Role of Outer Surface Protein D in the Borrelia burgdorferi Life Cycle

Bacterial strains and medium.Clone 5A11, a fully infectious isolate of B. burgdorferi strain B31 that lacks only lp5, was used as the wild-type B. burgdorferi strain throughout this study (11, 26). An isogenic ospD mutant was constructed from clone 5A11 through targeted mutagenesis as described below. Spirochetes were routinely cultured in BSK-H complete medium (Sigma) at 34°C unless otherwise stated. Escherichia coli strain TOP10 (Invitrogen) was used as the host strain for the transformation and maintenance of all plasmids. Luria broth was used to culture all E. coli strains.

DNA and RNA preparation and cDNA synthesis.DNA was extracted from mouse, tick, and spirochete samples with the DNeasy tissue kit (QIAGEN) by following the manufacturer's protocol. The mouse and tick samples used to measure ospD expression (Fig. 1) were the same as we had used previously (11). Briefly, bladder, heart, skin, and joint samples were collected at 21 days after the mice were each infected subcutaneously with 10⁵ wild-type B. burgdorferi spirochetes. Nymphs infected with wild-type B. burgdorferi were allowed to feed on naive mice, and ticks were collected (five ticks per group) before, during, and after tick feeding (on days 0, 1, 2, 3, 4, 7, and 11 after tick attachment). The time window for ticks to feed to repletion was between 66 and 96 h after attachment. Thus, the 1- and 2-day ticks were pulled off of mice before they fed to repletion. The collected mouse and tick samples were immediately frozen in liquid nitrogen and ground thoroughly prior to RNA extraction. Total RNA was extracted from mouse and tick samples with the TRIZOL reagent (Invitrogen) by following the manufacturer's protocol. All RNA samples were digested with RNase-free DNase I (Roche) and then cleaned up on RNeasy mini spin columns (QIAGEN). Total RNA was converted into first-strand cDNA with random hexamers and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's protocol.

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FIG. 1.

Differential ospD expression in B. burgdorferi during its life cycle in mice and ticks. The relative ospD expression level in each cDNA sample was calculated as the number of copies of the ospD transcript per 1,000 copies of the flaB transcript. In the left panel, the open circles represent values of individual mouse tissue samples and a black bar represent the mean value of each column. Shown in the right panel are the means and standard deviations of duplicate measurement of pooled tick samples (five ticks per time point), and the shaded area represents the time window during which the majority (>95%) of the ticks feed to repletion.

PCR and quantitative PCR (Q-PCR).The nucleotide sequences of the PCR primers used in this study are listed in Table 1. PCRs with primers P1 and P2, P3 and P4, and P13 and P14 were for cloning and were performed with Vent DNA polymerase (New England BioLabs Inc.). All cloned DNA fragments were sequenced to confirm that no mutations were introduced during PCR amplification. PCRs with primers P5 and P6, P7 and P8, P9 and P6, P9 and P8, and P15 and P16 were for detection and were performed with the Taq DNA polymerase (Roche). Q-PCR assays of DNA and cDNA samples were performed in an iCycler (Bio-Rad Laboratories) with high-fidelity Platinum Taq DNA polymerase (Invitrogen). The primers and probes used for Q-PCR analysis of B. burgdorferi flaB, mouse β-actin, and Ixodes scapularis actin were described in a previous study (11). The forward and reverse primers and probe used for Q-PCR analysis of the B. burgdorferi ospD gene were P10, P11, and P12, respectively. All Q-PCR probes were labeled at the 5′ end with 6-carboxyfluorescein and at the 3′ end with 5-carboxytetramethylrhodamine. Gene copy numbers of unknown DNA and cDNA samples were determined by the threshold cycle method according to 10-fold serial dilutions of standards. Genomic DNA of B. burgdorferi B31 clone 5A11 was used as the standard for Q-PCR analyses of the flaB and ospD genes. Standards for Q-PCR analysis of the mouse β-actin and I. scapularis actin genes were prepared from plasmid DNA containing the specific genes. The gene copy number in a solution of the standard was calculated on the basis of the molecular weight of the DNA molecule, and the DNA concentration was determined by sample absorbance at 260 nm. In each 96-well Q-PCR plate, six 10-fold serial dilutions of the standards were assayed in duplicate and used to calculate gene copy numbers of unknown samples. The theoretical limit of detection was one gene copy per reaction. Thus, we assigned a value of 1 instead of 0 to any samples that did not yield a positive Q-PCR result. This allowed us to plot all of the data points, including those below the detection limit, on a log scale.

Construction of the ospD mutant.An approximately 1.4-kb DNA fragment upstream of the ospD gene, termed the 5′ arm, was PCR amplified with primers P1 and P2 (Fig. 2A). An approximately 1.5-kb DNA fragment downstream of the ospD gene, termed the 3′ arm, was PCR amplified with primers P3 and P4 (Fig. 2A). The suicide plasmid used for homologous recombination to replace the ospD gene with a kanamycin resistance cassette was constructed as follows. First, the Borrelia-adapted kanamycin resistance cassette kanAn was excised from pTAkanAn (2) by EcoRI digestion and then cloned into the EcoRI site of pBluescript (Stratagene). Subsequently, the 5′ and 3′ arms were double digested with XbaI-BamHI and XhoI-KpnI, respectively, and then cloned sequentially into the corresponding sites of pBluescript. Therefore, in the final construct, designated pXLF10601-ospD, the kanAn cassette was flanked upstream by the 5′ arm and downstream by the 3′ arm. Electroporation-competent spirochetes of clone 5A11 were prepared as previously described (31, 32). Five microliters (25 μg) of the suicide plasmid was mixed with 50 μl of approximately 10⁹ competent spirochetes, placed in a 0.1-cm cuvette, and pulsed in a Gene Pulser apparatus (Bio-Rad) at settings of 1.8 kV, 25 μF, and 400 Ω. A total of 12 kanamycin-resistant transformants were obtained after selection in BSK-H medium containing kanamycin (350 μg/ml). Western blot analysis with murine sera raised against recombinant OspD (described below) confirmed that of these 12 transformants, 3 were negative for OspD expression. PCR analysis with kanAn-specific primers (P7 and P8) indicated that the nine OspD-positive transformants were negative for the kanAn cassette and therefore likely to be spontaneous kanamycin-resistant mutants. The three OspD-deficient kanamycin-resistant transformants were all PCR positive for the kanAn cassette. The plasmid content of these three OspD-deficient mutants was analyzed by a microarray assay (36), which indicated that one of the mutants had the same plasmid profile as wild-type clone 5A11. This OspD-deficient mutant was selected for all further studies and is referred to as the ospD mutant hereafter.

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FIG. 2.

Construction and characterization of the ospD mutant. (A) Schematic drawings of the wild-type B. burgdorferi strain (w) and the ospD mutant (m) near the ospD locus. The ospD (BBJ09) gene and its surrounding open reading frames, BBJ08 (8), BBJ10 (10), and BBJ11 (11), are illustrated as box arrows, the lengths and directions of which reflect the lengths and directions of the open reading frames. In the mutant, the ospD gene is replaced with the kanAn cassette. Primers P1 to P9 are illustrated as arrows with their positions and directions representing those of the primers. The positions of the two DNA fragments mediating the homologous recombination event (the 5′ and 3′ arms) are shown. (B) PCR analysis of the wild-type B. burgdorferi strain (w) and the ospD mutant (m). The primers used in each PCR are indicated above the gel. The molecular masses of the DNA standards are indicated on the right in base pairs. (C) Protein analysis of whole-cell lysates of the wild-type B. burgdorferi strain (w) and the ospD mutant (m). Borrelia lysates were separated by SDS-PAGE and then stained with Coomassie brilliant blue (left panel) or transferred to a membrane and probed with antibodies against OspD (right panel). An arrow points to the OspD protein. The molecular masses of the protein standards are shown on the right in kilodaltons.

Expression and purification of recombinant OspD, OspA, OspB, OspC, and Dps.The DNA sequences encoding the mature OspD peptide (minus the first 20 amino acid residues) were PCR amplified with primers P13 and P14. The PCR fragment was digested with BamHI and XhoI and cloned into the corresponding restriction sites of the expression vector pGEX-6P-2 (Amersham Biosciences). OspD was synthesized in E. coli TOP10 as a C-terminal fusion to glutathione S-transferase (GST). The fusion proteins were affinity purified with glutathione Sepharose 4B resin (Amersham Biosciences), and OspD was cleaved off of the GST tag with PreScission protease (Amersham Biosciences) by following the manufacturer's protocol. Recombinant OspD was further purified on a Superdex 200 column (Amersham Biosciences) and used to immunize mice to generate antisera. Recombinant OspA, OspB, OspC, and Dps proteins (6, 12, 20) were also expressed as GST fusions with the expression vector pGEX6p2 (Amersham Biosciences). Similarly, the GST tag was removed from the recombinant proteins during purification as described above.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. B. burgdorferi whole-cell lysates, prepared as previously described (11), were separated on a two-layer (3.75% stacking and 12.5% resolving) polyacrylamide gel. The separated proteins were either viewed directly by staining the gel with Coomassie brilliant blue or transferred to a polyvinylidene difluoride membrane and subjected to Western blot analysis. The membrane was incubated with a 1:5,000 dilution of the murine sera against recombinant OspD (see above), followed by a 1:2,000 dilution of anti-mouse immunoglobulin G (IgG)-horseradish peroxidase conjugate (Sigma), and developed with ECL Western blotting detection reagents (Amersham Biosciences).

Experimental mouse-tick model of Lyme disease.Four- to 6-week-old female C3H/HeN and C3H/HeJ mice were purchased from the National Cancer Institute and the Jackson Laboratories, respectively, and housed in the Yale Animal Resources Center according to the institutional guidelines for the care and use of laboratory animals. Larval, nymphal, and adult I. scapularis ticks were routinely maintained in our laboratory as described previously (11). Mice were subcutaneously inoculated with in vitro-grown B. burgdorferi at a dose of 10⁵, 10⁴, 10³, or 10² spirochetes per mouse. Fourteen days after inoculation, ear punch samples were collected and examined for the presence of spirochetes by flaB PCR with primers P15 and P16. When necessary, between 16 and 21 days after inoculation, larvae (200 to 300 per mouse) or naive nymphs (30 to 50 per mouse) were allowed to feed on spirochete-positive mice. Spirochete acquisition by ticks was studied by quantitative measurement of the spirochete burdens in ticks by both Q-PCR and immunofluorescence microscopy (described below). Twenty-one days after the initial spirochete inoculation, mice were sacrificed and examined for Borrelia infection by serology (serum IgG response to whole-cell lysates of in vitro-grown B. burgdorferi), by in vitro culturing of the bladder and the spleen, and by quantitative measurement of the spirochete burdens in the bladder, heart, skin, and joints. To study spirochete transmission, infected nymphs (obtained by feeding larvae on infected mice and letting them molt into nymphs) were allowed to feed on naive mice (five ticks per mouse). Twenty-one days after tick attachment, mice were sacrificed and assessed for Borrelia infection as described above. Q-PCR quantification of spirochete burdens in mice and ticks was performed on individual DNA samples.

Immunofluorescence microscopy.Spirochete burdens in ticks were also analyzed by immunofluorescence confocal microscopy as described previously (22, 37). Briefly, three to five guts from each group of nymphs were microscopically dissected in 20 μl of phosphate-buffered saline (PBS). Guts containing blood were isolated, placed on Silane-Prep slides (Sigma), and washed with PBS to remove luminal contents and unbound bacteria. The washed gut samples and luminal contents were isolated separately and processed for immunofluorescence confocal microscopy. Gut and luminal content samples were blocked with PBS containing Tween 20 (0.05%) and goat serum (5%) for 1 h at 37°C and then incubated for 1 h at 37°C with fluorescein isothiocyanate (FITC)-labeled anti-Borrelia antibody (KPL). Samples were subsequently stained with propidium iodide (20 μg/ml) for 3 min at 37°C and then mounted with the SlowFade-Antifade kit (Invitrogen). The samples were viewed with a Zeiss LSM 510 scanning laser confocal microscope equipped with an argon-krypton laser (Zeiss Incorporated).

In vitro tick gut extract (TGE) binding assay.Guts free of luminal content from flat nymphal ticks (40 ticks) and free of blood meal from fed nymphal ticks (25 ticks) were dissected in PBS as described above and homogenized on ice with a Kontes tissue grinder (VWR Scientific Products) in 100 μl of PBS containing protease inhibitor cocktail (Roche). Total protein concentrations in the TGE were determined with the Bio-Rad protein assay kit (Bio-Rad Laboratories Inc.). One hundred microliters of unfed TGE (5 μg/ml) or fed TGE (5 μg/ml) in PBS was used to coat the wells of 96-well plates (Nunc). Control wells were coated with 100 μl of a mouse b.End3 cell line extract (5 μg/ml). Mouse cell line extract was prepared by resuspending the b.End3 cell pellet in 100 μl of Cell Lytic reagent (Sigma) and processed as mentioned above. Coated plates were tightly covered with cellophane tape to prevent evaporation and incubated overnight at 4°C. Plates were then incubated with 100 μl of FITC-labeled OspD, OspA, OspB, OspC, or Dps (10 μg/ml) at 37°C for 1 h. FITC labeling of the individual proteins was performed with an EZ-label FITC labeling kit (Pierce) according to the manufacturer's instructions, and any unlabeled or residual FITC molecules were removed by centrifuging the samples in Amicon Ultra 10k filters (Millipore). Plates were then washed three times with PBS containing Tween 20 (0.05%). Binding was detected with anti-FITC IgG-horseradish peroxidase (Amersham Pharmacia Biotech), and TMB microwell peroxidase substrate (KPL) was used for color development. The reactions were stopped after 15 min of incubation with TMB stop solution (KPL), and optical density was read at a 450-nm wavelength.

Statistics.Statistical significance of differences observed in data sets was analyzed with GraphPad Instat version 3.05. In general, for data with small variation and normal distribution, the Student t test was performed to compare two means and one-way analysis of variance with Tukey's posttest was performed to compare multiple means; for data with large variation and/or nonparametric distribution, the Mann-Whitney test was performed to compare two medians and a Kruskal-Wallis test with Dunn's posttest was performed to compare multiple medians. Wherever necessary, two-tailed P values, along with the statistical test used, are reported.

ospD expression throughout the B. burgdorferi life cycle.Previous microarray studies showed that ospD expression in B. burgdorferi varied significantly in response to different culturing conditions (3, 19, 35). Here, we used Q-PCR to systematically analyze ospD expression by the spirochete during its life cycle through the murine host and the tick vector (Fig. 1). The copy numbers of the ospD and flaB transcripts were quantified for each cDNA sample (see Materials and Methods for details) and the relative expression level of ospD was calculated as the number of copies of ospD per 1,000 copies of flaB. Our data indicated that ospD expression was highly variable during the spirochete's life cycle, with an approximately 500-fold difference between the lowest level observed in the murine joint tissue and the highest level observed in the tick shortly after the blood meal (Fig. 1). The Tukey-Kramer multiple-comparison test was used to compare ospD expression levels in various murine tissues and in ticks at different feeding stages. The ospD expression levels in murine bladder, skin, heart, and joints were comparable to those in the feeding ticks (1 and 2 days after tick attachment) (P > 0.05) but were significantly lower than those in the unfed and fed ticks (0, 3, 4, 7, and 11 days after tick attachment) (P < 0.001). During tick feeding, we observed a significant decrease in ospD expression (P < 0.01), which is consistent with a previous finding that ospD expression decreases in response to the combined effects of blood and a temperature shift from 23°C to 35°C (35).

Construction and characterization of an ospD mutant in vitro.We constructed an isogenic ospD mutant from B. burgdorferi B31 clone 5A11 by replacing the ospD gene with the Borrelia-adapted kanamycin resistance cassette kanAn (2) through homologous recombination (Fig. 2A; see Materials and Methods for details). To verify the mutation, a series of PCRs were performed (Fig. 2B). The PCRs with primers P5 and P6 and primers P7 and P8 indicated the absence of the ospD gene and the presence of the kanamycin resistance cassette, respectively, in the ospD mutant. The location of the kanamycin resistance cassette in the ospD mutant was confirmed by PCR with primers P9 and P8. The migration of these PCR-amplified DNA fragments in the agarose gel is consistent with their predicted sizes (321, 283, 765, and 1,130 bp for DNA fragments amplified with primers P5 and P6, P7 and P8, P9 and P6, and P9 and P8, respectively). These PCR results support the idea that a double-crossover event had likely occurred in the mutant and resulted in replacement of the ospD gene with the kanamycin resistance cassette. SDS-PAGE analysis of whole-cell lysates indicated that there was no apparent difference between the overall protein profiles of the mutant and the wild-type strain (Fig. 2C, left side). Western blotting with murine sera against recombinant OspD showed that the most prominent immunoreactive protein in the wild-type B. burgdorferi strain was missing from the ospD mutant (Fig. 2C, right side). The apparent molecular mass of this protein is 28 kDa, the same as previously reported for OspD (18). Our data indicate that the ospD mutant is indeed OspD deficient.

Spirochete infectivity in mice.To determine whether the loss of OspD expression in the mutant had any adverse effect on its infectivity, we examined both the wild-type strain and the mutant in a laboratory mouse model of Lyme borreliosis originally described by Barthold and colleagues (1). Groups of C3H/HeN mice (five per group) were subcutaneously inoculated with in vitro-grown spirochetes, either the wild-type strain or the ospD mutant, at a dose of 10⁵ spirochetes per mouse (see Materials and Methods for details). On day 21 postinoculation, mice were sacrificed and assessed for spirochete infection by serology, by in vitro culturing of the bladder and the spleen, and by quantitative measurement of the spirochete burdens in various tissues. Results of two independent mouse experiments with a total of 20 mice, 10 infected with the wild-type strain and the other 10 infected with the mutant strain, indicated that all of the infected mice seroconverted and were culture positive for spirochetes in the bladder and the spleen, regardless of which spirochete strain they were infected with. Western blot analysis of spirochete cultures from mouse tissues indicated that the ospD mutant remained OspD deficient (data not shown). As shown in Fig. 3A, the spirochete burdens in a specific mouse tissue were comparable between mice infected with the wild-type strain and mice infected with the ospD mutant (P > 0.05, Dunn's multiple-comparison test). We also conducted a dose study to compare the infectivity of the ospD mutant and that of the wild-type strain in C3H/HeJ mice. As summarized in Table 2, there was no significant loss of infectivity of the ospD mutant even at the low inoculation dose of 10² spirochetes per mouse (P = 1, Fisher's exact test). In summary, our data demonstrate that the ospD mutant is fully infectious in mice.

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FIG. 3.

Quantification of spirochete burdens in mice and ticks. DNA samples from mice and ticks infected with the wild-type B. burgdorferi strain (w) or the ospD mutant (m) were subjected to Q-PCR analyses to determine the copy number of the B. burgdorferi flaB gene and that of the mouse or tick actin gene. The relative spirochete burden is shown as the number of copies of the B. burgdorferi flaB gene per 10⁶ copies of the mouse β-actin gene or per 10³ copies of the tick actin gene. (A) Spirochete burdens in bladder, heart, joint, and skin samples of mice at 21 days after syringe inoculation with spirochetes at a dose of 10⁵ spirochetes per mouse. (B) Spirochete burdens in larvae and nymphs after feeding on infected mice. (C) Spirochete burdens in infected nymphs before feeding (unfed) and during and after feeding (at 48, 72, and 96 h after tick attachment). (D) Spirochete burdens in bladder, heart, joint, and skin samples of mice at 21 days after being fed upon by infected nymphs. Individual circles indicate spirochete burdens in individual mouse tissue or tick samples, and a black bar represents the median of each column. The statistical significance of comparison of data is indicated by * (P < 0.005) or ** (P < 0.01).

Spirochete acquisition by ticks.To determine whether the deletion of ospD in the mutant had any adverse effect on spirochete acquisition by ticks, we fed larvae and naive nymphs on infected mice and quantified the spirochete burdens in fed ticks by Q-PCR analysis of individual ticks (see Materials and Methods for details). At 1 week after the larvae were fed to repletion, the spirochete burden was threefold greater in the larvae fed on mice infected with the wild-type strain than in those fed on mice infected with the ospD mutant (P = 0.007, Mann-Whitney test) (Fig. 3B). The spirochete burden in fed nymphs was analyzed shortly after the nymphs were fed to repletion (at 96 h after tick attachment), and it showed that the ticks fed on mice infected with the wild-type strain had fivefold greater spirochete burdens than the ticks fed on mice infected with the ospD mutant (Fig. 3B, P = 0.004, Mann-Whitney test).

We also examined spirochete colonization of the tick midgut by immunofluorescence microscopy. Representative confocal images of the blood meal and the midgut samples of the wild-type B. burgdorferi strain-infected and the ospD mutant-infected ticks are shown in Fig. 4A. The relative spirochete burdens in these samples were further quantified as the number of spirochetes per field (Fig. 4B), and our data indicated that whereas there were comparable numbers of spirochetes in the blood meals of these two groups of ticks (P > 0.05, Tukey-Kramer multiple-comparison test), there were threefold higher numbers of spirochetes in the midguts of ticks infected with the wild-type B. burgdorferi strain (P < 0.001, Tukey-Kramer multiple-comparison test). These results suggest that the loss of OspD expression may affect the spirochete's ability to fully colonize the tick midgut. We next examined whether OspD bound to TGE. The OspA and OspB proteins (6, 20) were used as positive controls, and the OspC and Dps proteins (12, 20) were used as negative controls. As shown in Fig. 5, similar to the OspA and OspB proteins, the OspD protein bound to unfed TGE and fed TGE at a level two- to threefold higher than its level of binding to a mouse cell line extract, whereas the OspC and Dps proteins did not preferentially bind to TGEs.

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FIG. 4.

Immunofluorescence microscopy analysis of ticks. Naive nymphs were fed on mice infected with the wild-type B. burgdorferi strain (w) or the ospD mutant (m). Shortly after tick feeding (at 96 h after tick attachment), ticks were dissected and the blood meal and tick midgut samples were separately isolated and processed for immunofluorescence microscopy as described in Materials and Methods. (A) Representative confocal images of gut and blood meal samples. Both samples were stained for B. burgdorferi with FITC-labeled anti-Borrelia antibody (green) and for tick cell nucleic acid with propidium iodide (red). Scale bar, 20 μm. (B) Quantitative assessment of the number of spirochetes from 15 random microscopic field observations. Values on the y axis are the numbers of spirochetes per microscopic field. Error bars define standard deviations from the mean. The statistical significance of comparison of data is indicated by *** (P < 0.001).

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FIG. 5.

In vitro TGE binding assay. The binding of FITC-labeled OspD, OspA, OspB, OspC, and Dps to TGE prepared from unfed (gray bar) and fed (black bar) ticks and to a mouse cell line extract (open bar) was assayed in triplicate or quadruplicate. The bars and error bars represent the means and standard deviations of three or four readings of optical density at a 450-nm wavelength.

Spirochete transmission by ticks.We next examined whether the reduced level of spirochete colonization of the tick midgut would affect spirochete transmission in a subsequent feeding. The remaining larvae from the acquisition experiment described above were allowed to molt into nymphs. Q-PCR analysis of individual ticks from these two groups of unfed nymphs indicated that the ticks infected with the wild-type B. burgdorferi strain had a threefold greater spirochete burden than the ticks infected with the ospD mutant (Fig. 3C, P = 0.05, Mann-Whitney test). To study spirochete transmission, we fed these infected nymphs on naive mice (5 to 12 ticks per mouse and 10 mice per group). Spirochete burdens in ticks were analyzed at 48, 72, and 96 h after tick attachment (Fig. 3C). Interestingly, the difference in spirochete burden observed between these two groups of ticks diminished during tick feeding; the spirochete burdens of these two groups of ticks were comparable at the 48- and 72-h time points (P > 0.05, Dunn's multiple-comparison test). Shortly after tick feeding, at the 96-h time point, ticks infected with the wild-type B. burgdorferi strain had a 2.7-fold greater spirochete burden than ticks infected with the ospD mutant (P < 0.05, Dunn's multiple-comparison test). We also examined whether the mice fed on by these infected nymphs were infected with spirochetes at 21 days after tick attachment and found that all 20 mice seroconverted and were culture positive for B. burgdorferi (data not shown). The spirochete burdens in a specific mouse tissue, quantified by Q-PCR, were comparable between these two groups of mice (Fig. 3D, P > 0.05, Dunn's multiple-comparison test). These results showed that although the ospD mutant colonized the tick midgut at a threefold reduced level compared with the wild-type B. burgdorferi strain, such a defect did not affect spirochete transmission to a new host during a subsequent blood meal.