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*Lyme Disease
The Journal of Immunology, 2001, 167: 3383-3390.
Copyright © 2001 by The American Association of Immunologists

Borrelia burgdorferi-Induced Inflammation Facilitates Spirochete Adaptation and Variable Major Protein-Like Sequence Locus Recombination1

Juan Anguita*,{dagger}, Venetta Thomas*, Swapna Samanta*, Rafal Persinski{dagger}, Carmen Hernanz*, Stephen W. Barthold{ddagger} and Erol Fikrig2,*

* Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; {dagger} Department of Biology, University of North Carolina, Charlotte, NC 28223; and {ddagger} Center for Comparative Medicine, University of California, Davis, CA 95616


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Spirochete adaptation in vivo is associated with preferential Borrelia burgdorferi gene expression. In this paper, we show that the administration of B. burgdorferi-immune sera to IFN-{gamma}R-deficient mice that have been infected with B. burgdorferi N40 for 4 days causes spirochete clearance. In contrast, immune sera-mediated clearance of B. burgdorferi N40 is not apparent in immunocompetent mice, suggesting a role for IFN-{gamma}-mediated responses in B. burgdorferi N40 host adaptation. B. burgdorferi-immune sera also induces clearance of B. burgdorferi N40 that have been passaged in vitro 75 times (B. burgdorferi N40-75), a derivative of B. burgdorferi N40 that does not rapidly adapt in vivo in immunocompetent mice. B. burgdorferi N40-75 produce lower levels of IFN-{gamma} and IL-12 in mice than does B. burgdorferi N40, and the administration of these cytokines to B. burgdorferi N40-75-infected mice results in an increased spirochetal burden, further indicating that IFN-{gamma}-mediated events promote B. burgdorferi survival. Differential immunoscreening and RT-PCR demonstrate that IFN-{gamma}-mediated signals facilitate spirochete recombination at the variable major protein like sequence locus, a site for early antigenic variation in vivo, and that recombination rates by B. burgdorferi N40 are lower in IFN-{gamma}R-deficient mice than in control animals. These results suggest that the murine immune response can promote the in vivo adaptation of B. burgdorferi.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lyme disease, caused by the spirochete Borrelia burgdorferi, can affect multiple organ systems including the skin, joints, heart, and nervous system (1, 2). Both spirochete virulence and the host response to B. burgdorferi can influence the outcome of infection (3, 4, 5, 6, 7, 8). CD4+ T cell differentiation affects the course of disease in humans and mice (4, 5, 8, 9, 10, 11, 12). Increased levels of IL-12 (4) and IFN-{gamma} (5, 8) correlate with the severity of murine Lyme arthritis, and Abs to these cytokines (4, 5, 8) can reduce the degree of joint inflammation. Moreover, the passage of B. burgdorferi in vitro resulted in selection of noninfectious spirochetes with alterations in plasmid and protein profiles (13, 14, 15, 16, 17). The ospD gene, present on the 38-kb linear plasmid (lp)3 38, was reported to be absent in noninfectious B. burgdorferi B31 (18). More recently, lp28-1 and lp25 have been associated with the infectivity of B. burgdorferi B31 (19, 20). Noninfectious B. burgdorferi N40 that have been passaged 120 times in vitro (B. burgdorferi N40-120) lack both lp28-1 and lp38 (21). The factors that contribute to spirochete infectivity and the pathogenesis of Lyme disease are thus clearly multifactorial, involving close interactions between B. burgdorferi and the host.

B. burgdorferi N40 that has been passaged in vitro 75 times (B. burgdorferi N40-75) demonstrate an intermediate phenotype between the highly infectious and pathogenic B. burgdorferi N40 and the noninfectious B. burgdorferi N40-120. B. burgdorferi N40-75 can infect mice but does not cause arthritis (22). B. burgdorferi N40-75 differs from B. burgdorferi N40 in its ability to rapidly adapt to the host (22). Whereas B. burgdorferi N40 promptly up-regulates the expression of diverse genes upon infection, preferential gene expression in vivo is delayed in B. burgdorferi N40-75 (22). This delay in in vivo gene expression results in an increased sensitivity of B. burgdorferi N40-75 to B. burgdorferi-immune sera compared with B. burgdorferi N40 (22). The passive administration of B. burgdorferi-immune sera to mice that have been infected with B. burgdorferi N40 for 4 days does not influence infection (22). In contrast, at 4 days, B. burgdorferi-immune sera can abort an ongoing infection with B. burgdorferi N40-75 (22). This difference is due to the inability of B. burgdorferi N40-75 to rapidly adapt to the host, thereby allowing the Abs in B. burgdorferi-immune sera to clear the infection (22).

To persist in mice, B. burgdorferi must actively evade the host immune response. One mechanism that may contribute to B. burgdorferi survival is recombination at the variable major protein-like sequence (vls) gene locus (23). This recombination could potentially help spirochetes avoid destruction by Ab responses to the VlsE (vls expression site) protein variants that arise during infection (23). The vls gene cluster has been characterized in B. burgdorferi B31. It consists of a vlsE located near the right telomere of the lp28-1 and 15 silent cassettes upstream. The vlsE encodes a surface-exposed protein of 34 kDa with three defined domains: two constant regions at the amino and carboxyl termini and an internal variable segment, which is composed of six invariable and six variable regions (23, 24, 25). Little is known about the signals that trigger recombination events at the vls locus, and alterations in temperature are not related to these changes (26). Recombination occurs in vivo as soon as 4 days after experimental infection of mice, but not in vitro (26), suggesting that the mammalian host provides the signal for vls recombination. We herein examine the ability of B. burgdorferi to adapt in vivo and undergo recombination at the vls locus in response to host immune responses, particularly IFN-{gamma}-mediated signals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C3H/HeNCr (C3H) mice were purchased from the Frederick Cancer Research Center (Frederick, MD). IFN-{gamma}R{alpha}-deficient mice on a 129/SvJ background and 129/SvJ control animals were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in filter-framed cages and euthanized with CO2.

B. burgdorferi and infections

A clonal isolate of B. burgdorferi N40 that is infectious and pathogenic in mice was used (27). B. burgdorferi N40-75 is a derivative of B. burgdorferi N40 that was isolated by in vitro passage. B. burgdorferi N40-75 is infectious to immunocompetent mice but does not cause arthritis or carditis and is therefore considered nonpathogenic (22). Infectious and pathogenic low passaged B. burgdorferi B31 were also used for some infection studies. Spirochetes were grown in Barbour-Stoenner-Kelly (BSK) II medium at 34°C.

Individual mice were inoculated with 102 or 104 spirochetes in the midline of the back by intradermal injection (4). Mice were assessed for infection by culturing specimens of the spleen, blood, urinary bladder, and skin (at the inoculation site) in BSK II medium at 33°C. Cultures were read after 14 days, a time period sufficient to allow a single spirochete to grow to stationary phase.

Formalin-fixed, paraffin-embedded hearts and joints (both knees and tibiotarsi) were examined microscopically for evidence of disease (4). Arthritis and carditis were assessed as previously described (4). All histopathologic assessments were made in a blinded fashion.

RNA purification and RT-PCR

At the time of sacrifice, whole splenocytes were depleted of RBCs, and RNA was extracted by the thioisocyanate method (28) using the reagents and protocol of the Micro RNA Isolation kit following the manufacturer’s instructions (Stratagene, La Jolla, CA). Five to 10 µg were used to obtain cDNA with an RT-PCR kit (Stratagene) using random primers. Semiquantitative PCR was performed as previously described (29). Briefly, 4-fold diluted (newly made) cDNA was used at 1/10, 1/100, and 1/500 dilutions to perform PCR using primer pairs specific for IFN-{gamma} and the p35 subunit of IL-12. Primers for hypoxanthine phosphoribosyltransferase (HPRT; control) insured that equal amounts of cDNA were used. Conditions of PCR were 25 cycles at 96°C for 1 min, 50°C for 30 s, and 72°C for 1 min (29). After the PCR, samples were run in a 2% agarose gel, transferred to a nylon filter (Hybond-N, Amersham Pharmacia Biotech, Piscataway, NJ), hybridized to internal oligonucleotide probes, and labeled using the 3'-oligonucleotide labeling kit (Amersham Pharmacia Biotech). Oligonucleotide primers and probes used were: HPRT forward, 5'-AGCGTCGTGATTAGCGATGATG-3'; HPRT backward, 5'-TTATAGCCCCCCTTGAGCACAC-3'; HPRT probe, 5'-CCTCATGGACTGATTATGGACAG-3'; IFN-{gamma} forward, 5'-TTTGGACCCTCTGACTTGAGACAG-3'; IFN-{gamma} backward, 5'-CGAATCAGCAGCGACTCCTTTTC-3'; IFN-{gamma} probe, 5'-TTGCAGCTCTTCCTCATGG-3'; IL-12 (p35) forward, 5'-ACCACAGATGACATGGTGAAGACGG-3'; IL-12 (p35) backward, 5'-TGCTTCTCCCACAGGAGGTTTC-3'; and IL-12 (p35) probe, 5'-CAGCACATTGAAGACCTGTTTACC-3'.

Quantification of IFN-{gamma} and IL-12

The levels of IFN-{gamma} and IL-12 in culture supernatants and murine sera were measured by ELISA as previously described (4). Briefly, 96-well ELISA plates (ICN Pharmaceuticals, Costa Mesa, CA) were coated with the capture Ab (2 µg/ml) overnight at 4°C. After blocking with PBS plus 10% FCS for 2 h at room temperature, samples were applied and incubated for 1 h at 37°C. The biotinylated detection Ab (1 µg/ml) was added after washing the plates with PBS plus 0.5% v/v Tween 20 (PBS/Tween 20). Quantification of cytokine levels was made after incubating the plates with HRP-conjugated avidin and adding the substrate for the enzyme (tetramethylbenzidine (TMB), Kirkegaard & Perry Laboratories, Gaithersburg, MD) and stop solution (TMB 1 Component Stop Solution, Kirkegaard & Perry Laboratories). The values indicated were calculated by comparing the values obtained with those derived using standard concentrations of recombinant mouse IFN-{gamma} (BD PharMingen, San Diego, CA) and IL-12 (R&D Systems, Minneapolis, MN).

In vitro stimulation of whole splenocytes

Whole splenocytes were obtained by mechanical disruption of the spleen, followed by hypotonic lysis of RBCs. A total of 106 cells/ml were incubated with 20 µg/ml of B. burgdorferi N40 or B. burgdorferi N40-75 extracts for 72 h, and the supernatants were collected and analyzed for levels of IFN-{gamma} by ELISA.

In vivo treatments

Recombinant murine IL-12 and IFN-{gamma} were purchased from R&D Systems. PBS plus 1% normal mouse serum (NMS) was used as a control solution. The cytokines were i.p. injected into mice starting on the day of spirochete inoculation, then daily for 5 days, and then every 2 days through day 21. The injection doses ranged from 1 to 5 µg of recombinant cytokines in 100 µl PBS plus 1% NMS.

CD4+ T cell restimulation in vitro

At the time of sacrifice, CD4+ T cells were purified by negative selection as previously described (4). A total of 106 CD4+ T cells/ml were stimulated with B. burgdorferi extracts (10 µg/ml) in the presence of 106 mitomycin C-treated (50 µg/ml for 40 min at 37°C) syngeneic spleen cells as APCs. The supernatants were collected after 40 h of incubation and analyzed by ELISA for the quantification of IFN-{gamma}.

In vivo sera treatments

Susceptibility of B. burgdorferi N40 to immune sera in the murine host was studied in IFN-{gamma}R{alpha}-deficient and control 129/SvJ mice as previously described (22, 30). Mice were i.p. administered 50 µl immune sera, a dose sufficient to eliminate nonadapted spirochetes from the murine host, at the time of spirochete inoculation and 4 days after infection (22, 30). Immune sera were obtained from 129/SvJ mice that had been infected with B. burgdorferi N40 for 21 days. The mice were necropsied 14 days after challenge and assessed for infection (22).

Immunoscreening of a genomic expression library

A B. burgdorferi N40 genomic expression library in {lambda}ZAP II (31) was differentially screened using immune sera from mice infected for 14 days with host-adapted (transplant) or in vitro-grown B. burgdorferi N40. Host-adapted immune sera were from mice implanted with a 5-mm skin biopsy from a B. burgdorferi N40-infected animal (27). In vitro immune sera were from mice infected with a syringe inoculum of 102 B. burgdorferi N40 cultured in BSK medium. Duplicate nitrocellulose filters were obtained for each plate, which contained ~104 plaques. The duplicate filters were screened with the immune sera, and clones that only reacted with sera from the mice exposed to host-adapted B. burgdorferi N40 were rescued for further screening. Potential specific clones were subjected to three rounds of screening. Filters were blocked for 2 h using PBS with 3% BSA (PBS/BSA) and incubated for 1 h with sera (1/100 dilution). Membrane filters were then washed three times using PBS/Tween 20 and were incubated for 30 min with goat anti-mouse IgG in PBS/BSA (1/1000 dilution) conjugated to alkaline phosphatase (Sigma, St. Louis, MO). After washing, the filters were incubated with TMB substrate (Kirkegaard & Perry Laboratories). Positive clones were recovered in sodium magnesium buffer with chloroform. Clones SS10, SS27, SS32, and SS34 were then excised and sequenced using standard protocols. DNA homology comparisons were performed using a BLAST search and ClustalW (32).

Assessment of recombination at the vls locus

IFN-{gamma}R{alpha}-/- and 129/SvJ control mice were infected with 104 B. burgdorferi B31, and 4 days later, the mice were sacrificed and skin biopsies at the inoculation site were cultured in BSK II medium for 2 wk. Spirochetal DNA was then isolated by standard methods and was used to perform PCR using the primers F4064 and R4066 (23). The PCR was then purified and used to ligate into the pCR 2.1 cloning vector (Invitrogen, Carlsbad, CA). Plasmids with insert (determined by EcoRI digestion) were sequenced using the primers M13 reverse and T7. A minimum of 15 different sequences were determined for each group of mice. Sequences were compared with the one from the parental isolate by multiple alignments using MegAlign software (DNASTAR, Madison, WI). Individual nucleotides that were different from the parental allele were counted as sequence changes and summed for each clone. Identical sequences from multiple clones were considered siblings and counted as a single clone. The sum of the sequence changes for each group of clones was divided by the total number of clones included to provide a measure of the average number of sequence changes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Infection with B. burgdorferi N40-75 results in lower proinflammatory cytokine production

To determine whether B. burgdorferi N40-75 differed from B. burgdorferi N40 in the capacity to induce proinflammatory cytokines in vivo, semiquantitative RT-PCR was performed on splenocytes from infected mice. B. burgdorferi N40-75-infected mice showed lower levels of mRNA for IFN-{gamma} and the small subunit of IL-12 (p35) compared with B. burgdorferi N40-infected animals (Fig. 1GoA). The production of IL-4 mRNA was also decreased in N40-75-infected mice, compared with N40-infected animals, suggesting that CD4+ T cell responses to B. burgdorferi were diminished in these mice (Fig. 1GoC). RT-PCR also demonstrated decreased IFN-{gamma} mRNA levels in the joints of B. burgdorferi N40-75-infected animals compared with B. burgdorferi N40-infected mice (Fig. 1GoB). Consistent with these results, the amounts of IFN-{gamma} and IL-12 (p70) in sera were also diminished in B. burgdorferi N40-75-infected mice compared with B. burgdorferi N40-infected animals (Fig. 1GoD). The amount of IFN-{gamma} produced by CD4+ T cells isolated from B. burgdorferi N40-75-infected mice that were stimulated in vitro with B. burgdorferi extracts was also reduced compared with B. burgdorferi N40-infected animals (Fig. 1GoE).



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  		FIGURE 1. Infection with B. burgdorferi N40-75 results in lower proinflammatory cytokine production and innate immune cell activation. A, Whole splenocytes from 3-wk-infected C3H mice were used to obtain total RNA. Expression of IFN-{gamma} and the small subunit of IL-12 were analyzed by RT-PCR and hybridization with specific internal probes. HPRT (control) was included to assure equal loads of RNA. B, Expression of IL-4 was also analyzed by the same method. C, IFN-{gamma} expression was measured in the joints of C3H mice infected with B. burgdorferi N40-75 or B. burgdorferi N40. Total RNA was extracted as described previously and analyzed by RT-PCR. HPRT (control) assured equal loads of RNA. D, The levels of IFN-{gamma} and IL-12 were measured by ELISA in the sera of mice infected with B. burgdorferi N40-75 or B. burgdorferi N40 for 3 wk. Results represent a pool of sera from five mice and are representative of five individual experiments. E, CD4+ T cell restimulation in response to B. burgdorferi Ags. CD4+ T cells were purified from the spleens of 3-wk-infected C3H mice and restimulated in vitro with 10 µg/ml of a B. burgdorferi extract. The data presented are representative of three separate experiments with similar results. F, B. burgdorferi N40-75 and B. burgdorferi N40 extracts were used to activate innate immune cells in vitro. Whole splenocytes from C3H mice were stimulated for 48 h with 20 µg/ml spirochetal extracts and analyzed for IFN-{gamma} production by capture ELISA.

 
The reduced production of IFN-{gamma} by B. burgdorferi N40-75 in vivo could be the result of an altered bacterial burden in mice, a diminished capacity of B. burgdorferi N40-75 to induce proinflammatory cytokine production in mice, or both. We have previously shown that B. burgdorferi N40-75 burdens are significantly reduced in immunocompetent mice at 2 wk of infection (22). Therefore, we assessed the ability of B. burgdorferi N40-75 to generate IFN-{gamma} in whole splenocytes in vitro. The cells were stimulated with extracts of B. burgdorferi N40 and B. burgdorferi N40-75, and the supernatants were analyzed 72 h later by ELISA. The amount of IFN-{gamma} produced by splenocytes that were stimulated with B. burgdorferi N40-75 was lower than the quantity produced by cells stimulated with B. burgdorferi N40 (Fig. 1GoF). These results suggest that infection with B. burgdorferi N40-75 results in lower proinflammatory cytokine production, due to the decreased capacity of these spirochetes to activate innate immune cells and the lowered bacterial burdens present in mice infected with high passaged spirochetes.

IL-12 and IFN-{gamma} treatment during infection restores pathogenicity of B. burgdorferi N40-75

To further demonstrate a correlation between a lower proinflammatory cytokine expression pattern during infection and the inability of B. burgdorferi N40-75 to induce disease, we infected mice with B. burgdorferi N40-75 and treated them with rIL-12 and rIFN-{gamma}. Administration of IL-12 induced levels of systemic IFN-{gamma} at 3 wk of infection (Fig. 2GoA) and augmented production of this cytokine by CD4+ T cells after in vitro restimulation with B. burgdorferi extracts (Fig. 2GoB), indicating stimulation of a Th1 response. Treatment with IL-12 also resulted in the development of arthritis in the B. burgdorferi N40-75-infected mice (Table IGo). As expected, arthritis was not evident in B. burgdorferi N40-75-infected mice that did not receive this cytokine or in uninfected mice that were treated with IL-12 (Table IGo), suggesting that inflammation was dependent on the presence of the spirochete. Arthritis also correlated with an increase in spirochetal DNA in the joints of the IL-12-treated, B. burgdorferi N40-75-infected mice compared with the control animals (Fig. 2GoC; Fischer’s exact test, p < 0.01, between PBS/NMS- and IL-12-treated groups).



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  		FIGURE 2. IL-12 or IFN-{gamma} increases Th1 responses and spirochetal burdens in B. burgdorferi N40-75-infected mice. Mice were treated with IL-12 (A, B, and C) or IFN-{gamma} (D and E) and infected with B. burgdorferi N40-75. Control mice were infected with B. burgdorferi N40 or treated with the recombinant cytokines without subsequent infection. At the time of sacrifice, the amount of IFN-{gamma} in the pooled sera of the mice was determined by capture ELISA (A and D). CD4+ T cells were purified from B. burgdorferi N40-75-infected, IL-12-treated mice, and controls and restimulated in vitro with B. burgdorferi extracts (B). The supernatants were analyzed for IFN-{gamma} by capture ELISA. The relative amount of spirochetal DNA in the joints of the infected mice was determined by PCR with primers corresponding to the flaB gene (C and E). Equal amounts of DNA were used to perform the PCR (500 ng/reaction). All results are representative of at least two independent experiments.

 

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  		Table I. IL-12 and IFN-{gamma} restore the pathogenicity of B. burgdorferi N40-751

 
To assess whether the effect of IL-12 was due to the increased production of IFN-{gamma}, we also treated B. burgdorferi N40-75-infected mice with IFN-{gamma}. As with IL-12, IFN-{gamma} raised the systemic levels of IFN-{gamma} during infection (Fig. 2GoD), a phenomenon not apparent in control animals. Similarly, the spirochete burden in the joints of the B. burgdorferi N40-75-infected, IFN-{gamma}-treated mice was increased compared with untreated, B. burgdorferi N40-75-infected animals (Fig. 2GoE), and joint inflammation became apparent (Table IGo). These data demonstrate that IL-12 and IFN-{gamma} increased Th1 responses and bacterial loads in the joints of the B. burgdorferi N40-75-infected animals.

B. burgdorferi N40 do not evade immune sera in the absence of IFN-{gamma}-mediated signals

The previous results suggest that the induction of proinflammatory responses during infection with B. burgdorferi N40-75 enables the spirochete to more efficiently colonize the host, even in the presence of an active humoral response. To further investigate whether proinflammatory cytokine production influenced the adaptation of B. burgdorferi, we performed passive transfer experiments (22, 30, 33) in mice deficient in IFN-{gamma} signaling. As expected, the administration of B. burgdorferi-immune sera 4 days after infection in 129/SvJ mice failed to clear B. burgdorferi N40 infection, as demonstrated by culture of the spirochete from tissues (Table IIGo). However, the administration of immune sera to the IFN-{gamma}R{alpha}-deficient mice significantly compromised the ability of B. burgdorferi N40 to establish infection (Table IIGo). Compared with wild-type controls treated with immune sera (13 of 14), only 6 of 14 immune sera-treated, IFN-{gamma}R{alpha}-deficient mice were culture-positive at 2 wk of infection (p < 0.01, Fisher’s exact test). Similarly, the number of tissue specimens that were culture-positive for B. burgdorferi was significantly reduced in IFN-{gamma}R{alpha}-deficient mice (52 vs 14% in 129/SvJ and IFN-{gamma}R{alpha}-deficient mice, respectively; p < 0.0005). IFN-{gamma} is an inducer of FcR expression (34), and therefore, the diminished protective capability of the immune sera could be a consequence of lower Fc-mediated opsonization of spirochetes. Thus, we also passively administered immune sera on the day of infection (22). The treatment prevented infection in both 129/SvJ and IFN-{gamma}R{alpha}-deficient mice (Table IIGo), strongly suggesting that mechanisms other than those mediated through FcRs are important in immune sera-mediated spirochetal killing. These data indicate that B. burgdorferi adaptation is compromised in the absence of IFN-{gamma}-induced signals.


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  		Table II. Immune sera evasion by B. burgdorferi N40 is compromised in the absence of IFN-{gamma}-mediated signals1

 
The difference between in vitro grown and "host-adapted" spirochetes resides within the vls locus

To identify antigenic differences between cultured and host-adapted spirochetes that could account for Abs that provide the protective capacity of the immune sera, we performed a differential immunoscreening procedure as previously described (22). Mice were infected with 102 in vitro-cultivated B. burgdorferi N40 (nonadapted, similar to the number of spirochetes that are present in the skin graft used to infect the mice by tissue transplant) or underwent ear tissue transplant from an immunodeficient donor (host adapted). Two weeks later, sera from these mice were used to probe a B. burgdorferi N40 genomic expression library to identify Ags that only react with sera from animals infected with host-adapted organisms (22). Four clones were obtained that selectively reacted with the sera from mice infected by tissue transplant. Analysis of the clones revealed that all four contained sequences that mapped to the vls locus (with >75% identity between the clones and the cassettes vls2 to vls16, Fig. 3Go). These results suggested a shift in antigenicity of the VlsE protein during infection of mice before transplant infection, resulting in different Ab specificities when immunocompetent mice were challenged by these two methods.



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  		FIGURE 3. Comparison of the sequences of the four clones identified by differential immunoscreening. Sera from mice infected by syringe (nonadapted spirochetes) and transplant (host-adapted spirochetes) were used to probe a B. burgdorferi expression library. Clones that selectively reacted with sera from mice infected with the host-adapted spirochetes were isolated. Gray boxes indicate identical residues. Gaps in the sequence are indicated by dashes.

 
Recombination at the vls locus is impaired in the absence of IFN-{gamma}-mediated signals

To characterize the relationship between proinflammatory cytokine production and vls recombination in vivo, IFN-{gamma}R{alpha}-deficient mice and wild-type controls (129Sv/J) were infected with B. burgdorferi. The sequence that is available for the vlsE gene corresponds to the B. burgdorferi B31 isolate, and reports have shown significant sequence divergence at this particular gene among different isolates (35, 36). Indeed, PCR using primers corresponding to the B31 sequence does not amplify the expected product in B. burgdorferi N40 (data not shown). Therefore, we used a low passage B. burgdorferi B31 isolate that is infectious and causes arthritis and carditis in mice. Four days after infection, skin biopsies at the inoculation site were cultured in BSK II medium, and the spirochetes were allowed to grow to stationary phase. DNA was then extracted, and PCR was performed using primers specific for the constant flanking region of the vls gene that results in the amplification of the variable domain of the gene (23). The analysis of 30 sequences obtained by this method revealed that B. burgdorferi recovered from the IFN-{gamma}R{alpha}-deficient mice had recombined with a significantly lower frequency compared with B. burgdorferi recovered from 129/SvJ mice (Fig. 4Go). Only 4 of 11 vls sequences contained changes compared with the parental clonal isolate in IFN-{gamma}R{alpha}-deficient mice, whereas 11 of 19 sequences were changed in wild-type animals (Fig. 4Go; average changes per clone: 129/SvJ mice, 4.4 ± 1.0; IFN-{gamma}R{alpha}-deficient mice, 1.7 ± 0.9; p = 0.015). These data indicate that vls recombination in B. burgdorferi is impaired in the absence of IFN-{gamma}-derived signals.



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  		FIGURE 4. Recombination is impaired in IFN-{gamma}R{alpha}-deficient mice. The 129/SvJ and IFN-{gamma}R{alpha}-deficient mice were infected with clonal B. burgdorferi B31, and 4 days later, the skin at the site of inoculation was incubated in BSK II medium. DNA was then extracted and amplified using primers flanking the variable domain of the vlsE gene. Only nucleotide changes are noted. Gaps in the sequence are marked with spaces. The six variable regions within the variable domain are boxed. These data represent cumulative changes found in 19 (129/SvJ mice) and 11 sequences (IFN-{gamma}R{alpha}-deficient mice), respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
B. burgdorferi must successfully adapt to diverse environments, including the tick vector and the mammalian host, to complete its natural life cycle. Some of the antigenic changes the spirochete undergoes throughout this process have been uncovered; one example is the down-regulation of outer surface protein A on B. burgdorferi during tick engorgement (37). The signals that cause the spirochete to change have not yet been fully discerned. Variations in temperature, bacterial density, and pH levels in vitro have been associated with differences in antigenic profiles of cultured B. burgdorferi, providing an initial framework for identifying important environmental cues (38, 39, 40, 41, 42). However, some genes, such as p21, are expressed relatively late (3 wk) during murine infection (43), suggesting that temperature or pH changes are not sufficient to account for many of the changes that B. burgdorferi experience. Recombination at the vls locus, which may be important in immune evasion, occurs in vivo but not in vitro (26), implying that the mammalian host provides additional signals that can influence B. burgdorferi.

A proinflammatory cytokine production phenotype is one of the factors that have been associated with Lyme arthritis in mice and humans (4, 5, 8, 9, 10, 11, 12). Our results herein demonstrate that the induction of IL-12 and IFN-{gamma} during infection facilitates the ability of the spirochete to persist in vivo. We previously showed that adaptation in mice was impaired in nonarthritogenic B. burgdorferi N40-75 (22). The diminished adaptation of these high passaged spirochetes resulted from deficient gene expression in vivo (22). Herein we also associate host adaptation with recombination at the vls locus and the inability of B. burgdorferi N40-75 to efficiently induce a proinflammatory immune response. The treatment of B. burgdorferi N40-75-infected mice with IL-12 or IFN-{gamma} resulted in an increased bacterial burden in the joints and the development of arthritis. Moreover, the adaptation process of B. burgdorferi N40, a virulent and arthritogenic spirochete, was significantly compromised in the absence of IFN-{gamma}-dependent signals. The changes in the spirochete that result in adaptation to the mammalian host are related to recombination events at the vlsE gene. Host-adapted B. burgdorferi N40 were able to escape the administration of immune sera even when administered the day of infection, in contrast to cultured B. burgdorferi N40 (22, 30). Therefore, our data strongly suggest that evasion of immune sera by spirochetes resides primarily, if not exclusively, in recombination events at the vls locus. The inability of B. burgdorferi N40 to circumvent immune sera at 4 days of infection in the absence of IFN-{gamma}-mediated signals is likely due to deficient recombination at the vls locus, resulting in the reduced generation of "escape" variants during infection.

Infectious agents have developed diverse ways to subvert the immune system. These strategies include the induction of cytokine production, suppression of cytokine synthesis or activity, cytokine degradation, binding to cytokines, and induction of cytokine receptor release (reviewed in Ref. 44). Cytokines can also affect the growth of both intracellular and extracellular bacteria. For example, IL-1{beta}, IL-2, GM-CSF, IL-6, TGF{beta}, and epidermal growth factor have been shown to stimulate the growth of Escherichia coli, Mycobacterium tuberculosis, M. avium, and Listeria monocytogenes (45, 46, 47, 48, 49, 50). Parasites also benefit from the production of certain cytokines by the host. For example, Trypanosoma cruzi infection of mammalian cells is dependent on an active TFG{beta} signal pathway (51, 52), and TNF-{alpha} enhances Schistosoma mansoni fertility and tyrosine uptake (53). Although the exact mechanisms used by these pathogens to respond to specific cytokines are not known, these studies indicate that microorganisms can manipulate the production of these effector molecules (i.e., through their induction by LPS-Toll-like receptors) and can respond to their presence. This ability is probably the result of coevolution between a pathogen and its host and represents a strategy for survival of an infectious agent in a hostile environment.

B. burgdorferi survive in the arthropod vector and reservoir host. Effective colonization by the spirochete is a multifactorial process, in which B. burgdorferi preferentially displays surface proteins with distinct function. Outer surface protein A, which is predominantly expressed in the unfed tick, facilitates B. burgdorferi-vector adherence (54), and decorin-binding protein A and BBK32, which are prominently expressed during mammalian infection, bind to host decorin and fibronectin, respectively (55, 56, 57, 58). During infection on the mammalian host, B. burgdorferi are exposed to a humoral and cellular immune response, particularly Ab production, that is important in immunity (33, 59, 60), and both preferential gene expression and vls recombination are likely to be mechanisms by which B. burgdorferi attempt to evade host defenses. Herein we show that B. burgdorferi use the host cytokine response to promote vls recombination, and that in the absence of these signals, B. burgdorferi survival is impaired. Our data indicate that the interplay between the host and invading spirochetes results in a cascade of signaling events that B. burgdorferi can use to facilitate persistent infection.


   Acknowledgments
 
We thank Debbie Beck for her excellent technical assistance.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (to J.A. and E.F.). E.F. is the recipient of a Clinical Scientist Award in Translational Research from the Burroughs Wellcome Fund. Back

2 Address correspondence and reprint requests to Dr. Erol Fikrig, Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, 06520-8031. E-mail address: erol.fikrig{at}yale.edu Back

3 Abbreviations used in this paper: lp, linear plasmid; B. Burgdorferi N40-120, B. Burgdorferi N40 that have been passaged 120 times in vitro; B. Burgdorferi N40-75, B. Burgdorferi N40 that have been passaged 75 times in vitro; vls, variable major protein-like sequence; vlsE, vls expression site; BSK, Barbour-Stoenner-Kelly; HPRT, hypoxanthine phosphoribosyltransferase; TMB, tetramethylbenzidine; NMS, normal mouse serum. Back

Received for publication May 23, 2001. Accepted for publication July 13, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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