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The Journal of Immunology, 2008, 180, 4858 -4864
Copyright © 2008 by The American Association of Immunologists, Inc.
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Complement Factor H-Binding Protein, a Putative Virulence Determinant of Borrelia hermsii, Is an Antigenic Target for Protective B1b Lymphocytes1

Matthew J. Colombo and Kishore R. Alugupalli2

Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Vaccination is the most effective way to control infectious diseases. A variety of microbial pathogens use antigenic variation, an immune evasion strategy that poses a challenge for vaccine development. To understand protective immune responses against such pathogens, we have been studying Borrelia hermsii, a bacterium that causes recurrent bacteremia due to antigenic variation. An IgM response is necessary and sufficient to control B. hermsii infection. We have recently found a selective expansion of B1b cells concurrent with the resolution of B. hermsii bacteremia. B1b cells from convalescent but not naive mice confer long-lasting immunity, but the Ag(s) driving the protective IgM responses is unknown. Herein we demonstrate that convalescent B1b cell-derived IgM recognizes complement factor H-binding protein (FhbA), a B. hermsii outer-surface protein and putative virulence factor that does not undergo antigenic variation and is expressed by all clinical isolates. A progressive increase in the IgM response to FhbA correlated with the kinetics of B1b cell expansion, diminished the severity of bacteremic episodes, and led to the eventual resolution of the infection. These data indicate that FhbA is a specific target for protective B1b cell responses. Ags recognized by B1b cells may be considered as an important component in vaccination strategies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The generation of B cell memory is a hallmark of the adaptive immune system, which provides a protective Ab response upon reexposure to the same Ag and is central to the concept of vaccination (1). Antigenic variation is an immune evasion strategy used by a variety of microbial pathogens and it poses a challenge for vaccine development, because these organisms randomly change the antigenic epitopes that are likely to provide the most robust and serotype-specific protective Ab response (2).

The hallmark of Borrelia hermsii infection is recurrent episodes of high-level bacteremia (~108 bacteria/ml blood), each caused by the emergence of serotypically distinct populations of bacteria (3, 4). Diverse serotypes are generated by DNA rearrangements of the genes encoding the variable major proteins (Vmps),3 the most abundant B. hermsii surface proteins, from a silent locus to an expression locus (5). The random expression of different Vmps during infection results in the outgrowth of new serotypes and allows evasion of the adaptive immune response. Recently, a strategy by which B. hermsii also evades the innate immune system has been proposed (6). This is based on the ability of B. hermsii to bind factor H, a serum complement regulatory protein. Bacteria-bound factor H can participate in the factor I-mediated cleavage of C3b deposited on bacterial surface, a process that could make B. hermsii resistant to the complement system and allow bacterial persistence independent of, or in addition to, an antigenic variation strategy.

Rodents are natural reservoirs for relapsing fever Borrelia spp. and murine infection recapitulates the critical pathophysiological aspects of the human disease (7, 8, 9, 10). To understand the immune mechanisms required for controlling a pathogen that employs antigenic variation, we have been studying the murine model of B. hermsii infection. Although mice infected with B. hermsii suffer recurrent episodes, the degree of bacteremia in each subsequent relapse is progressively less severe both in wild-type and in T cell-deficient mice (11, 12, 13). Moreover, by 4 wk both wild-type and T cell-deficient mice resolve the infection without any therapeutic intervention and become resistant to reinfection, indicating the generation of a novel T cell-independent memory response (14).

Humoral immunity is essential in controlling B. hermsii and other relapsing fever Borrelia spp. (12, 13, 15). The inability of mice deficient in secretory IgM and the ability of activation-induced cytidine deaminase (AID)-deficient mice, which secrete only IgM but not other Ig isotypes, to control B. hermsii infection demonstrates that IgM is the necessary and sufficient isotype for controlling B. hermsii bacteremia (13, 14). Mature B cells are divided into four subsets, namely follicular (FO), splenic marginal zone (MZ), B1a, and B1b (16). We found that IL-7–/– mice, which are deficient in FO B cells (17), and bone marrow chimeric mice deficient in B1a cells control B. hermsii bacteremia as efficiently as do wild-type mice, demonstrating that FO B and B1a subsets are dispensable in this infection system (13, 14). Severe bacterial burden in splenectomized mice during the primary bacteremic episode suggested that MZ B cells play a role in controlling B. hermsii (10, 13). Consistent with this, Bockenstedt and colleagues have recently demonstrated that MZ B cells mount anti-B. hermsii Ab responses (18). Nonetheless, the rapid control of bacteremia during secondary episodes in splenectomized mice suggested that MZ B cells are not the only subset that contributes to protection (13). Moreover, mice deficient in B1a, MZ, or FO B cells are resistant to B. hermsii reinfection (14), indicating that these subsets are not essential for long-lasting immunity, and suggesting that B1b cells may play a critical role in long-term immunity. Consistent with this possibility, we found a specific expansion of the B1b cell subset concurrent with the resolution of B. hermsii infection; this expansion persists indefinitely in convalescent mice (13, 14). Using adoptive transfer experiments we demonstrated a direct role for the expanded B1b cells (hereafter also referred to as convalescent B1b cells) in immunity to infection (14). Interestingly, while convalescent B1b cells confer complete protection when transferred to immunodeficient mice, naive B1b cells provide only partial and short-lived immunity, indicating that the convalescent B1b cells have acquired apparent immunological memory. Similar to memory B cells, these B1b cells do not mount an anti-B. hermsii response in the absence of specific stimulation, indicating that they maintain a quiescent state (14). However, upon challenge with B. hermsii, B1b cells rapidly differentiate into pathogen-specific IgM-secreting cells akin to conventional T cell-dependent memory B cells, but the Ag(s) driving the protective IgM responses is unknown.

Because B1b cells of convalescent mice confer long-lasting immunity to B. hermsii, in the present study we sought to identify protective Ags recognized by B1b-derived IgM. Herein we show that this IgM recognizes complement factor H-binding protein (FhbA), a putative virulence factor of B. hermsii that is implicated in serum resistance, suggesting that resolution of B. hermsii infection may occur independently of Vmps that undergo antigenic variation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, bacteria, and infections

The Institutional Animal Care and Use Committee have approved these studies. Mice were housed in microisolator cages with free access to food and water, and were maintained in a specific pathogen-free facility at Thomas Jefferson University. C57BL/6J (B6), C57BL/6J-Rag1tm1 Mom (Rag1–/–), and C57BL/6J-Tcr-βtm1 Mom x Tcr-{delta}tm1 Mom (TCR-βx{delta}–/–) were purchased from The Jackson Laboratory. Mice that lack the secreted form of IgM (sIgM–/–) (19) were provided by Dr. Jianzhu Chen. AID–/– mice (20) were provided by Dr. Tasuku Honjo. Mice deficient in Bruton’s tyrosine kinase (Btk) and MyD88, (Btk–/– x MyD88–/–) mice, have been described recently (21). B. hermsii strains were kindly provided by Dr. Tom Schwan (Rocky Mountain Laboratories). B. hermsii strain HS1 deficient in Vmp expression was described previously (22). B hermsii strains DAH, SWA, FRE, and OKA are clinical isolates from the western U.S. A fully virulent low-passage B. hermsii strain DAH (23) was used extensively in the present study. PCR and sequencing analysis of the Vmp expression locus (5) revealed that strains DAH, SWA, FRE, and OKA express Vmp2, Vmp24, Vmp4, and Vmp58, respectively. Spirochetes were grown in vivo because in vitro-grown B. hermsii express a distinct Vmp, termed Vmp33, which is not expressed in vivo. We have reported that B. hermsii grows to persistently high densities in blood of sIgM–/– or Btk–/– x MyD88–/– mice (13, 21). To obtain sufficient amounts of in vivo-adapted spirochetes, these mice were infected i.p., and bacteria from the blood were harvested on day 4 postinfection and stored at –80°C unless stated otherwise.

Generation of B1b cell-derived IgM

Rag1–/– mice were reconstituted with 2 x 105 B1b cells from either naive or convalescent (i.e., resolved B. hermsii DAH infection) mice as described (14). Because Rag1–/– mice reconstituted with convalescent B1b cells do not generate a specific IgM response without bacterial stimulation (14), they were infected i.v. with 5 x 104 bacteria of the B. hermsii strain DAH 1 day after adoptive transfer of B1b cells, and serum was collected 2 or 10 days postinfection. Although naive B1b cells do not confer protection (14), Rag1–/– mice reconstituted with naive B1b cells were also infected in parallel and serum was collected 2 or 10 days postinfection.

ELISA

Specific IgM levels in blood were measured by ELISA, according to the manufacturer’s instructions (Bethyl Laboratories). The B. hermsii-specific IgM was determined by coating 96-well plates (ICN Biomedicals) with in vivo (sIgM–/– mice) grown B. hermsii DAH-Vmp2, HS1-Vmp33, or HS1-Vmp– strain (105 wet bacteria/well) and the specific Ab levels were interpreted as nanograms per microliter using IgM standards.

Preparation of naive or immune serum

B6, TCR-βx{delta}–/–, or AID–/– mice were infected with 5 x 104 bacteria of the B. hermsii strain DAH, and blood was collected at days 0, 7, 10, 14, 20, and 23, 24, or 25 postinfection.

Proteinase K treatment of B. hermsii

A selective cleavage of outer-surface proteins of B. hermsii has been described previously (24). B. hermsii DAH cells freshly harvested from infected mouse blood were washed and resuspended in PBS (pH 7.4) supplemented with 5 mM MgCl2 (PBS-Mg) at 109 bacteria/ml. Intact bacterial cells were digested with increasing concentrations of proteinase K (Roche) for 1 h at room temperature, and the proteolysis was stopped by adding phenylmethanesulfonyl fluoride (Sigma-Aldrich) to a final concentration of 1 mM. Bacterial cells were washed three times with PBS-Mg and suspended in 100 µl tricine sample buffer (Bio-Rad), boiled 5 min, and subjected to SDS-PAGE and Western blot analyses.

B. hermsii cell-surface biotinylation

Surface biotinylation of bacterial cells has been described previously (25). Intact B. hermsii DAH cells were washed three times and resuspended at a concentration of 5 x 108 bacteria/ml PBS (pH 8.0). Bacteria were incubated with or without 4 µg/ml EZ-Link Sulfo-NHS-LC-Biotin (Pierce) for 10 min at room temperature. Bacteria were centrifuged at 16,000 x g for 6 min and resuspended in PBS supplemented with 100 mM glycine to quench unbound biotin, then washed three times with PBS. The bacterial pellet was resuspended in 100 µl tricine sample buffer, boiled for 5 min, and subjected to SDS-PAGE and Western blot analyses.

Immunoblotting

B. hermsii strain DAH cells were solubilized in Laemmli sample buffer for resolution on 4–15% polyacrylamide Tris-HCl Ready Gels (Bio-Rad) or in tricine sample buffer for resolution on 10–20% Tris-Tricine Ready Gels (Bio-Rad). Resolved proteins were transferred to polyvinylidene difluoride membrane (Millipore) and blocked in 2% BSA in TBS containing 0.05% Tween 20 (TBST). Immune sera were diluted 1/10,000 in 2% BSA/TBST and were used to probe the membrane. B. hermsii-specific IgM was detected with goat anti-mouse IgM conjugated to alkaline phosphatase (SouthernBiotech), followed by development with the bromochloroindolyl phosphate/nitroblue tetrazolium phosphatase substrate system (KPL).

Immunoprecipitation and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis of tryptic peptides

B. hermsii DAH cells were washed three times with PBS-Mg and resuspended (109 bacteria/ml) in immunoprecipitation buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 0.05% NaN3, 0.1% Nonidet P-40 (pH 7.4)) supplemented with 50 µM PMSF. Bacteria were vortexed and lysed on ice for 1 h, then centrifuged at 16,000 x g for 15 min at 4°C. Twenty-four-day-postinfected serum was diluted (1/200) into the clarified lysate and incubated for 2 h at 4°C on a tilting rocker. B. hermsii Ag-IgM complexes were immobilized by addition of anti-IgM-conjugated agarose beads (Zymed Laboratories) and were incubated for 1 h at 4°C on a tilting rocker. Beads were washed with immunoprecipitation buffer, and immunoprecipitated bacterial Ags were recovered with 40 µl tricine sample buffer. Products were resolved by SDS-PAGE with 10–20% tricine gels and stained with Coomassie blue. An IgM-reactive band corresponding to molecular mass of 18 kDa was excised, and tryptic peptide analysis was performed by Tufts University Core Facility (Boston, MA) on a fee-for-service basis as previously described (26). The LC-MS/MS spectra were searched against the National Center for Biotechnology Information nonredundant protein sequence database using the SEQUEST computer algorithm (27).

Recombinant expression of FhbA1 or FhbA2 of B. hermsii

PCR and ligation-independent cloning of FhbA were performed as described by Marconi and colleagues (6). Plasmids were transformed into Escherichia coli NovaBlue cells (Novagen), and sequence analysis of the inserts was performed by Thomas Jefferson University’s Nucleic Acid Core Facility. Plasmids from clones carrying either the FhbA1 insert (from strain DAH) or the FhbA2 insert (from B. hermsii strain CMC) and one clone missing an insert were purified and transformed into isopropyl β-D-thiogalactoside (IPTG)-inducible BL21(DE3) E. coli. Induction of log-phase recombinant E. coli was performed using 25 µM IPTG in Luria-Bertani ampicillin broth (50 µg/ml ampicillin; Mediatech) for 2 h at 37°C. The recombinant protein is generated as a 35-kDa fusion protein with N-terminal Trx, S-, and His6 tags, while the empty-vector (EV) control recombinant protein (molecular mass 22 kDa) is missing only FhbA. Recombinant proteins were recovered by sonication of IPTG-induced bacteria in the presence of 287 µM PMSF followed by centrifugation at 100,000 x g for 70 min at 4°C. Recombinant proteins were purified to >95% homogeneity by affinity (Ni column) chromatography and gel-filtration.

PCR analysis of fhba1 and fhba2 genotype

PCR analysis and PCR primers have been described previously by Marconi and colleagues (28).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Vmp-independent IgM response during B. hermsii infection

In naive wild-type mice, the primary bacteremic episode of a given B. hermsii strain is of short duration (3 days at most), independent of its serotypically distinct Vmp expression (10). Relapses persist for even shorter periods—no more than 2 days—a time period that precludes generation of a Vmp-specific, high-affinity Ab response by somatic hypermutation of Ig variable regions. Although most of the rapid Ab response is directed at the abundantly expressed Vmps, a number of other minor B. hermsii surface proteins and lipoproteins (i.e., non-Vmp Ags) may also induce specific IgM Ab responses that could play a role in the generation of protective immunity in addition to the anti-Vmp response.

To determine the extent of a non-Vmp-specific response, IgM generated in response to infection with B. hermsii strain DAH expressing Vmp2 was tested for reactivity to strain HS1 expressing Vmp33. It has been reported that the Vmp33 of B. hermsii is expressed in either ticks or in vitro-cultured bacteria but not in in vivo-adapted bacteria (23). Despite this, IgM generated in wild-type mice during DAH-Vmp2 infection unexpectedly bound DAH-Vmp2 and HS1-Vmp33 equally well (Fig. 1). Because the ELISA assay using intact bacteria measures all surface-exposed Ags of B. hermsii including Vmps, we assessed the extent of a possible Vmp crossreactivity by testing the IgM specificity for HS1-Vmp–. This is a noninfectious, high passage derivative of HS1, which has been previously shown to lack Vmp expression (22). The HS1-Vmp– derivative also bound efficiently to IgM generated during the primary episode of DAH-Vmp2 infection (Fig. 1). Importantly, the DAH-Vmp2-induced IgM response does not recognize unrelated bacterial pathogens (21). Collectively, these data indicate that the IgM response to B. hermsii includes non-Vmp Ags. Consistent with the lack of a role for somatic hypermutation (14), DAH-Vmp2-infected AID–/– mouse IgM recognized both Vmp-deficient and Vmp-sufficient B. hermsii (Fig. 1).


Figure 1
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  		FIGURE 1. Vmp-independent IgM response during B. hermsii infection. C57BL/6 (wild-type) or AID–/– mice were infected with 5 x 104 DAH-Vmp2, and specific IgM responses to plates coated with the same strain (DAH-vmp2) or other strains (HS1-vmp33 or HS1-vmp–) were measured by ELISA (see Materials and Methods).

 
A T cell-independent response to a non-Vmp Ag of ~18 kDa during B. hermsii infection

Typically the relapse of B. hermsii bacteremia is less severe than the preceding bacteremic episode, and infection with B. hermsii is self-limiting in the murine model by 3–4 wk postinfection (13, 14). This indicates that, despite the bacterium’s ability to use antigenic variation using an elaborate repertoire of vmp gene rearrangement, there is a progressive development of host resistance toward B. hermsii. As Vmp-specific antisera typically do not react with other Vmps (29), crossreactivity with B. hermsii Vmps is unlikely to be accountable for the decreased severity of relapses. Because the IgM response tested above measured Ab binding to intact bacteria, we aimed to identify potential non-Vmp Ags recognized by the immune serum obtained at four time points that temporally corresponded to the resolution of bacteremic episodes.

Immunoblots of B. hermsii DAH-Vmp2 lysates revealed that the IgM of the infected mice bound to a number of bacterial molecules. The IgM response to Vmp2 peaked rapidly during the 1st bacteremic episode but gradually declined concurrent with the resolution of recurrent episodes (Fig. 2B). Interestingly, the IgM response to a band at ~18 kDa (Fig. 2A; p18) progressively increased during the course of infection, and the intensity of this reaction temporally corresponded to the convalescence of each of the four sequential bacteremic episodes (Fig. 2B). This increased intensity of IgM reactivity also occurred to a comparable degree in TCR-β x {delta}–/– mice, demonstrating that p18 is a T-independent Ag (Fig. 2C). We have previously shown that the resolution of all episodes of bacteremia and the kinetics of B. hermsii-specific IgM in AID–/– mice are indistinguishable from those in wild-type mice (14) (Fig. 1). Consistent with this, we also found an increased reactivity of IgM from AID–/– mice to p18 (Fig. 2D). Thus, the increased reactivity of IgM is not due to an increase in Ab affinity by somatic hypermutation of Ig variable regions, but it is likely due to an expansion of p18 Ag-specific B cells. Because the resolution of B. hermsii infection is concurrent with a selective expansion of B1b but not B1a or B2 cells, we tested whether convalescent B1b cells generate IgM specific for p18. Immunoblot analysis using sera from Rag1–/– mice reconstituted with purified B1b cells from convalescent mice (see Materials and Methods) revealed IgM reactivity to p18 (Fig. 2E), demonstrating that p18-specific IgM can be generated by B1b cells.


Figure 2
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  		FIGURE 2. A progressively enhanced IgM reactivity to a non-Vmp Ag of ~18 kDa. Bacterial lysates (strain DAH-Vmp2; 1.25 x 106 spirochetes/well) were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Resolved proteins were stained with (A) Coomassie blue or probed with (B) wild-type mouse serum, (C) TCR-β x {delta}–/– mouse serum, or (D) AID–/– mouse serum from indicated days postinfection (dpi), and the IgM response was detected. E, Rag1–/– mice were reconstituted with convalescent B1b cells, and serum samples from these mice were taken at 2 and 10 days postinfection and used to probe SDS-PAGE-resolved bacterial lysates, and IgM response was detected. Protein bands corresponding to flagellin, Vmp2, and p18 are indicated.

 
p18 is a B. hermsii outer-surface protein

If p18 is indeed a potential target of a B1b-mediated IgM response, we expected that p18 should be exposed on the cell surface for accessibility to IgM. To test this possibility, intact DAH-Vmp2 cells were treated with increasing concentrations of proteinase K to degrade all surface proteins. Flagellin, which is present in the periplasmic space and is inaccessible to proteinase K, was not digested (Fig. 3A). As expected, proteinase K degraded the abundant outer-surface protein Vmp2 in a concentration-dependent manner (Fig. 3, A and B). Similarly, the less abundant protein p18 was also degraded, as revealed by immunoblot analysis using convalescent wild-type mouse serum IgM (Fig. 3B) or convalescent B1b-derived IgM (Fig. 3C), indicating that p18 is expressed on the cell surface. Because p18 appears to be expressed at very low levels (it is visible by immunoblot analysis but not readily by Coomassie stain; see Fig. 3, A or D vs B or C), we sought to confirm its surface accessibility by an independent approach. Intact B. hermsii cells were treated with membrane-impermeable biotinylation reagent Sulfo-NHS-LC-Biotin to label surface-exposed bacterial molecules. As expected, immunoblots probed with anti-biotin revealed a number of surface molecules that were biotinylated (Fig. 3E), including those corresponding to Vmp2 and p18 as detected by immunoblot analysis with convalescent wild-type serum IgM (Fig. 3F) or convalescent B1b cell-derived IgM (Fig. 3G), but not to the abundant flagellin protein (Fig. 3E). Taken together, these data demonstrate that p18 is a bacterial cell-surface protein.


Figure 3
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  		FIGURE 3. p18 is a B. hermsii cell surface-exposed protein. In vivo-adapted B. hermsii cells (strain DAH-Vmp2) were subjected to proteinase K digestion of cell-surface proteins. Lysates (106 spirochetes/well) were resolved by SDS-PAGE on 10–20% Tris-tricine gels and transferred to polyvinylidene difluoride. Membranes were stained with (A) Coomassie blue or probed with (B) convalescent wild-type mouse serum (24 days postinfection serum) or (C) immune sera from a Rag1–/– mouse reconstituted with convalescent B1b cells, and the IgM response was detected. B. hermsii cells treated with (+) or without (–) biotin and the bacterial lysates were resolved and transferred to polyvinylidene difluoride membranes and stained with (D) Coomassie blue or probed with (E) anti-biotin Abs, (F) convalescent wild-type mouse serum (24 days postinfection serum), or (G) immune sera from a Rag1–/– mouse reconstituted with convalescent B1b cells (10 days postinfection serum), and the IgM response was detected.

 
p18 of B. hermsii is FhbA

To determine the identity of the B. hermsii Ag with the molecular mass of 18 kDa (i.e., p18) recognized by IgM, convalescent serum from DAH-infected AID–/– mice, which produce IgM that recognizes p18 (see Fig. 2D), was used as a capture Ab to enrich p18 by immunoprecipitation (14). Tryptic peptides of p18 were analyzed by LC-MS/MS. The results from the peptide fragment analysis are summarized in Table I. Three B. hermsii proteins matched at least six peptides obtained from LC-MS/MS: a Vmp, FhbA, and flagellin. We eliminated Vmp and flagellin as the identity of p18, because their molecular masses are 22 and 35 kDa, respectively, and because we have detected them as distinct bands by Western blot with specific Abs (data not shown).


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  		Table I. B. hermsii proteins identified by LC-MS/MS analysis of tryptic peptides derived from immunoprecipitated bacterial lysatesa

 
To confirm the identity of p18 as FhbA, we expressed FhbA from strain DAH as a recombinant fusion protein in an inducible E. coli expression system. The recombinant proteins contained 17-kDa N-terminal fusions with Trx, S, and His tags for a final molecular mass of 35 kDa (rFhbA). A 22-kDa EV fusion protein expressing only the N-terminal tags was also generated (Fig. 4A). As expected, immunoblots revealed that S-protein was reactive with both the 22-kDa EV control protein as well as the 35-kDa rFhbA (Fig. 4B). In contrast, probing immunoblots with convalescent wild-type mouse IgM (24 days postinfection serum) or convalescent B1b cell-derived IgM revealed reactivity to the 35-kDa FhbA-containing full-length fusion protein but not to the 22-kDa tag portion of the protein (Fig. 4, C and D). As expected, neither naive wild-type serum IgM (data not shown) nor naive B1b cell-derived IgM recognized rFhbA (Fig. 4E). Moreover, purified rFhbA competitively inhibited the binding of both convalescent wild-type mouse IgM (Fig. 4F) as well as convalescent B1b cell-derived IgM (Fig. 4G) to p18 of strain DAH, indicating that p18 is FhbA. These data demonstrate that anti-FhbA IgM is generated in a progressive manner during relapsing fever in wild-type mice, and that the convalescent B1b cell repertoire recognizes this putative virulence factor of B. hermsii.


Figure 4
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  		FIGURE 4. p18 of B. hermsii is factor H-binding protein. An IPTG-inducible E. coli strain BL21(DE3) was transformed with pET32-FhbA or the empty-vector control pET32-EV. Transformed cells were with held IPTG or induced in the presence of 25 µM IPTG. Bacterial cells were lysed and the cytoplasmic contents were resolved on 12.5% SDS-PAGE gel and blotted onto polyvinylidene difluoride membranes. Blots were either stained with (A) Coomassie blue or probed with (B) S protein-alkaline phosphatase, (C) convalescent wild-type mouse serum (24 days postinfection serum), (D) 10-day postinfection sera from a Rag1–/– mouse reconstituted with convalescent B1b cells, or (E) 10-day postinfection sera from a Rag1–/– mouse reconstituted with naive B1b cells (see Materials and Methods), and the IgM response was detected. F and G, IgM reactivity was also tested in the presence of 0.5 or 10 µg/ml purified rFhbA1 or rFhbA2 as competitor.

 
The gene for FhbA is encoded on a 220-kb linear plasmid of B. hermsii. Two genetically distinct FhbA types, designated fhbA1 and fhbA2, have been described (28). Although the factor H-binding capabilities of the two isoforms are similar, FhbA2 is slightly larger than FhbA1 due to a tandem repeat (LLKTLDN) within the N-terminal region of FhbA2 (30). Sequence analysis of the fhba gene from the strain DAH used extensively in our experimental infection system revealed its homology to FhbA1 (GenBank accession EU330200), in part due to the lack of the LLKTLDN repeat. PCR analysis of the DAH fhba gene using primers specific for fhbA1 (28) amplified the anticipated 172-bp product (Fig. 5A), demonstrating that the B. hermsii strain DAH expresses FhbA1 but not FhbA2. IgM from B1b cells isolated from mice recovered from DAH infection specifically recognized other B. hermsii strains expressing FhbA1, but not OKA, which expresses FhbA2 (Fig. 5B). Furthermore, the same B1b-derived IgM recognized rFhbA1, but not rFhbA2. Purified rFhbA1 but not rFhbA2 competitively inhibited the binding of convalescent B1b-derived IgM to p18 (Fig. 4G). These data together demonstrate that FhbA is a specific antigenic target for B1b cells.


Figure 5
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  		FIGURE 5. B1b cell response is specific to FhbA1 but not to FhbA2. A, PCR was performed on clinical isolates FRE, SWA, DAH, and OKA using FhbA variant-specific primers, and PCR products were resolved by 2.5% agarose gel electrophoresis. B, Clinical isolates FRE, SWA, DAH, and OKA, as well as rFhbA1 and rFhbA2, were resolved by SDS-PAGE on 12.5% gels and probed with convalescent B1b cell-derived IgM. Blots were probed with anti-flagellin Abs or S-protein alkaline phosphatase as loading controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Resistance to reinfection is the reflection of the development of B cell memory. Although naive mice suffer recurrent episodes of bacteremia due to antigenic variation, convalescent mice are resistant to reinfection by B. hermsii. A novel T cell-independent IgM memory mediated by B1b cells confers this protective immunity (14). Using a Rag1–/– reconstitution experimental system, we show that IgM derived from convalescent but not naive B1b cells specifically recognizes the B. hermsii surface Ag FhbA, a putative virulence factor implicated in serum resistance (6). This is the first example of a bacterial protein Ag driving a likely protective B1b cell response during an infection. FhbA is present in all B. hermsii clinical isolates and is not associated with antigenic variation, suggesting that the development of an Ag-specific B1b cell response to this Ag may help the immune system to overcome not only the Vmp-mediated immune evasion strategy but also complement resistance.

Although the mechanism by which convalescent, but not naive, B1b cells confers protection against B. hermsii infection is yet not known (14), an attractive possibility may be an increase in the precursor frequency of Ag-specific B1b cells in B. hermsii-infected mice. Consistent with such a possibility, we found a specific expansion of B1b cell numbers in convalescent mice (13, 14) that correlates with a progressive increase in FhbA reactivity (Fig. 1). Because IgM derived from convalescent but not naive B1b cells recognizes FhbA of B. hermsii, it is likely that this bacterial protein drives Ag-specific B1b expansion by stimulating the BCR of potential B1b cell precursors. It is known that stronger BCR stimulation is required for the generation of both B1a and B1b cell subsets than for FO B cells (31). Critical in mediating BCR signaling is Btk. Thus, X-linked immunodeficient (xid) mice, which have a mutation in the gene encoding Btk, are severely deficient in B1a and B1b cells (32, 33). Despite this deficiency, B1b cells are dramatically expanded in convalescent xid mice with absolute cell numbers comparable to wild-type levels (13). It is known that xid B cells are impaired in homeostatic (Ag-independent) expansion (34) but not in Ag-driven B cell expansion when costimulatory signals are provided (35). We have also found that serum from convalescent xid mice specifically recognizes B. hermsii (21) and FhbA as efficiently as wild-type convalescent B1b-derived IgM (data not shown). Thus, the identification of B. hermsii FhbA as a B1b cell-specific Ag supports the notion that B1b expansion during this infection is an Ag-driven process and that the enhanced protection by convalescent B1b cells is likely to be due to an increase in Ag-specific B1b cell numbers, as described for conventional T cell-dependent B cell memory.

Convalescent but not naive serum IgM confers passive protection to relapsing fever Borrelia spp. (12, 36, 37, 38). Although the mechanism for IgM-mediated protection is not known, several hypotheses have been proposed. Specific IgM or their F(ab')2 fragments have the capacity to destroy relapsing fever spirochetes in vitro, suggesting that this novel mechanism could be responsible for B. hermsii clearance in vivo (38). The high-affinity IgM receptor Fc{alpha}µR (39) that is capable of promoting phagocytosis of IgM-opsonized bacteria in vitro has also been suggested to facilitate the clearance of relapsing fever spirochetes in vivo. Consistent with this possibility, immune serum, but not preimmune serum, significantly increased phagocytosis of B. hermsii by polymorphonuclear leukocytes (40). To promote any of these effector functions mediated by IgM requires specific interaction with the bacterial surface. For example, the specific binding of monoclonal IgM to Vmp7 has been shown to confer protection both in vitro and in vivo against B. hermsii expressing Vmp7 but not other Vmps (12). Unlike Vmp-specific IgM, the IgM derived from convalescent B1b cells isolated from strain DAH-Vmp2-infected mice specifically recognizes a common Ag, FhbA, found in other clinical isolates regardless of their distinct Vmp expression, indicating that B1b-mediated protection could be independent of Vmp expression. Future studies involving passive immunization experiments using FhbA-specific IgM mAbs derived from B1b cell hybridomas may provide a better evaluation of the potential cross-protection of other B. hermsii serotypes and the mechanisms of IgM-mediated protection.

Successful establishment of infection in the blood may also depend on the evasion of the complement cascade. Many pathogenic organisms, including Borrelia burgdorferi and Bordetella pertussis, express binding proteins for soluble serum complement regulators factor H- and C4-binding protein, respectively (41). The expression of complement regulator-acquiring surface protein 1 (CRASP-1), a factor H-binding protein on B. burgdorferi, has been correlated with the inactivation of the complement system by this bacterium (42). Deletion of the B. burgdorferi gene encoding for CRASP-1 results in a dramatic increase in serum sensitivity, while genetic complementation of CRASP-1 promotes serum resistance (43). Analogous to the complement resistance property exhibited by the related pathogen B. burgdorferi, expression of FhbA may facilitate B. hermsii in achieving serum resistance (6, 28, 30, 44). Because the constitutive expression of FhbA is presumably vital for B. hermsii survival in the host, such molecules provide an attractive antigenic target for the host immune system. Our identification of FhbA as a target for B1b cells suggests that specific IgM binding to the bacterial surface may not only enhance the above-mentioned IgM-mediated clearance mechanisms, but it may also make B. hermsii susceptible to complement-mediated mechanisms by preventing the interaction of factor H with the bacterium.

The functionality of B1b cells in protective immunity is only beginning to be understood (35). Ags driving B1b cell responses appear to be heterogeneous. For example, FhbA of B. hermsii is a protein implicated in virulence of this pathogen (6). In the case of Streptococcus pneumoniae, it is the capsular polysaccharide, a well-known T cell-independent Ag (45), whose expression is also implicated in a variety of immune evasion strategies. Thus, the recognition of biochemically distinct Ags (e.g., carbohydrates, proteins, and haptens such as 4-hydroxy-3-nitrophenyl acetyl-conjugated Ficoll (46)) by B1b cells clearly indicates that the B1b repertoire is capable of responding to a wide spectrum of Ags. Unlike pneumococcal polysaccharide or Ficoll, B. hermsii induces functional (i.e., protective) B cell responses including specific IgM secretion (21) and B1b cell expansion, indicating that B. hermsii engages protective mechanisms distinct from previously described T cell-independent Ags (35). Although the exact mechanism is not clear, B. hermsii stimulates not only BCR signaling but also TLR signaling pathways that are critical for the induction of a specific IgM response (21). Unlike the T cell-independent Ags mentioned above, FhbA is lipidated (6) and is likely to be recognized by TLR2.

Among the mature B cell subsets, the biology of B1b cells is the least studied, and a functional role for B1b cells had not been described until recently (35). Although it is unknown whether control of B. hermsii infection is dependent on human phenotypic equivalents of mouse B1b cells, there could exist functional equivalents of B1b cell in humans. The murine model of relapsing fever recapitulates a number of aspects of human disease, including the recurrent episodes of bacteremia as well as antigenic variation. Thus, a functionally similar Ab response might play a role in controlling bacteremic episodes in both species. Indeed, serological analysis revealed that both infected mouse sera and sera of patients with relapsing fever recognized FhbA similarly (28). In addition to providing novel approaches to overcome Ag variation, the identification of FhbA as an antigenic target for B1b cells will aid in the development of molecular tools to help define the biology of B1b cells, including the formation an Ag-specific B1b cell memory.


   Acknowledgments
 
We thank Dr. Tom G. Schwan for providing B. hermsii strains; Drs. Jianzhu Chen and Tasuku Honjo for providing sIgM–/– and AID–/– mice, respectively; Dr. Tim Manser, Dr. Laurence Eisenlohr, and Kathy Reinersmann for a critical review of the manuscript; and Guizhi Sun, Natasha Zaveri, and Sasidhar Madugula for Vmp serotyping the B. hermsii strains used in the study.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant RO1 AI065750 (to K.R.A.). Back

2 Address correspondence and reprint requests to Dr. Kishore R. Alugupalli, Department of Microbiology and Immunology, Thomas Jefferson University, 233 South 10th Street, BLSB 726, Philadelphia, PA 19107. E-mail address: kishore.alugupalli{at}mail.jci.tju.edu Back

3 Abbreviations used in this paper: Vmp, variable major protein; AID, activation-induced cytidine deaminase; Btk, Bruton’s tyrosine kinase; CRASP-1, complement regulator-acquiring surface protein 1; EV, empty vector; FhbA, complement factor H-binding protein A; FO, follicular; IPTG, isopropyl β-D-thiogalactoside; LC-MS/MS, liquid chromatography/mass spectrometry/mass spectrometry; MZ, marginal zone; rFhbA, recombinant FhbA; xid, X-linked immunodeficient. Back

Received for publication December 11, 2007. Accepted for publication January 28, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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