Borrelia burgdorferi Lacking BBK32, a Fibronectin-Binding Protein, Retains Full Pathogenicity
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感染与免疫杂志 2006年第6期
Sections of Rheumatology Allergy and Immunology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8031
ABSTRACT
BBK32, a fibronectin-binding protein of Borrelia burgdorferi, is one of many surface lipoproteins that are differentially expressed by the Lyme disease spirochete at various stages of its life cycle. The level of BBK32 expression in B. burgdorferi is highest during infection of the mammalian host and lowest in flat ticks. This temporal expression profile, along with its fibronectin-binding activity, strongly suggests that BBK32 may play an important role in Lyme pathogenesis in the host. To test this hypothesis, we constructed an isogenic BBK32 deletion mutant from wild-type B. burgdorferi B31 by replacing the BBK32 gene with a kanamycin resistance cassette through homologous recombination. We examined both the wild-type strain and the BBK32 deletion mutant extensively in the experimental mouse-tick model of the Borrelia life cycle. Our data indicated that B. burgdorferi lacking BBK32 retained full pathogenicity in mice, regardless of whether mice were infected artificially by syringe inoculation or naturally by tick bite. The loss of BBK32 expression in the mutant had no adverse effect on spirochete acquisition (mouse-to-tick) and transmission (tick-to-mouse) processes. These results suggest that additional B. burgdorferi proteins can complement the function of BBK32, fibronectin binding or otherwise, during the natural spirochete life cycle.
INTRODUCTION
Borrelia burgdorferi is the etiologic agent of Lyme disease, the most common arthropod-borne disease in the United States (9, 35). In nature, B. burgdorferi is maintained in an enzootic cycle that involves an Ixodes tick and a vertebrate host (53). Larval and nymphal ticks feed mainly on small rodents, such as white-footed mice, and adult ticks commonly feed on deer. Humans are accidental hosts that do not play a meaningful role in the B. burgdorferi life cycle.
Shuttling between its reservoir host and tick vector, B. burgdorferi is highly adaptable. Even before the genome of B. burgdorferi was revealed, researchers in the field of Lyme disease had long recognized that many of the spirochete's surface lipoproteins are differentially expressed in response to various environmental cues, such as a blood meal in the tick, entering the mammalian host, etc. (15, 30, 51). The genome sequence of B. burgdorferi only underscores the enormity of its lipoprotein repertoire, which accounts for more than 10% of the whole genome (11, 23). This information touched off an explosion of microarray studies, which further confirmed that most of the surface lipoproteins are differentially expressed in spirochetes cultured under various conditions in vitro, cultured in dialysis membrane chambers implanted in rats, or isolated from tissues of infected mice or nonhuman primates (5, 29, 33, 34, 46, 57).
Significant advances in the molecular techniques for constructing B. burgdorferi mutants lacking a specific gene have made it possible for researchers to study the functions of individual spirochete gene products (47). There have been a remarkable number of targeted mutagenesis studies of B. burgdorferi in recent years (8, 10, 13, 16, 22, 24, 27, 28, 31, 32, 36, 37, 39, 45, 55, 56, 60, 61). Some of these studies have directly tested the hypothesis that differentially expressed lipoproteins have important functions at the stage(s) of the B. burgdorferi life cycle at which their expression is turned on.
Outer surface protein A (OspA) and OspC are two of the most notable examples of lipoproteins that are differentially expressed by the spirochete at various stages of its life cycle (50). OspA is expressed by spirochetes residing in the midgut of unfed ticks but not by spirochetes infecting the mammalian host, whereas the reverse is true for OspC. When a tick starts feeding, spirochetes residing in the midgut turn off the expression of OspA and start making OspC, although the two events may not be directly related. Thus, spirochetes migrating to tick salivary glands and subsequently entering the mammalian host mostly express OspC and not OspA. Although spirochetes rarely express OspA when they are infecting a mammalian host, shortly after being acquired by a tick through a blood meal, they turn on expression of OspA but not expression of OspC. These expression patterns are consistent with the role of OspA in spirochete colonization of the tick gut and the role of OspC in spirochete migration to tick salivary glands and infection of the mammalian host, as defined by mutagenesis studies (24, 39, 61). Further studies of the functions of OspA and OspC have revealed that OspA binds a tick receptor to promote spirochete attachment to the tick midgut and OspC sequesters a tick salivary protein with immune-suppressive functions to facilitate spirochete infection in the mammalian host (38, 44).
BBK32 is another surface lipoprotein that is differentially expressed by the spirochete. BBK32 was first identified as an antigen that is expressed by spirochetes infecting mice but not by spirochetes grown in vitro, and antibodies against BBK32 elicited protective immunity in mice (17, 54). Further studies showed that BBK32 was expressed in feeding ticks but not in unfed ticks and that antibodies against BBK32 blocked spirochete transmission (18). Serological studies indicated that an immunoglobulin G (IgG) antibody response to BBK32 is a common early response in patients with Lyme arthritis, and higher IgG antibody responses to BBK32 correlated with less severe disease (1). These results indicated the diagnostic value of BBK32 and its potential as a vaccine candidate, both of which were demonstrated experimentally (6, 26). Studies by Johnson and colleagues identified BBK32 as a fibronectin-binding protein (41, 42). Therefore, it was proposed that BBK32-mediated attachment to fibronectin, an extracellular matrix component, is important for the pathogenesis of Lyme borreliosis. To directly test this hypothesis, we constructed an isogenic BBK32 deletion mutant from a wild-type strain of B. burgdorferi and examined this mutant extensively in a laboratory tick-mouse model of the Borrelia life cycle.
MATERIALS AND METHODS
Bacterial strains and media. Clone 5A11 was a fully infectious clone of B. burgdorferi strain B31 that was obtained from Steve Norris, and it contained all 20 plasmids analyzed (43). The presence of lp5 (plasmid T) in 5A11 was unclear until PCR primers specific for lp5 were generated (59). The PCR results indicated that 5A11 had lost lp5. However, the loss of lp5 apparently had no effect on the infectivity of 5A11. We therefore used 5A11 as the parent strain to construct a BBK32 deletion mutant. Spirochetes were routinely cultured in BSK-H complete medium (Sigma) at 34°C unless indicated otherwise. Escherichia coli strain TOP10 (Invitrogen) was used as the host strain for transformation and maintenance of all plasmids. Luria broth was used to culture all E. coli strains.
Ixodes scapularis ticks. Larval, nymphal, and adult I. scapularis ticks were routinely maintained in our laboratory. Adult female ticks, in the presence of male ticks, were fed on New Zealand White rabbits to produce egg masses. Hatched larvae were fed either on nave C3H mice to generate nave nymphs or on B. burgdorferi-infected C3H mice to generate infected nymphs. Acquisition of spirochetes by the ticks was studied by feeding larvae or nave nymphs on infected mice. Transmission of spirochetes by the ticks was studied by feeding infected nymphs on nave mice. All ticks were incubated in a humidified chamber maintained at 25°C with 85 to 90% relative humidity and a photoperiod consisting of 14 h of light and 10 h of darkness.
DNA and RNA preparation and cDNA synthesis. DNA was extracted from mouse, tick, and spirochete samples using a DNeasy tissue kit (QIAGEN) according to the manufacturer's protocol. Total RNA was extracted from frozen mouse, tick, and spirochete samples using TRIZOL reagent (Invitrogen) according to the manufacturer's protocol. Prior to TRIZOL extraction, the frozen mouse and tick samples were ground thoroughly in liquid nitrogen. The RNA samples were digested with RNase-free DNase I (Roche) and then cleaned on RNeasy mini spin columns (QIAGEN). RNA was converted into first-strand cDNA using random hexamers and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's protocol.
Q-PCR analysis. All quantitative PCR (Q-PCR) assays were performed with an iCycler (Bio-Rad Laboratories) using high-fidelity Platinum Taq DNA polymerase (Invitrogen), gene-specific primers, and TaqMan probes with a program consisting of an initial denaturing step of 3 min at 95°C and 50 amplification cycles consisting of 30 s at 95°C followed by 1 min at 60°C. The forward and reverse primers and probe used for Q-PCR analysis of flaB were 5'-AGC TGA AGA GCT TGG AAT GC-3', 5'-TTG GTT TGC TCC AAC ATG AA-3', and 5'-TCC AAG ACG CTT GAG ACC CTG AAA-3', respectively. The forward and reverse primers and probe used for Q-PCR analysis of BBK32 were 5'-CAA CAA AGC TAA CCC AAA TGT AT-3', 5'-CTT TTG TAA ACT TTG CAG CTT CT-3', and 5'-ACG CCT TGA CAA CTT TGC TAA AGC C-3', respectively. The forward and reverse primers and probe used for Q-PCR analysis of mouse beta-actin were 5'-AGA GGG AAA TCG TGC GTG AC-3', 5'-CAA TAG TGA TGA CCT GGC CGT-3', and 5'-CAC TGC CGC ATC CTC TTC CTC CC-3', respectively. The forward and reverse primers and probe used for Q-PCR analysis of tick actin were 5'-GAT CAT GTT CGA GAC CTT CA-3', 5'-CGA TAC CCG TGG TAC GA-3', and 5'-CCA TCC AGG CCG TGC TCT C-3', respectively. All TaqMan probes were labeled at the 5' end with 6-carboxyfluorescein and at the 3' end with 5-carboxytetramethylrhodamine. Genomic DNA of B. burgdorferi B31 clone 5A11 was used as a standard for the flaB and BBK32 genes. Standards for the mouse beta-actin gene and the tick actin gene were prepared from plasmid DNA containing the specific gene. Gene copy numbers of the standards were calculated based on the molecular weight of the DNA molecule, and the DNA concentration was determined by sample absorbance at 260 nm. Gene copy numbers of unknown samples were determined by the threshold cycle method, using serial dilutions of the standards.
Sample collection for differential BBK32 gene expression. Eight mice were subcutaneously inoculated on the back with B. burgdorferi B31 clone 5A11 at a dose of 105 spirochetes/mouse. At 3 weeks postinoculation, the mice were sacrificed, an approximately 4-cm2 piece of the abdominal skin (distal from the inoculation site), the bladder, the heart, and both tibia tarsal joints were collected from each mouse, frozen in liquid nitrogen immediately, and then stored at –80°C. Infected nymphs (molted from larvae fed on mice infected with B. burgdorferi B31 clone 5A11) were fed on nave mice. Five ticks were collected for each time at 24, 48, 72, 96, 168, and 264 h after attachment. The time window for ticks to feed to repletion was between 66 and 96 h after attachment. Thus, the 24- and 48-h ticks were pulled off mice before they were fed to repletion. Five unfed ticks were also collected to represent zero time. All seven groups of ticks (five ticks/group) were frozen immediately in liquid nitrogen at the time of collection. A mid-log-phase culture of B. burgdorferi B31 clone 5A11 grown at 34°C was used to inoculate six 3-ml BSK-H complete medium tubes (initial density, 5 x 105 spirochetes/ml), three of which were incubated at 25°C and three of which were incubated at 37°C. All six cultures were grown to a density of approximately 5 x 107 spirochetes/ml, and spirochetes were collected by centrifugation and frozen immediately in liquid nitrogen. All frozen mouse, tick, and spirochete samples were processed for total RNA extraction (see above).
Construction of the BBK32 deletion mutant. First, a Borrelia-adapted kanamycin resistance cassette, kanAn, was excised from pTAkanAn (3) by EcoRI digestion and cloned into the EcoRI site of pBluescript (Stratagene). The resulting construct, designated pXLF10601, had two multiple-cloning sites flanking the kanAn cassette, which allowed efficient cloning of the two DNA fragments (the 5' and 3' arms) required for homologous recombination. To replace the BBK32 gene (base pairs 20389 to 21453 of lp36) and additional 97-bp downstream sequences with the kanAn cassette, primers P1 (5'-GCG GAG CTC AGA AAA TTT TAG AAG AAA ACA AGC T-3'), P2 (5'-CGC TCT AGA GCT TTC TCT CCT TTA AAG TTA ATA CT-3'), P3 (5'-GCG CTC GAG TGG ATA AGA AAA TGG ATT TCG-3'), and P4 (5'-CGC GGA TCC CCT CGC GGG ATA GAT AGT AG-3') were designed to PCR amplify the 5' arm (base pairs 18781 to 20388 of lp36) and the 3' arm (base pairs 21550 to 23030 of lp36). The primer sequences and base pair numbers were obtained from the GenBank sequence of B. burgdorferi lp36 (accession no. NC_001855). Both DNA fragments were PCR amplified from the genomic DNA of B. burgdorferi B31 clone 5A11 using high-fidelity Vent DNA polymerase (New England BioLabs Inc.) and a program consisting of an initial denaturing step of 5 min at 95°C and 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 90 s. The restriction sites in the primers (underlined) allowed directional cloning of the 5' arm into the SacI-XbaI site and of the 3' arm into the XhoI-KpnI site of pXLF10601. All restriction enzymes were purchased from New England BioLabs Inc. The final construct, designated pXLF10601-bbk32, was sequenced to confirm that no mutation was introduced into the 5' and 3' arms during PCR amplification.
Plasmid DNA of pXLF10601-bbk32 (25 μg) was electroporated into B. burgdorferi B31 clone 5A11 by using the protocol described in detail by Samuels et al. (48, 49). Transformants were selected in BSK-H complete medium (Sigma) containing kanamycin (350 μg/ml) and were examined by PCR to confirm the presence of the kanAn cassette and by Western blot analysis to confirm the loss of BBK32 expression. In two separate experiments, a total of 17 kanamycin-resistant transformants were obtained. PCR and Western blot analyses indicated that 14 of the 17 transformants were PCR positive for the kanAn cassette and BBK32 deficient, which resulted from a double-crossover event (data not shown). The remaining three kanamycin-resistant transformants were PCR negative for the kanAn cassette and thus likely to be spontaneous mutants. Single-crossover events, which would have resulted in a kanAn-positive and BBK32-positive transformant, were not detected. The plasmid contents of the BBK32-deficient mutants were determined by an array-based assay (59), which indicated that 3 of the 14 mutants had the same plasmid content as the parental 5A11 clone (data not shown). We randomly selected one of these three mutants for further study. Although the construct pXLF10601-bbk32 contained the ampicillin resistance marker (carried on the vector pBluescript), no ampicillin-resistant transformants were ever selected for. All BBK32 deletion mutants generated after transformation were PCR negative for the BBK32 gene and lacked BBK32 expression, and therefore they clearly resulted from a double-crossover event rather than a single-crossover event between pXLF10601-bbk32 and lp36 and did not contain the ampicillin resistance marker. B. burgdorferi lacking BBK32 and wild-type B. burgdorferi were also tested for sensitivity to ampicillin; both spirochetes were highly susceptible to ampicillin, and no differences between the wild-type and mutant spirochetes were observed. Experiments to genetically manipulate and transform B. burgdorferi with vector DNA containing antibiotic resistance markers were approved by the Yale University Biosafety Committee.
PCR analyses for confirmation of the BBK32 deletion. Primers P5 (5'-AGG CAA AAG CAA GGT TTC TA-3'), P6 (5'-ACT TTC AAT AGG CTC CTC CA-3'), P7 (5'-GTA CTC CTG ATG ATG CAT GG-3'), P8 (TGC ATT TCT TTC CAG ACT TG-3'), and P9 (5'-TCA GCA TAT GAT AAG ATG GTA GCT-3') were designed to verify the BBK32 deletion in the mutant by PCR. Primers P5, P6, and P9 were specific for B. burgdorferi lp38 (GenBank accession no. NC_001855), and primers P7 and P8 were specific for the kanamycin resistance cassette derived from pOK12 (GenBank accession no. AF223639). All PCRs were performed using Taq DNA polymerase (Roche), 1 ng of genomic DNA of wild-type clone 5A11 or the BBK32 deletion mutant, and a program consisting of an initial denaturing step of 5 min at 95°C and 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s.
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Western blot analysis, and fibronectin-binding assays. Wild-type clone 5A11 and the BBK32 deletion mutant were cultured at 34°C in BSK-H complete medium (Sigma) to a density of approximately 108 spirochetes/ml. Spirochetes were harvested and washed three times with Dulbecco's phosphate-buffered saline with centrifugation (5 min at 5,000 x g). B. burgdorferi pellets were suspended in SDS sample buffer (62.5 mM Tris-HCl [pH 6.8], 5% -mercaptoethanol, 2% SDS, 0.02% bromophenol blue, 10% glycerol) to a density of 107 spirochetes/μl and then denatured for 15 min at 100°C. B. burgdorferi whole-cell lysates (10 μl each) were separated on a two-layer polyacrylamide gel (3.75% polyacrylamide stacking gel and 10% polyacrylamide resolving gel). The separated proteins were either viewed directly by staining the gel with Coomassie brilliant blue or transferred to a polyvinylidene fluoride membrane and subjected to Western blot analysis or examined in a fibronectin-binding assay.
For Western blot analysis, the membrane was incubated with a 1:5,000 dilution of rabbit sera raised against BBK32 (17), followed by a 1:2,000 dilution of anti-rabbit IgG-horseradish peroxidase (HRP) conjugate. For the fibronectin-binding assay, the membrane was incubated first with human fibronectin (5 μg/ml), then with a 1:500 dilution of affinity-purified rabbit antibodies against human fibronectin, and finally with a 1:2,000 dilution of anti-rabbit IgG-HRP conjugate. To visualize the biotinylated molecular weight standards on the membrane, a 1:5,000 dilution of anti-biotin-HRP conjugate was also added in the final incubation. Except for the antibodies against BBK32, all other reagents, including human fibronectin, molecular weight standards, and antibodies, were purchased from Sigma. Prior to the first incubation, membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T). Between incubations, membranes were washed three times with Tris-buffered saline containing 0.1% Tween 20. Membranes were developed using ECL Western blotting detection reagents (Amersham Biosciences).
Experimental mouse model of Lyme disease. Four- to six-week-old female C3H/HeN mice were purchased from the National Cancer Institute and housed in the Yale Animal Resources Center according to the institutional guidelines for care and use of laboratory animals. Mice were inoculated with B. burgdorferi either by syringe injection of in vitro-grown spirochetes (105 spirochetes/mouse) subcutaneously on the back or by tick feeding using B. burgdorferi-infected nymphs (5 to 12 ticks/mouse). Fourteen days after inoculation, ear punch samples were collected and examined by PCR for the presence of spirochetes using flaB primers (forward primer 5'-GCT CAA ATA AGA GGT TTG TC-3' and reverse primer 5'-ATT CCA AGC TCT TCA GCT G-3'). When necessary, between 16 and 21 days after inoculation, nave ticks were allowed to feed on spirochete-positive mice to study Borrelia acquisition by ticks (see below). Twenty-one days after 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.
Experimental tick model of Borrelia acquisition and transmission. For Borrelia acquisition studies, nave ticks, either larvae (200 to 300 larvae/mouse) or nymphs (30 to 50 nymphs/mouse), were fed on infected mice. The spirochete burden in individual larvae was determined by Q-PCR at the following times: immediately, 1 week after larvae were fed to repletion, and 2 to 3 weeks after larvae molted into nymphs. The spirochete burden in individual nymphs was determined by Q-PCR at the following times: during feeding (48 h after attachment), shortly after feeding (72 and 96 h after attachment), and 3 days after feeding (168 h after attachment). For Borrelia transmission studies, infected nymphs (five nymphs /mouse), obtained by feeding larvae on infected mice, were fed on nave mice. To determine the success of Borrelia transmission, 21 days after tick attachment, mice were sacrificed and examined for Borrelia infection as described above.
Statistics. The statistical significance of differences observed in data sets was analyzed using GraphPad Instat, version 3.05. For data with small variations and normal distribution, such as levels of BBK32 expression in cDNA samples, a two-tailed Student's 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 variations and/or a nonparametric distribution, such as the spirochete burdens in individual mouse and tick samples, a two-tailed Mann-Whitney test was performed to compare two medians and a Kruskal-Wallis test with Dunn's posttest was performed to compare multiple medians.
RESULTS
Differential BBK32 gene expression by B. burgdorferi. Previous studies in our laboratory showed that BBK32 was differentially expressed by B. burgdorferi at different stages of its life cycle and was primarily expressed in mice and engorged ticks and not in flat ticks (17, 18). Microarray studies of in vitro-grown spirochetes also showed that BBK32 expression increased 1.7-fold upon a shift from 25°C to 35°C and increased another 2.3-fold at 35°C in the presence of blood, conditions mimicking the environment of a feeding tick (34, 57). Here, we systematically analyzed BBK32 expression in spirochetes grown in vitro or in infected mice or residing in ticks by Q-PCR (Fig. 1). The copy numbers of the BBK32 and flaB transcripts were determined for each cDNA sample (see Materials and Methods for details), and the relative level of expression of BBK32 was expressed as the number of copies of BBK32 per 1,000 copies of flaB. Our data (Fig. 1) not only confirmed all previous reports but also provided additional and more quantitative information on BBK32 differential expression. BBK32 expression varied over a remarkably wide range, and there was a nearly 1,000-fold increase from the lowest level in flat (unfed) ticks to the highest level in mouse tissues. BBK32 expression also varied significantly in different mouse tissues; the level of BBK32 expression in the joints was two- to threefold lower than that in the bladder, the skin, and the heart (P < 0.001), and the level of BBK32 expression in the heart was also significantly lower than that in the bladder and the skin (P < 0.05). There was a 2.8-fold increase in BBK32 expression in in vitro-grown spirochetes upon a shift from 25°C to 37°C (P < 0.0001). However, this increase was much too small to account for the difference between the feeding ticks and the flat (unfed) ticks. BBK32 expression in ticks varied significantly during feeding (P < 0.0001); it increased steadily after tick attachment and during engorgement and then decreased slowly after feeding to repletion. The level of BBK32 expression in 48-, 72-, and 96-h feeding ticks was 6- to 48-fold higher than that in flat (unfed) and 24-h feeding ticks (P < 0.001). Even at the highest level (in 96-h feeding ticks), the BBK32 expression in ticks was still 6- to 29-fold lower than that in various mouse tissues, suggesting that BBK32 may play a more important role in spirochete pathogenesis in mice.
Construction and characterization of a BBK32 deletion mutant in vitro. We constructed a BBK32 deletion mutant from B. burgdorferi B31 clone 5A11 by replacing the BBK32 gene with a Borrelia-adapted kanamycin resistance cassette, kanAn, through homologous recombination (Fig. 2A) (3). Two approximately 1.5-kb DNA fragments, one upstream (the 5' arm) and the other downstream (the 3' arm) of the BBK32 gene, were PCR amplified using primers P1 and P2 and primers P3 and P4, respectively (see Materials and Methods for details). These two DNA fragments (the 5' and 3' arms) mediated the homologous recombination event necessary to generate the mutant. The BBK32 deletion mutants were selected in medium containing kanamycin, their plasmid contents were analyzed, and one of the BBK32 deletion mutants that retained all 20 plasmids that the parent 5A11 clone had was used for further study (see Materials and Methods for details).
To verify the mutation, a series of PCRs were performed (Fig. 2B). The PCR using primers P5 and P6 indicated that the BBK32 gene was absent in the mutant, and the PCR using primers P7 and P8 indicated that the kanamycin resistance cassette was present in the mutant. The location of the kanamycin resistance cassette in the mutant was confirmed by PCR using primers P9 and P8. The sizes of all PCR-amplified fragments were the same as the predicted sizes. These PCR results conclusively showed that a double-crossover event had occurred in the mutant and resulted in replacement of the BBK32 gene with the kanamycin resistance cassette.
We also examined the BBK32 deletion mutant at the protein level (Fig. 2C). There was no apparent difference between the mutant and the wild-type strain as determined by the overall protein profiles generated by Coomassie brilliant blue staining of the whole-cell lysates separated by SDS-PAGE (Fig. 2C, panel a). Western blotting using antibodies against BBK32 indicated that the mutant was truly BBK32 deficient (Fig. 2C, panel b). Besides the most dominant immunoreactive band, which migrated as an approximately 45- kDa protein, there were four other bands that were absent in the mutant. These minor immunoreactive bands migrated faster than the predicted BBK32 protein migrated, suggesting that they might be degradation products of BBK32. A fibronectin-binding assay of the whole-cell lysates (Fig. 2C, panel c) indicated that the major fibronectin-binding protein in the wild-type strain was absent in the BBK32 deletion mutant, confirming that BBK32 was the major fibronectin-binding protein expressed by in vitro-grown B. burgdorferi. We noticed that of the four minor BBK32 antibody-reactive bands, only the two larger bands bound fibronectin, suggesting that the fibronectin-binding domain was cleaved off in the two smaller degradation products of BBK32. The nonspecific bands that reacted with the anti-rabbit IgG-HRP conjugate used for both the Western blot and the fibronectin overlay blot are shown in Fig. 2C, panel d.
Pathogenicity of the BBK32 deletion mutant in syringe-inoculated mice. To determine whether the loss of BBK32 expression in the mutant had any adverse effect on its pathogenicity, we examined both the wild-type strain and the mutant in the experimental mouse model of Lyme disease originally described by Barthold and colleagues (2). Groups of C3H/HeN mice (five mice/group) were subcutaneously inoculated with in vitro-grown spirochetes, either the wild-type strain or the BBK32 deletion mutant, at a dose of 105 spirochetes/mouse (see Materials and Methods for details). On day 21 postinoculation, mice were sacrificed and spirochete infection was assessed by serology and by in vitro culturing of the bladder and the spleen. The results of two independent mouse experiments using a total of 20 mice, 10 infected with the wild-type strain and the other 10 infected with the mutant strain, indicated that all infected mice seroconverted and were culture positive for spirochetes in the bladder and the spleen, regardless of which spirochete strain they were infected with (data not shown). The spirochete burden in mouse tissues was measured by Q-PCR analyses and was expressed as the number of copies of the B. burgdorferi flaB gene per 106 copies of the mouse actin gene. The Q-PCR results for two independent mouse experiments, shown in Fig. 3A, indicated that the spirochete burdens in a specific mouse tissue were comparable for mice infected with the wild-type strain and mice infected with the BBK32 deletion mutant (P > 0.05). It was also found that for both groups of mice, the spirochete burden in the joints was approximately fivefold higher than that in the bladder (P < 0.01) and three- to fivefold higher than that in the heart (P < 0.05).
Acquisition of the BBK32 deletion mutant by ticks. To determine whether the loss of BBK32 expression in the mutant had any adverse effects on its ability to be acquired by ticks, we fed larvae and nave nymphs on infected mice (see Materials and Methods for details). Larvae from one egg mass were evenly divided among four mice, two infected with the wild-type strain and the other two infected with the mutant strain (for each mouse infection was confirmed by positive flaB PCR results for an ear punch biopsy), and approximately 200 to 300 fed larvae were fed and collected from each mouse. One week after feeding, 10 individual ticks from each mouse were subjected to Q-PCR analyses. The spirochete burden in larvae, expressed as the number of copies of the flaB gene per 103 copies of the tick actin gene, was 2.3-fold higher (P = 0.02) in the larvae fed on mice infected with the wild-type strain than in larvae fed on mice infected with the mutant strain (Fig. 3B). To confirm that this small difference was real, we repeated the acquisition experiment using nave nymphs. Engorged nymphs were collected 96 h after attachment. Spirochete burdens in individual ticks were measured by Q-PCR (Fig. 3B). Ticks fed on mice infected with the BBK32 deletion mutant acquired numbers of spirochetes comparable to the numbers acquired by ticks fed on mice infected with the wild-type strain (P = 0.64). We also analyzed several other times, from as early as 72 h to as late as 168 h after attachment, and there was no significant difference between the ability of the mutant strain to be acquired by ticks from infected mice and the ability of the wild-type strain to be acquired by ticks from infected mice (data not shown).
Transmission of the BBK32 deletion mutant by ticks and its pathogenicity in tick-inoculated mice. To determine whether the loss of BBK32 expression in the mutant had any adverse effects on its transmission by ticks and/or its pathogenicity in mice infected by tick bites, we fed infected nymphs on nave mice (see Materials and Methods for details). The infected nymphs molted from larvae that were fed on infected mice (see above). The transmission experiment was repeated twice; each time we used two groups of mice (five mice/group), one fed on by the wild-type strain-infected nymphs and the other fed on by the BBK32 deletion mutant-infected nymphs. In the first experiment, 12 ticks were allowed to feed on each mouse; at 48 h after attachment, three ticks were pulled off each mouse before they fed to repletion, and the remaining ticks were collected after they were fully engorged, at 72 and 96 h after attachment. In the second experiment, five ticks were allowed to feed to repletion on each mouse.
The spirochete burdens in flat ticks and 48-, 72-, and 96-h feeding ticks were measured by Q-PCR (Fig. 3C). Overall, ticks infected with the mutant strain had a lower spirochete burden than ticks infected with the wild-type strain, although the difference was not statistically significant (P > 0.05). The small difference may have resulted from the difference observed between the two groups of ticks when they were engorged larvae (see above). Despite this difference, the spirochete populations in these two groups of ticks underwent similar expansions during feeding; there was a 3.3-fold increase for the wild-type strain and a 3.4-fold increase for the BBK32 deletion mutant.
All 20 mice inoculated by tick bite with either the wild-type strain or the BBK32 deletion mutant developed Lyme disease, as shown by positive IgG responses to B. burgdorferi lysates and positive cultures of spirochetes from the bladder and the spleen (data not shown). Spirochete burdens in bladder, heart, joint, and skin samples of all infected mice were determined by Q-PCR (Fig. 3D), and there were no significant differences between mice infected with the wild-type strain and mice infected with the BBK32 deletion mutant (P = 0.25).
The mutation in the BBK32 deletion mutant was stable even in the absence of antibiotic selection when spirochetes were cultured in vitro. To rule out the possibility that the mutant somehow reverted back to the wild type and expressed BBK32 when it infected mice, we performed a Western blot analysis with spirochetes cultured from infected mouse tissues, and our data indicated that the mutant was still BBK32 deficient and therefore that no reversion had occurred during infection (data not shown).
DISCUSSION
We found that BBK32 is differentially expressed by the Lyme spirochete at various stages of its life cycle. The expression pattern of BBK32 is very similar to that of OspC (50). Both proteins are expressed at the lowest level in flat ticks and at the highest level in mice, and the expression of both proteins increases during tick engorgement and decreases after ticks feed to repletion. This expression profile and the fibronectin-binding activity of BBK32 prompted us to hypothesize that BBK32 may play a role in promoting spirochete infection of the mammalian host. We tested this hypothesis by constructing a BBK32 deletion mutant by targeted mutagenesis and examining the mutant in an experimental tick-mouse model of the Borrelia life cycle. However, we failed to identify any defect of the BBK32 deletion mutant. Compared with the parental wild-type strain, the mutant is equally pathogenic in mice and equally competent in acquisition and transmission by ticks. These results lead to the inevitable conclusion that the function of BBK32 is dispensable to the spirochete, adding BBK32 to a list of nonessential genes, defined by targeted mutagenesis studies, of B. burgdorferi that includes luxS, celC/chbC, and BBA36 (27, 45, 56).
Bacterial adhesion to host tissues is a common theme in microbial pathogenesis (4, 19). To gain entry into the host through mucosal surfaces, bacterial pathogens express fimbrial and/or afimbrial adhesins that bind to various abundant sugar moieties of glycolipids and glycoproteins on epithelial cells. Mucosal colonization by pathogens often results in inflammation and tissue damage that exposes the underlying extracellular matrix. Binding to integrin and components of the extracellular matrix, such as fibronectin, collagen, and selectin, by pathogens not only promotes bacterial colonization but also facilitates bacterial invasion into deeper tissues, which may lead to systemic dissemination.
Several adhesion molecules that bind to integrin and components of the extracellular matrix have been identified in the Lyme spirochete (14). In addition to the fibronectin-binding protein BBK32, there are the decorin-binding proteins DbpA and DbpB, the integrin-binding protein P66, and the glycosaminoglycan-binding protein Bgp (12, 25, 40, 41). It is interesting, however, that to date the significance of these functions for B. burgdorferi pathogenesis has not been directly evaluated by targeted mutagenesis studies. Most studies have been limited to demonstrating the in vitro binding activities of these spirochete proteins (13, 21). Studies using decorin-deficient mice have indirectly demonstrated that the decorin-binding proteins are important to, but not essential for, the development of Lyme disease (7).
Our targeted mutagenesis study further confirmed that BBK32 is a fibronectin-binding protein. The importance of such an activity to Lyme disease pathogenesis, however, cannot be established using the BBK32 deletion mutant. The nonessential nature of the BBK32 function could be due to the fact that B. burgdorferi expresses many adherence factors. It is common for bacterial pathogens to express many adhesins, some with different tissue tropisms and some that are simply redundant. The genome of uropathogenic E. coli strain CFT073, for example, contains 12 distinct fimbria gene clusters (58). It has been difficult to define a role for a particular fimbria using mutagenesis studies because mutants lacking one adhesin often compensate by increasing expression of another adhesin (52).
Similarly, the loss of BBK32 function in the mutant could be compensated for either by the adhesins already identified, such as DbpA, DbpB, P66, and Bgp, or by other adherence factors yet to be discovered. A recent study indicated that BBK32 also bound to glycosaminoglycans (20), a function redundant with the function of Bgp. Although BBK32 is the most dominant fibronectin-binding protein as revealed by the in vitro blot overlay assay, it may not be the only or even the most dominant fibronectin-binding protein expressed by the Lyme spirochete due to technical limitations of the assay. First, proteins are separated on a denaturing SDS-PAGE gel before they are transferred to a membrane for fibronectin overlay. Thus, any fibronectin-binding activity involving secondary and tertiary protein conformation would be destroyed during the denaturing process. Second, whole-cell lysates are prepared from spirochetes cultured in vitro, conditions in which the expression of some potential fibronectin-binding proteins may not be optimal. A putative candidate fibronectin-binding protein, based on sequence homology, is encoded by the chromosome gene BB0347 (23). However, whether and when BB0347 is expressed by the spirochete and whether it truly binds to fibronectin have not been investigated.
B. burgdorferi is a remarkably adaptable microbe that can survive in environments that are as diverse as various arthropod vectors and numerous vertebrate reservoir hosts. Spirochete gene products that are preferentially expressed throughout the Borrelia life cycle have been implicated in pathogenesis, both directly through analysis of mutant B. burgdorferi and indirectly via binding studies and specific functional assays. The current data demonstrate that the major microbial protein that binds to fibronectin is not required for spirochete survival in ticks or mice and that B. burgdorferi has developed multiple, redundant mechanisms to persist in vivo.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institutes of Health. Xin Li is the recipient of an Arthritis Foundation Investigator Award. Erol Fikrig is the recipient of a Burroughs Wellcome Clinical Scientist Award in Translational Research.
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ABSTRACT
BBK32, a fibronectin-binding protein of Borrelia burgdorferi, is one of many surface lipoproteins that are differentially expressed by the Lyme disease spirochete at various stages of its life cycle. The level of BBK32 expression in B. burgdorferi is highest during infection of the mammalian host and lowest in flat ticks. This temporal expression profile, along with its fibronectin-binding activity, strongly suggests that BBK32 may play an important role in Lyme pathogenesis in the host. To test this hypothesis, we constructed an isogenic BBK32 deletion mutant from wild-type B. burgdorferi B31 by replacing the BBK32 gene with a kanamycin resistance cassette through homologous recombination. We examined both the wild-type strain and the BBK32 deletion mutant extensively in the experimental mouse-tick model of the Borrelia life cycle. Our data indicated that B. burgdorferi lacking BBK32 retained full pathogenicity in mice, regardless of whether mice were infected artificially by syringe inoculation or naturally by tick bite. The loss of BBK32 expression in the mutant had no adverse effect on spirochete acquisition (mouse-to-tick) and transmission (tick-to-mouse) processes. These results suggest that additional B. burgdorferi proteins can complement the function of BBK32, fibronectin binding or otherwise, during the natural spirochete life cycle.
INTRODUCTION
Borrelia burgdorferi is the etiologic agent of Lyme disease, the most common arthropod-borne disease in the United States (9, 35). In nature, B. burgdorferi is maintained in an enzootic cycle that involves an Ixodes tick and a vertebrate host (53). Larval and nymphal ticks feed mainly on small rodents, such as white-footed mice, and adult ticks commonly feed on deer. Humans are accidental hosts that do not play a meaningful role in the B. burgdorferi life cycle.
Shuttling between its reservoir host and tick vector, B. burgdorferi is highly adaptable. Even before the genome of B. burgdorferi was revealed, researchers in the field of Lyme disease had long recognized that many of the spirochete's surface lipoproteins are differentially expressed in response to various environmental cues, such as a blood meal in the tick, entering the mammalian host, etc. (15, 30, 51). The genome sequence of B. burgdorferi only underscores the enormity of its lipoprotein repertoire, which accounts for more than 10% of the whole genome (11, 23). This information touched off an explosion of microarray studies, which further confirmed that most of the surface lipoproteins are differentially expressed in spirochetes cultured under various conditions in vitro, cultured in dialysis membrane chambers implanted in rats, or isolated from tissues of infected mice or nonhuman primates (5, 29, 33, 34, 46, 57).
Significant advances in the molecular techniques for constructing B. burgdorferi mutants lacking a specific gene have made it possible for researchers to study the functions of individual spirochete gene products (47). There have been a remarkable number of targeted mutagenesis studies of B. burgdorferi in recent years (8, 10, 13, 16, 22, 24, 27, 28, 31, 32, 36, 37, 39, 45, 55, 56, 60, 61). Some of these studies have directly tested the hypothesis that differentially expressed lipoproteins have important functions at the stage(s) of the B. burgdorferi life cycle at which their expression is turned on.
Outer surface protein A (OspA) and OspC are two of the most notable examples of lipoproteins that are differentially expressed by the spirochete at various stages of its life cycle (50). OspA is expressed by spirochetes residing in the midgut of unfed ticks but not by spirochetes infecting the mammalian host, whereas the reverse is true for OspC. When a tick starts feeding, spirochetes residing in the midgut turn off the expression of OspA and start making OspC, although the two events may not be directly related. Thus, spirochetes migrating to tick salivary glands and subsequently entering the mammalian host mostly express OspC and not OspA. Although spirochetes rarely express OspA when they are infecting a mammalian host, shortly after being acquired by a tick through a blood meal, they turn on expression of OspA but not expression of OspC. These expression patterns are consistent with the role of OspA in spirochete colonization of the tick gut and the role of OspC in spirochete migration to tick salivary glands and infection of the mammalian host, as defined by mutagenesis studies (24, 39, 61). Further studies of the functions of OspA and OspC have revealed that OspA binds a tick receptor to promote spirochete attachment to the tick midgut and OspC sequesters a tick salivary protein with immune-suppressive functions to facilitate spirochete infection in the mammalian host (38, 44).
BBK32 is another surface lipoprotein that is differentially expressed by the spirochete. BBK32 was first identified as an antigen that is expressed by spirochetes infecting mice but not by spirochetes grown in vitro, and antibodies against BBK32 elicited protective immunity in mice (17, 54). Further studies showed that BBK32 was expressed in feeding ticks but not in unfed ticks and that antibodies against BBK32 blocked spirochete transmission (18). Serological studies indicated that an immunoglobulin G (IgG) antibody response to BBK32 is a common early response in patients with Lyme arthritis, and higher IgG antibody responses to BBK32 correlated with less severe disease (1). These results indicated the diagnostic value of BBK32 and its potential as a vaccine candidate, both of which were demonstrated experimentally (6, 26). Studies by Johnson and colleagues identified BBK32 as a fibronectin-binding protein (41, 42). Therefore, it was proposed that BBK32-mediated attachment to fibronectin, an extracellular matrix component, is important for the pathogenesis of Lyme borreliosis. To directly test this hypothesis, we constructed an isogenic BBK32 deletion mutant from a wild-type strain of B. burgdorferi and examined this mutant extensively in a laboratory tick-mouse model of the Borrelia life cycle.
MATERIALS AND METHODS
Bacterial strains and media. Clone 5A11 was a fully infectious clone of B. burgdorferi strain B31 that was obtained from Steve Norris, and it contained all 20 plasmids analyzed (43). The presence of lp5 (plasmid T) in 5A11 was unclear until PCR primers specific for lp5 were generated (59). The PCR results indicated that 5A11 had lost lp5. However, the loss of lp5 apparently had no effect on the infectivity of 5A11. We therefore used 5A11 as the parent strain to construct a BBK32 deletion mutant. Spirochetes were routinely cultured in BSK-H complete medium (Sigma) at 34°C unless indicated otherwise. Escherichia coli strain TOP10 (Invitrogen) was used as the host strain for transformation and maintenance of all plasmids. Luria broth was used to culture all E. coli strains.
Ixodes scapularis ticks. Larval, nymphal, and adult I. scapularis ticks were routinely maintained in our laboratory. Adult female ticks, in the presence of male ticks, were fed on New Zealand White rabbits to produce egg masses. Hatched larvae were fed either on nave C3H mice to generate nave nymphs or on B. burgdorferi-infected C3H mice to generate infected nymphs. Acquisition of spirochetes by the ticks was studied by feeding larvae or nave nymphs on infected mice. Transmission of spirochetes by the ticks was studied by feeding infected nymphs on nave mice. All ticks were incubated in a humidified chamber maintained at 25°C with 85 to 90% relative humidity and a photoperiod consisting of 14 h of light and 10 h of darkness.
DNA and RNA preparation and cDNA synthesis. DNA was extracted from mouse, tick, and spirochete samples using a DNeasy tissue kit (QIAGEN) according to the manufacturer's protocol. Total RNA was extracted from frozen mouse, tick, and spirochete samples using TRIZOL reagent (Invitrogen) according to the manufacturer's protocol. Prior to TRIZOL extraction, the frozen mouse and tick samples were ground thoroughly in liquid nitrogen. The RNA samples were digested with RNase-free DNase I (Roche) and then cleaned on RNeasy mini spin columns (QIAGEN). RNA was converted into first-strand cDNA using random hexamers and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's protocol.
Q-PCR analysis. All quantitative PCR (Q-PCR) assays were performed with an iCycler (Bio-Rad Laboratories) using high-fidelity Platinum Taq DNA polymerase (Invitrogen), gene-specific primers, and TaqMan probes with a program consisting of an initial denaturing step of 3 min at 95°C and 50 amplification cycles consisting of 30 s at 95°C followed by 1 min at 60°C. The forward and reverse primers and probe used for Q-PCR analysis of flaB were 5'-AGC TGA AGA GCT TGG AAT GC-3', 5'-TTG GTT TGC TCC AAC ATG AA-3', and 5'-TCC AAG ACG CTT GAG ACC CTG AAA-3', respectively. The forward and reverse primers and probe used for Q-PCR analysis of BBK32 were 5'-CAA CAA AGC TAA CCC AAA TGT AT-3', 5'-CTT TTG TAA ACT TTG CAG CTT CT-3', and 5'-ACG CCT TGA CAA CTT TGC TAA AGC C-3', respectively. The forward and reverse primers and probe used for Q-PCR analysis of mouse beta-actin were 5'-AGA GGG AAA TCG TGC GTG AC-3', 5'-CAA TAG TGA TGA CCT GGC CGT-3', and 5'-CAC TGC CGC ATC CTC TTC CTC CC-3', respectively. The forward and reverse primers and probe used for Q-PCR analysis of tick actin were 5'-GAT CAT GTT CGA GAC CTT CA-3', 5'-CGA TAC CCG TGG TAC GA-3', and 5'-CCA TCC AGG CCG TGC TCT C-3', respectively. All TaqMan probes were labeled at the 5' end with 6-carboxyfluorescein and at the 3' end with 5-carboxytetramethylrhodamine. Genomic DNA of B. burgdorferi B31 clone 5A11 was used as a standard for the flaB and BBK32 genes. Standards for the mouse beta-actin gene and the tick actin gene were prepared from plasmid DNA containing the specific gene. Gene copy numbers of the standards were calculated based on the molecular weight of the DNA molecule, and the DNA concentration was determined by sample absorbance at 260 nm. Gene copy numbers of unknown samples were determined by the threshold cycle method, using serial dilutions of the standards.
Sample collection for differential BBK32 gene expression. Eight mice were subcutaneously inoculated on the back with B. burgdorferi B31 clone 5A11 at a dose of 105 spirochetes/mouse. At 3 weeks postinoculation, the mice were sacrificed, an approximately 4-cm2 piece of the abdominal skin (distal from the inoculation site), the bladder, the heart, and both tibia tarsal joints were collected from each mouse, frozen in liquid nitrogen immediately, and then stored at –80°C. Infected nymphs (molted from larvae fed on mice infected with B. burgdorferi B31 clone 5A11) were fed on nave mice. Five ticks were collected for each time at 24, 48, 72, 96, 168, and 264 h after attachment. The time window for ticks to feed to repletion was between 66 and 96 h after attachment. Thus, the 24- and 48-h ticks were pulled off mice before they were fed to repletion. Five unfed ticks were also collected to represent zero time. All seven groups of ticks (five ticks/group) were frozen immediately in liquid nitrogen at the time of collection. A mid-log-phase culture of B. burgdorferi B31 clone 5A11 grown at 34°C was used to inoculate six 3-ml BSK-H complete medium tubes (initial density, 5 x 105 spirochetes/ml), three of which were incubated at 25°C and three of which were incubated at 37°C. All six cultures were grown to a density of approximately 5 x 107 spirochetes/ml, and spirochetes were collected by centrifugation and frozen immediately in liquid nitrogen. All frozen mouse, tick, and spirochete samples were processed for total RNA extraction (see above).
Construction of the BBK32 deletion mutant. First, a Borrelia-adapted kanamycin resistance cassette, kanAn, was excised from pTAkanAn (3) by EcoRI digestion and cloned into the EcoRI site of pBluescript (Stratagene). The resulting construct, designated pXLF10601, had two multiple-cloning sites flanking the kanAn cassette, which allowed efficient cloning of the two DNA fragments (the 5' and 3' arms) required for homologous recombination. To replace the BBK32 gene (base pairs 20389 to 21453 of lp36) and additional 97-bp downstream sequences with the kanAn cassette, primers P1 (5'-GCG GAG CTC AGA AAA TTT TAG AAG AAA ACA AGC T-3'), P2 (5'-CGC TCT AGA GCT TTC TCT CCT TTA AAG TTA ATA CT-3'), P3 (5'-GCG CTC GAG TGG ATA AGA AAA TGG ATT TCG-3'), and P4 (5'-CGC GGA TCC CCT CGC GGG ATA GAT AGT AG-3') were designed to PCR amplify the 5' arm (base pairs 18781 to 20388 of lp36) and the 3' arm (base pairs 21550 to 23030 of lp36). The primer sequences and base pair numbers were obtained from the GenBank sequence of B. burgdorferi lp36 (accession no. NC_001855). Both DNA fragments were PCR amplified from the genomic DNA of B. burgdorferi B31 clone 5A11 using high-fidelity Vent DNA polymerase (New England BioLabs Inc.) and a program consisting of an initial denaturing step of 5 min at 95°C and 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 90 s. The restriction sites in the primers (underlined) allowed directional cloning of the 5' arm into the SacI-XbaI site and of the 3' arm into the XhoI-KpnI site of pXLF10601. All restriction enzymes were purchased from New England BioLabs Inc. The final construct, designated pXLF10601-bbk32, was sequenced to confirm that no mutation was introduced into the 5' and 3' arms during PCR amplification.
Plasmid DNA of pXLF10601-bbk32 (25 μg) was electroporated into B. burgdorferi B31 clone 5A11 by using the protocol described in detail by Samuels et al. (48, 49). Transformants were selected in BSK-H complete medium (Sigma) containing kanamycin (350 μg/ml) and were examined by PCR to confirm the presence of the kanAn cassette and by Western blot analysis to confirm the loss of BBK32 expression. In two separate experiments, a total of 17 kanamycin-resistant transformants were obtained. PCR and Western blot analyses indicated that 14 of the 17 transformants were PCR positive for the kanAn cassette and BBK32 deficient, which resulted from a double-crossover event (data not shown). The remaining three kanamycin-resistant transformants were PCR negative for the kanAn cassette and thus likely to be spontaneous mutants. Single-crossover events, which would have resulted in a kanAn-positive and BBK32-positive transformant, were not detected. The plasmid contents of the BBK32-deficient mutants were determined by an array-based assay (59), which indicated that 3 of the 14 mutants had the same plasmid content as the parental 5A11 clone (data not shown). We randomly selected one of these three mutants for further study. Although the construct pXLF10601-bbk32 contained the ampicillin resistance marker (carried on the vector pBluescript), no ampicillin-resistant transformants were ever selected for. All BBK32 deletion mutants generated after transformation were PCR negative for the BBK32 gene and lacked BBK32 expression, and therefore they clearly resulted from a double-crossover event rather than a single-crossover event between pXLF10601-bbk32 and lp36 and did not contain the ampicillin resistance marker. B. burgdorferi lacking BBK32 and wild-type B. burgdorferi were also tested for sensitivity to ampicillin; both spirochetes were highly susceptible to ampicillin, and no differences between the wild-type and mutant spirochetes were observed. Experiments to genetically manipulate and transform B. burgdorferi with vector DNA containing antibiotic resistance markers were approved by the Yale University Biosafety Committee.
PCR analyses for confirmation of the BBK32 deletion. Primers P5 (5'-AGG CAA AAG CAA GGT TTC TA-3'), P6 (5'-ACT TTC AAT AGG CTC CTC CA-3'), P7 (5'-GTA CTC CTG ATG ATG CAT GG-3'), P8 (TGC ATT TCT TTC CAG ACT TG-3'), and P9 (5'-TCA GCA TAT GAT AAG ATG GTA GCT-3') were designed to verify the BBK32 deletion in the mutant by PCR. Primers P5, P6, and P9 were specific for B. burgdorferi lp38 (GenBank accession no. NC_001855), and primers P7 and P8 were specific for the kanamycin resistance cassette derived from pOK12 (GenBank accession no. AF223639). All PCRs were performed using Taq DNA polymerase (Roche), 1 ng of genomic DNA of wild-type clone 5A11 or the BBK32 deletion mutant, and a program consisting of an initial denaturing step of 5 min at 95°C and 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s.
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Western blot analysis, and fibronectin-binding assays. Wild-type clone 5A11 and the BBK32 deletion mutant were cultured at 34°C in BSK-H complete medium (Sigma) to a density of approximately 108 spirochetes/ml. Spirochetes were harvested and washed three times with Dulbecco's phosphate-buffered saline with centrifugation (5 min at 5,000 x g). B. burgdorferi pellets were suspended in SDS sample buffer (62.5 mM Tris-HCl [pH 6.8], 5% -mercaptoethanol, 2% SDS, 0.02% bromophenol blue, 10% glycerol) to a density of 107 spirochetes/μl and then denatured for 15 min at 100°C. B. burgdorferi whole-cell lysates (10 μl each) were separated on a two-layer polyacrylamide gel (3.75% polyacrylamide stacking gel and 10% polyacrylamide resolving gel). The separated proteins were either viewed directly by staining the gel with Coomassie brilliant blue or transferred to a polyvinylidene fluoride membrane and subjected to Western blot analysis or examined in a fibronectin-binding assay.
For Western blot analysis, the membrane was incubated with a 1:5,000 dilution of rabbit sera raised against BBK32 (17), followed by a 1:2,000 dilution of anti-rabbit IgG-horseradish peroxidase (HRP) conjugate. For the fibronectin-binding assay, the membrane was incubated first with human fibronectin (5 μg/ml), then with a 1:500 dilution of affinity-purified rabbit antibodies against human fibronectin, and finally with a 1:2,000 dilution of anti-rabbit IgG-HRP conjugate. To visualize the biotinylated molecular weight standards on the membrane, a 1:5,000 dilution of anti-biotin-HRP conjugate was also added in the final incubation. Except for the antibodies against BBK32, all other reagents, including human fibronectin, molecular weight standards, and antibodies, were purchased from Sigma. Prior to the first incubation, membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T). Between incubations, membranes were washed three times with Tris-buffered saline containing 0.1% Tween 20. Membranes were developed using ECL Western blotting detection reagents (Amersham Biosciences).
Experimental mouse model of Lyme disease. Four- to six-week-old female C3H/HeN mice were purchased from the National Cancer Institute and housed in the Yale Animal Resources Center according to the institutional guidelines for care and use of laboratory animals. Mice were inoculated with B. burgdorferi either by syringe injection of in vitro-grown spirochetes (105 spirochetes/mouse) subcutaneously on the back or by tick feeding using B. burgdorferi-infected nymphs (5 to 12 ticks/mouse). Fourteen days after inoculation, ear punch samples were collected and examined by PCR for the presence of spirochetes using flaB primers (forward primer 5'-GCT CAA ATA AGA GGT TTG TC-3' and reverse primer 5'-ATT CCA AGC TCT TCA GCT G-3'). When necessary, between 16 and 21 days after inoculation, nave ticks were allowed to feed on spirochete-positive mice to study Borrelia acquisition by ticks (see below). Twenty-one days after 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.
Experimental tick model of Borrelia acquisition and transmission. For Borrelia acquisition studies, nave ticks, either larvae (200 to 300 larvae/mouse) or nymphs (30 to 50 nymphs/mouse), were fed on infected mice. The spirochete burden in individual larvae was determined by Q-PCR at the following times: immediately, 1 week after larvae were fed to repletion, and 2 to 3 weeks after larvae molted into nymphs. The spirochete burden in individual nymphs was determined by Q-PCR at the following times: during feeding (48 h after attachment), shortly after feeding (72 and 96 h after attachment), and 3 days after feeding (168 h after attachment). For Borrelia transmission studies, infected nymphs (five nymphs /mouse), obtained by feeding larvae on infected mice, were fed on nave mice. To determine the success of Borrelia transmission, 21 days after tick attachment, mice were sacrificed and examined for Borrelia infection as described above.
Statistics. The statistical significance of differences observed in data sets was analyzed using GraphPad Instat, version 3.05. For data with small variations and normal distribution, such as levels of BBK32 expression in cDNA samples, a two-tailed Student's 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 variations and/or a nonparametric distribution, such as the spirochete burdens in individual mouse and tick samples, a two-tailed Mann-Whitney test was performed to compare two medians and a Kruskal-Wallis test with Dunn's posttest was performed to compare multiple medians.
RESULTS
Differential BBK32 gene expression by B. burgdorferi. Previous studies in our laboratory showed that BBK32 was differentially expressed by B. burgdorferi at different stages of its life cycle and was primarily expressed in mice and engorged ticks and not in flat ticks (17, 18). Microarray studies of in vitro-grown spirochetes also showed that BBK32 expression increased 1.7-fold upon a shift from 25°C to 35°C and increased another 2.3-fold at 35°C in the presence of blood, conditions mimicking the environment of a feeding tick (34, 57). Here, we systematically analyzed BBK32 expression in spirochetes grown in vitro or in infected mice or residing in ticks by Q-PCR (Fig. 1). The copy numbers of the BBK32 and flaB transcripts were determined for each cDNA sample (see Materials and Methods for details), and the relative level of expression of BBK32 was expressed as the number of copies of BBK32 per 1,000 copies of flaB. Our data (Fig. 1) not only confirmed all previous reports but also provided additional and more quantitative information on BBK32 differential expression. BBK32 expression varied over a remarkably wide range, and there was a nearly 1,000-fold increase from the lowest level in flat (unfed) ticks to the highest level in mouse tissues. BBK32 expression also varied significantly in different mouse tissues; the level of BBK32 expression in the joints was two- to threefold lower than that in the bladder, the skin, and the heart (P < 0.001), and the level of BBK32 expression in the heart was also significantly lower than that in the bladder and the skin (P < 0.05). There was a 2.8-fold increase in BBK32 expression in in vitro-grown spirochetes upon a shift from 25°C to 37°C (P < 0.0001). However, this increase was much too small to account for the difference between the feeding ticks and the flat (unfed) ticks. BBK32 expression in ticks varied significantly during feeding (P < 0.0001); it increased steadily after tick attachment and during engorgement and then decreased slowly after feeding to repletion. The level of BBK32 expression in 48-, 72-, and 96-h feeding ticks was 6- to 48-fold higher than that in flat (unfed) and 24-h feeding ticks (P < 0.001). Even at the highest level (in 96-h feeding ticks), the BBK32 expression in ticks was still 6- to 29-fold lower than that in various mouse tissues, suggesting that BBK32 may play a more important role in spirochete pathogenesis in mice.
Construction and characterization of a BBK32 deletion mutant in vitro. We constructed a BBK32 deletion mutant from B. burgdorferi B31 clone 5A11 by replacing the BBK32 gene with a Borrelia-adapted kanamycin resistance cassette, kanAn, through homologous recombination (Fig. 2A) (3). Two approximately 1.5-kb DNA fragments, one upstream (the 5' arm) and the other downstream (the 3' arm) of the BBK32 gene, were PCR amplified using primers P1 and P2 and primers P3 and P4, respectively (see Materials and Methods for details). These two DNA fragments (the 5' and 3' arms) mediated the homologous recombination event necessary to generate the mutant. The BBK32 deletion mutants were selected in medium containing kanamycin, their plasmid contents were analyzed, and one of the BBK32 deletion mutants that retained all 20 plasmids that the parent 5A11 clone had was used for further study (see Materials and Methods for details).
To verify the mutation, a series of PCRs were performed (Fig. 2B). The PCR using primers P5 and P6 indicated that the BBK32 gene was absent in the mutant, and the PCR using primers P7 and P8 indicated that the kanamycin resistance cassette was present in the mutant. The location of the kanamycin resistance cassette in the mutant was confirmed by PCR using primers P9 and P8. The sizes of all PCR-amplified fragments were the same as the predicted sizes. These PCR results conclusively showed that a double-crossover event had occurred in the mutant and resulted in replacement of the BBK32 gene with the kanamycin resistance cassette.
We also examined the BBK32 deletion mutant at the protein level (Fig. 2C). There was no apparent difference between the mutant and the wild-type strain as determined by the overall protein profiles generated by Coomassie brilliant blue staining of the whole-cell lysates separated by SDS-PAGE (Fig. 2C, panel a). Western blotting using antibodies against BBK32 indicated that the mutant was truly BBK32 deficient (Fig. 2C, panel b). Besides the most dominant immunoreactive band, which migrated as an approximately 45- kDa protein, there were four other bands that were absent in the mutant. These minor immunoreactive bands migrated faster than the predicted BBK32 protein migrated, suggesting that they might be degradation products of BBK32. A fibronectin-binding assay of the whole-cell lysates (Fig. 2C, panel c) indicated that the major fibronectin-binding protein in the wild-type strain was absent in the BBK32 deletion mutant, confirming that BBK32 was the major fibronectin-binding protein expressed by in vitro-grown B. burgdorferi. We noticed that of the four minor BBK32 antibody-reactive bands, only the two larger bands bound fibronectin, suggesting that the fibronectin-binding domain was cleaved off in the two smaller degradation products of BBK32. The nonspecific bands that reacted with the anti-rabbit IgG-HRP conjugate used for both the Western blot and the fibronectin overlay blot are shown in Fig. 2C, panel d.
Pathogenicity of the BBK32 deletion mutant in syringe-inoculated mice. To determine whether the loss of BBK32 expression in the mutant had any adverse effect on its pathogenicity, we examined both the wild-type strain and the mutant in the experimental mouse model of Lyme disease originally described by Barthold and colleagues (2). Groups of C3H/HeN mice (five mice/group) were subcutaneously inoculated with in vitro-grown spirochetes, either the wild-type strain or the BBK32 deletion mutant, at a dose of 105 spirochetes/mouse (see Materials and Methods for details). On day 21 postinoculation, mice were sacrificed and spirochete infection was assessed by serology and by in vitro culturing of the bladder and the spleen. The results of two independent mouse experiments using a total of 20 mice, 10 infected with the wild-type strain and the other 10 infected with the mutant strain, indicated that all infected mice seroconverted and were culture positive for spirochetes in the bladder and the spleen, regardless of which spirochete strain they were infected with (data not shown). The spirochete burden in mouse tissues was measured by Q-PCR analyses and was expressed as the number of copies of the B. burgdorferi flaB gene per 106 copies of the mouse actin gene. The Q-PCR results for two independent mouse experiments, shown in Fig. 3A, indicated that the spirochete burdens in a specific mouse tissue were comparable for mice infected with the wild-type strain and mice infected with the BBK32 deletion mutant (P > 0.05). It was also found that for both groups of mice, the spirochete burden in the joints was approximately fivefold higher than that in the bladder (P < 0.01) and three- to fivefold higher than that in the heart (P < 0.05).
Acquisition of the BBK32 deletion mutant by ticks. To determine whether the loss of BBK32 expression in the mutant had any adverse effects on its ability to be acquired by ticks, we fed larvae and nave nymphs on infected mice (see Materials and Methods for details). Larvae from one egg mass were evenly divided among four mice, two infected with the wild-type strain and the other two infected with the mutant strain (for each mouse infection was confirmed by positive flaB PCR results for an ear punch biopsy), and approximately 200 to 300 fed larvae were fed and collected from each mouse. One week after feeding, 10 individual ticks from each mouse were subjected to Q-PCR analyses. The spirochete burden in larvae, expressed as the number of copies of the flaB gene per 103 copies of the tick actin gene, was 2.3-fold higher (P = 0.02) in the larvae fed on mice infected with the wild-type strain than in larvae fed on mice infected with the mutant strain (Fig. 3B). To confirm that this small difference was real, we repeated the acquisition experiment using nave nymphs. Engorged nymphs were collected 96 h after attachment. Spirochete burdens in individual ticks were measured by Q-PCR (Fig. 3B). Ticks fed on mice infected with the BBK32 deletion mutant acquired numbers of spirochetes comparable to the numbers acquired by ticks fed on mice infected with the wild-type strain (P = 0.64). We also analyzed several other times, from as early as 72 h to as late as 168 h after attachment, and there was no significant difference between the ability of the mutant strain to be acquired by ticks from infected mice and the ability of the wild-type strain to be acquired by ticks from infected mice (data not shown).
Transmission of the BBK32 deletion mutant by ticks and its pathogenicity in tick-inoculated mice. To determine whether the loss of BBK32 expression in the mutant had any adverse effects on its transmission by ticks and/or its pathogenicity in mice infected by tick bites, we fed infected nymphs on nave mice (see Materials and Methods for details). The infected nymphs molted from larvae that were fed on infected mice (see above). The transmission experiment was repeated twice; each time we used two groups of mice (five mice/group), one fed on by the wild-type strain-infected nymphs and the other fed on by the BBK32 deletion mutant-infected nymphs. In the first experiment, 12 ticks were allowed to feed on each mouse; at 48 h after attachment, three ticks were pulled off each mouse before they fed to repletion, and the remaining ticks were collected after they were fully engorged, at 72 and 96 h after attachment. In the second experiment, five ticks were allowed to feed to repletion on each mouse.
The spirochete burdens in flat ticks and 48-, 72-, and 96-h feeding ticks were measured by Q-PCR (Fig. 3C). Overall, ticks infected with the mutant strain had a lower spirochete burden than ticks infected with the wild-type strain, although the difference was not statistically significant (P > 0.05). The small difference may have resulted from the difference observed between the two groups of ticks when they were engorged larvae (see above). Despite this difference, the spirochete populations in these two groups of ticks underwent similar expansions during feeding; there was a 3.3-fold increase for the wild-type strain and a 3.4-fold increase for the BBK32 deletion mutant.
All 20 mice inoculated by tick bite with either the wild-type strain or the BBK32 deletion mutant developed Lyme disease, as shown by positive IgG responses to B. burgdorferi lysates and positive cultures of spirochetes from the bladder and the spleen (data not shown). Spirochete burdens in bladder, heart, joint, and skin samples of all infected mice were determined by Q-PCR (Fig. 3D), and there were no significant differences between mice infected with the wild-type strain and mice infected with the BBK32 deletion mutant (P = 0.25).
The mutation in the BBK32 deletion mutant was stable even in the absence of antibiotic selection when spirochetes were cultured in vitro. To rule out the possibility that the mutant somehow reverted back to the wild type and expressed BBK32 when it infected mice, we performed a Western blot analysis with spirochetes cultured from infected mouse tissues, and our data indicated that the mutant was still BBK32 deficient and therefore that no reversion had occurred during infection (data not shown).
DISCUSSION
We found that BBK32 is differentially expressed by the Lyme spirochete at various stages of its life cycle. The expression pattern of BBK32 is very similar to that of OspC (50). Both proteins are expressed at the lowest level in flat ticks and at the highest level in mice, and the expression of both proteins increases during tick engorgement and decreases after ticks feed to repletion. This expression profile and the fibronectin-binding activity of BBK32 prompted us to hypothesize that BBK32 may play a role in promoting spirochete infection of the mammalian host. We tested this hypothesis by constructing a BBK32 deletion mutant by targeted mutagenesis and examining the mutant in an experimental tick-mouse model of the Borrelia life cycle. However, we failed to identify any defect of the BBK32 deletion mutant. Compared with the parental wild-type strain, the mutant is equally pathogenic in mice and equally competent in acquisition and transmission by ticks. These results lead to the inevitable conclusion that the function of BBK32 is dispensable to the spirochete, adding BBK32 to a list of nonessential genes, defined by targeted mutagenesis studies, of B. burgdorferi that includes luxS, celC/chbC, and BBA36 (27, 45, 56).
Bacterial adhesion to host tissues is a common theme in microbial pathogenesis (4, 19). To gain entry into the host through mucosal surfaces, bacterial pathogens express fimbrial and/or afimbrial adhesins that bind to various abundant sugar moieties of glycolipids and glycoproteins on epithelial cells. Mucosal colonization by pathogens often results in inflammation and tissue damage that exposes the underlying extracellular matrix. Binding to integrin and components of the extracellular matrix, such as fibronectin, collagen, and selectin, by pathogens not only promotes bacterial colonization but also facilitates bacterial invasion into deeper tissues, which may lead to systemic dissemination.
Several adhesion molecules that bind to integrin and components of the extracellular matrix have been identified in the Lyme spirochete (14). In addition to the fibronectin-binding protein BBK32, there are the decorin-binding proteins DbpA and DbpB, the integrin-binding protein P66, and the glycosaminoglycan-binding protein Bgp (12, 25, 40, 41). It is interesting, however, that to date the significance of these functions for B. burgdorferi pathogenesis has not been directly evaluated by targeted mutagenesis studies. Most studies have been limited to demonstrating the in vitro binding activities of these spirochete proteins (13, 21). Studies using decorin-deficient mice have indirectly demonstrated that the decorin-binding proteins are important to, but not essential for, the development of Lyme disease (7).
Our targeted mutagenesis study further confirmed that BBK32 is a fibronectin-binding protein. The importance of such an activity to Lyme disease pathogenesis, however, cannot be established using the BBK32 deletion mutant. The nonessential nature of the BBK32 function could be due to the fact that B. burgdorferi expresses many adherence factors. It is common for bacterial pathogens to express many adhesins, some with different tissue tropisms and some that are simply redundant. The genome of uropathogenic E. coli strain CFT073, for example, contains 12 distinct fimbria gene clusters (58). It has been difficult to define a role for a particular fimbria using mutagenesis studies because mutants lacking one adhesin often compensate by increasing expression of another adhesin (52).
Similarly, the loss of BBK32 function in the mutant could be compensated for either by the adhesins already identified, such as DbpA, DbpB, P66, and Bgp, or by other adherence factors yet to be discovered. A recent study indicated that BBK32 also bound to glycosaminoglycans (20), a function redundant with the function of Bgp. Although BBK32 is the most dominant fibronectin-binding protein as revealed by the in vitro blot overlay assay, it may not be the only or even the most dominant fibronectin-binding protein expressed by the Lyme spirochete due to technical limitations of the assay. First, proteins are separated on a denaturing SDS-PAGE gel before they are transferred to a membrane for fibronectin overlay. Thus, any fibronectin-binding activity involving secondary and tertiary protein conformation would be destroyed during the denaturing process. Second, whole-cell lysates are prepared from spirochetes cultured in vitro, conditions in which the expression of some potential fibronectin-binding proteins may not be optimal. A putative candidate fibronectin-binding protein, based on sequence homology, is encoded by the chromosome gene BB0347 (23). However, whether and when BB0347 is expressed by the spirochete and whether it truly binds to fibronectin have not been investigated.
B. burgdorferi is a remarkably adaptable microbe that can survive in environments that are as diverse as various arthropod vectors and numerous vertebrate reservoir hosts. Spirochete gene products that are preferentially expressed throughout the Borrelia life cycle have been implicated in pathogenesis, both directly through analysis of mutant B. burgdorferi and indirectly via binding studies and specific functional assays. The current data demonstrate that the major microbial protein that binds to fibronectin is not required for spirochete survival in ticks or mice and that B. burgdorferi has developed multiple, redundant mechanisms to persist in vivo.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institutes of Health. Xin Li is the recipient of an Arthritis Foundation Investigator Award. Erol Fikrig is the recipient of a Burroughs Wellcome Clinical Scientist Award in Translational Research.
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