted from aliquots of the blood using the Qiagen DNeasy tissue kit and Qiagen RNeasy kit, respectively. For all PCR-based methods, standardization approaches and data analyses were conducted as previously described (61). In brief, real-time PCR was performed in triplicate using Sybr green PCR master mix as instructed by the supplier (Applied Biosystems). The primer pairs were designed to amplify 100- to 150-bp segments of the target gene. Standard curves were generated using serial dilutions of a known concentration of genomic DNA. For the mRNA analyses, the absence of DNA in the RNA preparations was confirmed by reverse transcriptase PCR (RT-PCR) analyses using 16S rRNA-targeting primers. cDNA was generated using Superscript II RT and methods recommended by the supplier (Invitrogen) as previously described (61). The cDNA was then used as a template in real-time PCR analyses. Standardization was achieved using primers targeting the mouse nidogen gene as described previously (60).


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RESULTS
 
Identification of FhbA2 alpha helices with high coiled-coil formation probability that are required for FH binding. Coiled-coil domains have been implicated as being key determinants in the formation of the FH-binding pocket of multiple spirochetal FH-binding proteins (38, 42, 51). In addition, these structural domains are also involved in the formation of conformational or discontinuous epitopes that are presented at the cell surface during infection. Recent analyses of FhbA sequences from a large panel of B. hermsii isolates revealed that FhbA is ~67% alpha helical. Four alpha helices within FhbA2 and two within FhbA1 have significant predictive probability of coiled-coil formation (28). The putative coiled coils of FhbA2 are indicated in Fig. 1, and the predictive probability of coiled-coil formation of each one is listed in Fig. 2. Although FhbA1 sequences lack the two N-terminal coiled-coil domains (referred to as cc1 and cc2) the C-terminal coiled-coil elements (cc3 and cc4) are present and are highly conserved. To assess the contribution of each of the four coiled-coil elements in ligand binding, site-directed mutagenesis was performed to individually disrupt coiled-coil formation probability. The residues targeted for substitution were identified through computer-assisted structural analyses (35). Depending on the specific alpha helix being analyzed, 2 to 6 amino acid substitutions were required to decrease or eliminate coiled-coil formation probability (Fig. 2A). The substitutions consisted of the replacement of the coiled-coil heptad repeat "a" and "d" position residues with charged or polar residues. Controls for these analyses consisted of the introduction of neutral substitutions that are not predicted to influence coiled-coil formation probability. The panel of recombinant proteins was tested for FH binding using the ALBI assay format and for infection-induced antibody binding using an immunoblot format. Equal protein loading of the membrane-immobilized recombinant proteins was confirmed by screening an identical blot with HRP-conjugated S protein. Mutations that disrupted cc3 and cc4 completely abolished the binding of human FH and the binding of infection-induced antibody derived from mice (Fig. 2B). In contrast, the disruption of cc1 and cc2 had no impact on FH or antibody binding, indicating that these N-terminal alpha helices are not involved in the formation of the FH-binding domain or in the presentation of the epitopes that are recognized during infection. It is important to note that all recombinant proteins with substitutions that do not alter the predictive probability of coiled-coil formation retained strong FH and infection-induced antibody binding.


Figure 1
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  		FIG. 1. FhbA amino acid sequences and domains predicted to form coiled-coil structural elements. Shown are the FhbA2 and FhbA1 amino acid sequences derived from B. hermsii YOR and FRE, respectively. The four alpha helices of FhbA2 with coiled-coil formation probability (designated cc1 to cc4) are indicated by boxing. Note that the cc1 and cc2 domains of FhbA1 have a low probability of coiled-coil formation. The signal peptide is not included in the aligned sequences.


Figure 2
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  		FIG. 2. Demonstration that the C-terminal coiled-coil domains of FhbA2 are required for factor H binding and detection by infection-induced antibody. A series of FhbA2 coiled-coil mutants were generated using a site-directed mutagenesis approach. (A) Amino acid changes introduced to create each coiled-coil mutant. The COILs program was also used to predict the probability of coiled-coil formation for each mutant. Shown are the probabilities of coiled-coil formation using windows of 14 or 21 amino acid residues. (B) The purified recombinant FhbA2 coiled-coil mutants were fractionated by SDS-PAGE, immunoblotted, and screened with HRP-conjugated S protein to assess loading levels. Each mutant was then tested for FH binding using the FH ALBI assay and detection by infection-induced antibody with B. hermsii (B.h.) YOR infection (inf.) serum. The mutants in which a coiled coil (cc) was disrupted are indicated as m1, and the control mutants with a conserved coiled coil are designated m2. WT, wild type.

Analysis of the role of a conserved serine-rich loop in FH and infection-induced-antibody binding. cc3 and cc4 are separated by a 14-amino-acid nonstructured domain. The antiparallel interaction of cc3 and cc4 would present this unique serine-rich domain as a loop element. To determine if the loop serves as an interaction site for FH or infection-induced antibody, site-directed mutagenesis analyses in which loop residues were substituted with residues of differing properties (loop mutants 1 through 8) were performed (Fig. 3A). Additional substitutions in which specific amino acids were replaced with residues of similar properties (loop mutants 9 through 11) were introduced (Fig. 3A). Lastly, one variant in which the 14-residue loop was replaced with 6 alanine residues (loop mutant 12) was generated. Equal loading of the recombinant proteins on the immunoblots was demonstrated by screening one membrane with HRP-conjugated S protein. FH binding was assessed using the ALBI assay. Infection-induced antibody binding was assessed by screening an immunoblot with serum collected at week 8 from YOR-infected mice (Fig. 3B). Loop mutants 1 through 11 retained human FH-binding ability, although in some cases, binding was attenuated. Loop mutant 12 completely lost its FH-binding ability. In contrast to that observed for FH binding, all of the loop mutants were immunoreactive with infection serum, indicating that the epitopes targeted during infection are separable from the FH-binding site.


Figure 3
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  		FIG. 3. Mutational analysis of the FhbA2 loop element and its role in factor H and infection-induced antibody binding. A series of FhbA2 loop mutants was generated using a site-directed mutagenesis approach. The amino acid changes introduced into the loop for each mutant are indicated in boldface type (A). The purified recombinant FhbA2 loop mutants were fractionated by SDS-PAGE, immunoblotted, and screened with HRP-conjugated S protein to assess loading levels (B). The mutants were then tested for FH binding and detection by infection-induced antibody using the FH ALBI assay and B. hermsii (B.h.) YOR infection (inf.) serum, respectively. WT, wild type.

Comparative analysis of the binding of FhbA1 and FhbA2 to human and mouse serum proteins. FH-binding proteins of Borrelia spp. have been demonstrated to bind to several human serum proteins including members of the FH protein family, plasminogen, and an additional unidentified 60-kDa protein (30, 52). To determine if the serum protein binding patterns differ for FhbA orthologs, recombinant FhbA1 and recombinant FhbA2 were used to screen membrane-immobilized serum proteins obtained from healthy mice and humans. The serum proteins were fractionated under nonreducing conditions. FhbA2 bound to FH, 75-kDa, and ~60-kDa proteins present in both mouse and human sera (Fig. 4). FhbA1 displayed significant differences in its patterns of binding to human serum proteins compared to its patterns of binding to mouse serum proteins. FhbA1 bound to FH from both mammals but displayed an inverse binding pattern with the ~75-kDa and 60-kDa proteins (Fig. 4B and C). A recent study provided strong evidence that the ~75-kDa proteins are plasminogen/plasmin (52). This was confirmed by screening an identical blot of the mouse and human serum samples with anti-human plasminogen antiserum. The identity of the 60-kDa protein remains to be determined (Fig. 4B).


Figure 4
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  		FIG. 4. Comparative analyses of the abilities of recombinant FhbA1 and FhbA2 to bind FH, plasminogen, and other serum proteins from mice and humans. Serum proteins from mice and humans (indicated above each lane) were separated by SDS-PAGE with 7.5% precast Criterion gels under nonreducing conditions. The immunoblots were incubated with individual recombinant FhbA proteins, recombinant FhbA site-directed mutants, or anti-human plasminogen antiserum as indicated below each panel. Detection of bound protein or antibody was done through chemiluminescence. WT, wild type.

The ability of the FhbA site-directed mutants described above to bind to serum proteins was also assessed using the reverse ALBI approach. These analyses were focused on FhbA2. FhbA2 cc1m1 displayed binding to FH and plasminogen similar to that of the wild-type protein (Fig. 4B). Interestingly, the cc1 mutant bound more strongly to the 60-kDa protein than the wild type. The basis for this is not clear. FhbA2 cc2m1 retained full binding to the 60-kDa protein, but binding to FH and plasminogen was attenuated (data not shown). The FhbA2 cc3m1 mutant displayed no binding to FH, and the binding to plasminogen and the 60-kDa protein was significantly reduced in both mice and humans. FhbA2 loop mutant 12 (Fig. 4) and FhbA2 cc4m1 (data not shown) lost binding to FH and plasminogen but not to the 60-kDa protein. These results and their significance are described in detail below.

Comparative analysis of serum sensitivity. Approaches for the genetic manipulation of relapsing fever spirochetes are not yet fully developed. As an alternative approach for investigating the contribution of FhbA to pathogenesis, the serum sensitivity of the FhbA+ YOR isolate and the FhbA– REN isolate was assessed. Each isolate was incubated with NHS or hiNHS (final concentration of 50%), and percent survival was determined at 0, 2, and 6 h using the Live/Dead BacLight bacterial viability kit (Molecular Probes) staining approach (Fig. 5). Serum-resistant B. burgdorferi B31MI clone 5A4 and serum-sensitive B. garinii strain G25 served as controls. Nearly complete killing of B. garinii strain G25 was observed by 2 h when exposed to NHS but not to hiNHS. B. burgdorferi was not significantly affected by treatment with either NHS or hiNHS. For B. hermsii YOR, 79% of the spirochetes remained viable after 6 h of treatment, while only 46% of the FhbA– REN spirochetes survived.


Figure 5
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  		FIG. 5. Analysis of serum sensitivities of relapsing fever strains that are FhbA+ or FhbA–. Spirochetes were incubated at 37°C with NHS (dashed lines) or hiNHS (solid lines). Survival was assessed after 0, 2, and 6 h using the Live/Dead BacLight bacterial viability kit. The mean percent survivals of 15 40x fields were calculated for each time point.

Comparative analysis of infectivity. To compare the abilities of serum-sensitive and serum-resistant strains of B. hermsii to infect and persist in mice, strains REN and YOR were needle inoculated into C3H-HeJ mice. Blood was collected from each mouse at days 2, 4, and 7, and dilutions were examined for the presence of spirochetes using dark-field microscopy. At days 2 and 4, spirochetes were readily apparent in the blood of mice infected with either strain. However, at day 7, spirochetes were observed in the YOR-infected mice but not in the REN-infected mice. To quantify the numbers of spirochetes in the blood at the 4- and 7-day time points, DNA was extracted from the blood, and real-time PCR was performed. The number of B. hermsii genome equivalents was determined based on the 16S rRNA gene copy number, with standardization performed using the mouse nidogen gene. The data are expressed as the number of spirochete genomes per 1,000 copies of the mouse nidogen gene (Fig. 6A). Interestingly, a greater number of spirochetes were detected at day 4 in the REN-infected mouse than in the YOR-infected mice. However, at day 7, the number of REN isolates decreased to almost zero, indicating clearance of the infection. These data suggest that while both strains can establish infection, the REN strain is not able to persist and is quickly cleared.


Figure 6
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  		FIG. 6. Analysis of the ability of FhbA+ and FhbA– strains to infect and persist in mice and quantification of fhbA expression levels in blood during infection. (A) Mice were infected with either B. hermsii YOR (black bars) or B. hermsii REN (hatched bars). Real-time PCR was performed with samples collected at days 4 and 7. The data are presented as the number of spirochetes per ml of blood. (B) RNA was collected from infected mice, and the levels of fhbA transcript were assessed at days 4 and 7 postinfection by real-time RT-PCR. For comparative purposes, the data from each time point are presented as relative units. No transcript was detected in blood collected from the mice infected with the REN isolate. The data were standardized as described above, and error bars are shown.

Demonstration that fhbA is expressed at high levels during infection. To further assess the correlation between FhbA production and persistence, fhbA mRNA levels were determined at days 4 and 7 after inoculation of mice. As expected, no fhbA mRNA was detected in RNA extracted from the blood of REN-infected mice at any time point. However, fhbA mRNA was readily detected in the blood extracted from the YOR-infected mice at day 4, and expression was dramatically upregulated at day 7 (Fig. 6B). It is important to note that this upregulation of fhbA occurred while the number of spirochetes were decreasing.


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DISCUSSION
 
Infection with B. hermsii is characterized by recurring high-level spirochetemia. The ability to thrive in the blood and persist indicates that the spirochetes possess mechanisms that allow the circumvention of both acquired and innate immune defenses. The binding of FH and other serum proteins to B. hermsii may contribute to several aspects of the host-pathogen interaction. Here, we investigate the molecular interaction and differential binding of B. hermsii FhbA variants with FH, plasminogen, other serum proteins, and infection-induced antibody.

It has been demonstrated for some spirochetal FH-binding proteins that the formation of the FH- and/or FHL-1-binding pocket or antibody recognition domains is dependent on hydrophobic interactions between alpha helices that have a significant predictive probability of coiled-coil formation (28, 38, 39, 45, 51). Two C-terminal, highly conserved, putative coiled-coil elements are present in both FhbA1 and FhbA2 (28). To determine if the individual coiled-coil elements of FhbA2 are involved in its interaction with antibody or ligand, site-directed mutagenesis was performed, and ligand binding was assessed. These analyses revealed that cc3 and cc4 (but not cc1 or cc2) are important determinants for FH binding and antibody recognition. Site-directed mutation of residues within these alpha helices that decreased coiled-coil formation probability impacted the binding to some of the ligands. In contrast, recombinant proteins with amino acid substitutions that did not decrease the predicted probability of coiled-coil formation did not lose their binding abilities. These findings suggest that conformation and structure, as opposed to a simple, contiguous, primary sequence, are the key determinants in the binding interaction. The conclusion that conformational elements are required for the binding of FH and infection antibody to recombinant proteins fractionated under SDS-PAGE conditions may seem paradoxical. However, coiled coils are highly stable, hydrophobicity-based interactions that can withstand and/or rapidly reform after treatment with SDS and boiling (17, 31, 59).

cc3 and cc4 are separated by a nonstructured sequence of 14 amino acids. An antiparallel interaction between cc3 and cc4 would present this 14-amino-acid domain as a loop element. The loop is unique in that it has a high serine content (36% in the loop versus 5.7% in the protein overall). Random mutagenesis analyses of B. burgdorferi OspE proteins provided evidence that one or more serine residues are involved in the interaction with FH (42). To determine if the FhbA loop domain and specific residues within contribute to ligand binding, one or more residues within the loop were substituted. The resulting recombinant proteins were tested for FH binding and infection antibody recognition. While all of the loop mutants were recognized by infection antibody, several displayed significantly attenuated FH binding (mutants 4, 5, 8, 9, and 12). The complete loss of FH binding was observed with loop mutant 12 in which the entire loop was replaced with a hexa-alanine tract. Analysis of these mutants with the COILs program indicated that the mutated or shortened loop sequences do not decrease the coiled-coil formation probability of either cc3 or cc4. The impact of the wholesale replacement of the loop with the Ala tract on the potential antiparallel interaction of cc3 and cc4 was assessed using the ROSETTA program for three-dimensional structure prediction. Replacement of the loop is predicted to spatially separate cc3 and cc4 to a degree that would most likely inhibit the formation of an antiparallel coiled coil (computer predictions not shown). The fact that antibody recognition can be abolished by the mutation of either cc3 or cc4 suggests that the epitope(s) of FhbA that is recognized during infection is formed by the interaction of two alpha helices.

It has recently been demonstrated that the OspE paralogs of Lyme disease spirochetes bind to serum proteins from a diverse range of mammals (30). In addition to their binding to FH, the OspE paralog BBL39 bound primarily to serum proteins that were approximately 75 kDa in size (plasminogen), while BBN38 bound serum proteins that were 60 kDa and 75 kDa in size. To determine if FhbA orthologs display differences in their serum protein binding patterns, recombinant FhbA1 and recombinant FhbA2 proteins were employed in reverse ALBI assays. The interactions of these proteins with both human- and murine-derived serum proteins were assessed. The serum protein binding patterns were qualitatively similar but differed quantitatively. For example, FhbA1 consistently bound human plasminogen more readily than mouse plasminogen. However, it remains to be determined if this is due to differing expression levels of plasminogen in these mammals or if it reflects higher-affinity binding to human plasminogen by FhbA. It is noteworthy that the overall ligand binding patterns observed for FhbA paralleled those observed for the OspE BBN38 paralog (30). These proteins have no discernible primary sequence homology. The interaction of Borrelia species with plasminogen has important implications for understanding the nature of the host-pathogen interaction. It has been established that the binding of plasminogen to Borrelia facilitates dissemination and correlates with enhanced invasive capabilities (11, 12, 33, 46). Hence, based on the ability of FhbA to bind FH, plasminogen, and yet-to-be identified serum proteins, it is clear that FhbA may contribute to virulence through multiple mechanisms. Future studies will seek to identify the remaining serum proteins that bind FhbA and determine the contributions of these interactions to Borrelia pathogenesis.

The data presented here suggest that the B. hermsii relapsing fever murine model is an excellent system for evaluating and dissecting the different functional roles of FhbA in pathogenesis. Here, we demonstrate that FhbA binds to both FH and plasminogen derived from both mice and humans. In addition, FhbA also binds to the unidentified 60-kDa serum protein that we previously demonstrated is produced by a wide range of mammals. This 60-kDa protein is also bound by the B. burgdorferi OspE paralog BBN38 (27). Interestingly, some of the FH-binding proteins of Lyme disease spirochetes do not bind or bind weakly to mouse FH (1, 30, 39). Others such as CspA (BBA68) are not expressed during infection of humans or mice (39).

Gene inactivation and complementation have traditionally served as the "gold standard" for assessing the contribution of specific proteins to bacterial pathogenesis. However, at present, genetic manipulation approaches are not sufficiently developed to allow the inactivation of FhbA and subsequent analysis in vivo. Pending the refinement of genetic manipulation in relapsing fever spirochetes, we assessed the complement sensitivity, infective potential, and persistence capabilities of strains that are either FhbA+ or FhbA–. We previously demonstrated that REN (a high-passage isolate) lacks an approximately 30-kb segment of the 200-kb linear plasmid that carries fhbA (29). Regarding the comparative analyses of REN and YOR presented here, an important caveat at the outset is that these isolates are not isogeneic, and the degree to which they may vary at other potentially important loci is unknown. Nonetheless, here, we demonstrate that REN is significantly more sensitive to serum than the FhbA+ YOR strain. The serum-sensitive phenotype of REN is consistent with the "intermediate" serum sensitivity that has been reported previously for some Borrelia afzelii strains (2). The fact that complete killing was not observed with REN may indicate that there are additional factors produced by relapsing fever spirochetes that contribute to serum resistance. The animal model studies conducted here also support the correlation between FhbA production and pathogenesis. Both REN and YOR were able to establish infection in mice, but by day 7, infection with REN was largely cleared. A striking upregulation of fhbA expression was also observed in the YOR-infected mice at day 7 versus day 4, indicating that high-level expression is important for persistence. Consistent with the high levels of fhbA transcript detected in spirochetes in mice, we previously demonstrated that a robust IgG response to FhbA is elicited during early infection (29).

In summary, we have demonstrated that the cc3 and cc4 domains of FhbA and the loop between these alpha helices are involved to various degrees in the interaction of FhbA with FH, infection antibody, plasminogen, and an unidentified 60-kDa serum protein. The differences observed in the abilities of FhbA site-directed mutants to bind to these different ligands indicate that the binding sites are not identical. This important observation suggests that it may be possible to assess the individual role of each ligand binding activity in pathogenesis. The interaction of FhbA with multiple ligands in mice supports the application of the murine model to study the role of FhbA in vivo. Toward this goal, in future studies, we will seek to develop a genetic manipulation system for B. hermsii. Once in place, it may be possible to replace the wild-type fhbA gene of infectious strains with fhbA harboring site-directed mutations that abolish one or more of its specific binding activities. Such analyses would allow a detailed dissection of the contribution of the multiple ligand binding activities of FH-binding proteins to pathogenesis.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, 1112 E. Clay St., Richmond, VA 23298-0678. Phone: (804) 828-3779. Fax: (804) 828-9946. E-mail: rmarconi{at}hsc.vcu.edu Back

{triangledown} Published ahead of print on 25 February 2008. Back

Editor: W. A. Petri, Jr.


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Infection and Immunity, May 2008, p. 2113-2122, Vol. 76, No. 5
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  • Frederick, J. R., Rogers, E. A., Marconi, R. T. (2008). Analysis of a Growth-Phase-Regulated Two-Component Regulatory System in the Periodontal Pathogen Treponema denticola. J. Bacteriol. 190: 6162-6169 [Abstract] [Full Text]  

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