Whole-Genome DNA Array Analysis of the Response of Borrelia burgdorferi to a Bactericidal Monoclonal Antibody

Experimental design.Differential gene expression of B. burgdorferi was analyzed in response to a sublethal concentration of CB2. Various amounts of CB2 were analyzed for an optimal sublethal concentration. RNA from B. burgdorferi was isolated at various time points up to 1 h (5, 20, 25, and 60 min) and used to create cDNA for use on a whole Borrelia genome DNA array membrane (54). Array results were validated by quantitative real-time PCR of selected differentially expressed genes and randomly chosen stable genes for controls.

B. burgdorferi strains, culture conditions, and antibodies. B. burgdorferi strain B31 (high passage) was grown in BSK-H medium (Sigma, St. Louis, Mo.) at 33°C and was enumerated by dark-field microscopy. The plasmid content of the B31 strain used for all experiments was determined by PCR with previously designed primers (33). The following plasmids are present in this strain: lp54, cp26, lp17, lp28-1, lp38, lp5, and cp32-1-3-4-6-8. Affinity-purified murine MAb CB2, an IgG1κ to OspB, was used and has been described previously (19, 21, 22, 34, 51).

Antibody treatment and subsequent RNA isolation.Borreliae were harvested from a 20-ml culture in the mid to late logarithmic phase of growth (7 × 10⁷ cells per ml) at 7,000 × g and washed once with BSK-H. Spirochetes were resuspended in 1 ml of BSK-H at 33°C; two 400-μl aliquots served as an untreated control and a CB2-treated experimental fraction. Equivalent aliquots (5.6 × 10⁸ spirochetes) received CB2 at final concentrations of 2, 20, or 200 μg/ml. Untreated control samples received comparable amounts of phosphate buffer alone (0.02 M sodium phosphate [pH 7.0]). After CB2 was added, samples were incubated at 33°C for 5, 20, and 60 min. RNA was isolated from the spirochetes in the 400-μl sample by using TRI-Reagent LS (Molecular Research Center, Inc., Cincinnati, Ohio). TRI-Reagent LS was designed for immediate lysis of spirochetes in liquid samples, hence the small (400 μl) volume. The RNA was run through an RNeasy Mini-Column (Qiagen, Inc., Valencia, Calif.) and resuspended in 30 μl of RNase-free water. RNA integrity was verified by agarose gel electrophoresis and quantified by spectrophotometry. A₂₆₀/A₂₈₀ ratios consistently ranged between 1.8 and 2.0. Separate RNA isolations were completed for each individual experiment.

In other experiments, B. burgdorferi was grown to a concentration of 10⁸ cells per ml in a volume of 50 ml of BSK-H each for untreated control and for CB2-treated (20 μg/ml) samples. Both untreated and CB2-treated samples were incubated at 33°C for 25 min. RNA was isolated by using TRI-Reagent (Molecular Research Center, Inc.). RNA was verified as described previously.

Direct enumeration of spirochetes after antibody treatment was done by assessing motility under dark-field microscopy. In addition, samples were recultured in BSK-H to determine viability after 60 min of CB2 treatment. Borreliae were allowed to recover at 33°C for 5 days. Bacteria were enumerated daily thereafter to assess recovery.

HS and FT procedures.The spirochete pellet was resuspended in 5 ml of prewarmed 33°C BSK-H. Twelve 400-μl aliquots served as untreated control, heat shock (HS) and freeze-thaw (FT) fractions (carried out in quadruplicate). Untreated control samples were placed at 33°C for 60 min. HS samples were placed at 60°C for 60 min. FT samples were placed in liquid nitrogen for 10 s, followed by 5 min at 37°C. The FT procedure was repeated nine times over 60 min.

PCR amplification and slot blot. flaB DNA was obtained from B. burgdorferi MedImmune genomic DNA (a gift from Patricia Rosa, Rocky Mountain Laboratory, National Institutes of Health). Oligonucleotide primers for flaB (BB0147) were described previously (54). All PCRs were completed in 200-μl tubes in a GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk, Conn.). Each 50-μl final volume reaction contained 1 μl of DNA template (100 ng/μl); 1× PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl₂, 50 mM KCl [pH 8.3]); dATP, dCTP, dGTP, and dTTP (10 mM each); BB0147FWD and BB0147REV (1 μM each); and 2 U of TaqDNA polymerase (Roche, Indianapolis, Ind.). PCR conditions included 1 cycle at 95°C for 5 min and 30 cycles of denaturation at 95°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min, followed by a second extension cycle at 72°C for 15 min. Amplified flaB PCR product was cleaned and concentrated via the Wizard PCR Preps DNA purification system (Promega, Madison, Wis.). For construction of the slot blot (Schleicher & Schuell, Keene, N.H.), a charged nylon membrane was initially soaked in 0.4 M Tris-HCl (pH 7.5) for 5 min. DNA was denatured for 10 min at room temperature in a solution of 0.25 N NaOH-0.5 M NaCl. Each slot received 76 ng of flaB DNA. The membrane was neutralized in a solution of 0.5 M NaCl-0.5 M Tris-HCl (pH 7.5). DNA was fixed to the membrane in a UV StrataLinker 1800 (Stratagene, La Jolla, Calif.). The membrane was dried at room temperature and stored until probed with ³³P-labeled cDNA.

cDNA probe synthesis.A cDNA probe was created for hybridization to the DNA array membranes as described previously, with some modifications (54). Briefly, for both untreated and CB2-treated conditions, a 15-μl reaction mixture contained 5 μg of RNA and 1 μl of 3′ open reading frame (ORF) B. burgdorferi cDNA labeling primers (Sigma-Genosys, Inc., Woodlands, Tex.). Amplifications were carried out in a PTC-1152 MiniCycler (MJ Research, Waltham, Mass.) with the parameters set at 90°C for 2 min, followed by a ramp to 42°C over 20 min. A final volume of 30 μl containing 1× reaction buffer (50 mM Tris-HCl, 8 mM MgCl₂, 30 mM KCl, 1 mM dithiothreitol [pH 8.5]), 50 U of avian myeloblastosis virus reverse transcriptase, deoxynucleoside triphosphates (333 μM dCTP, 333 μM dGTP, and 333 μM dTTP; Roche), and 20 μCi of [α-³³P]dATP (ICN, Costa Mesa, Calif.) was added to the reaction mixture. This reaction was incubated at 42°C for 2.5 h in the MiniCycler. Unincorporated radiolabeled nucleotide was removed from the labeled cDNA by use of a Panorama Sephadex G-25 column, based on the manufacturer's protocol (Sigma-Genosys). Radioactivity was measured in duplicate, both before and after removal of unincorporated label, in a Wallac 1409 DSA liquid scintillation counter (Perkin-Elmer, Gaithersburg, Md.), and the incorporation was calculated according to the following formula: percent incorporation = (incorporated disintegrations per minute × volume/total disintegrations per minute × volume) × 100.

Membrane hybridization and array image analysis.The B. burgdorferi whole-genome DNA array used for these experiments was created by a consortium of laboratories in conjunction with Sigma-Genosys (54). Membranes were neutralized in 35-by-300-mm hybridization bottles (Robbins Scientific, Sunnyvale, Calif.) in 250 ml of 2× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA [pH 7.7]) for 25 min and in subsequent experiments membranes were neutralized for 5 min. Membranes were prehybridized in 5 ml of 65°C hybridization solution (Sigma-Genosys) containing 100 μg of salmon testes DNA (Sigma-Genosys)/ml for 60 min. The entire radiolabeled cDNA sample was added to 10 ml of hybridization solution containing 100 μg of salmon testes DNA/ml, denatured in a 95°C water bath for 10 min, and then added to the membranes and allowed to hybridize for 16 to 18 h at 65°C in a model 2000 Micro-Hybridization incubator (Robbins Scientific). The cDNA was removed from the bottles, and 40 ml of wash solution (0.5× SSPE, 2% sodium dodecyl sulfate [SDS]) was added. Bottles were inverted 10 times at room temperature, and the wash solution was decanted. This step was repeated twice. After the third wash, 80 ml of wash solution was added, and the bottles were incubated at room temperature in the hybridization oven for 20 min. Membranes were exposed to a 35- by 43-cm PhosphorImager screen (Molecular Dynamics, Sunnyvale, Calif.) for 2 to 3 days depending on the percent incorporation of ³³P label in the cDNA. Screens were viewed in a Storm 860 PhosphorImager (Molecular Dynamics) by using ImageQuant 1.2 software. Pixel densities for each spot were generated with ArrayVision software version 6.0.

Membranes were stripped in roller bottles initially with 250 ml of 0.4 M NaOH, followed by incubation with stripping solution (250 ml of 200 mM Tris-HCl-0.1× SSPE-0.2% SDS [pH 7.0]) each for 30 min at 45°C with rotation. This procedure was followed by incubation with 250 ml of stripping solution at room temperature in the hybridization oven for 30 min with moderate rotation. Membranes were exposed to the PhosphorImager screen (as before) overnight to ensure that the stripping was successful, so membranes could be reprobed.

Data processing.ArrayVision software (Imaging Research, St. Catharines, Ontario, Canada) was used to generate raw data from each untreated and CB2-treated replicate (54). Briefly, an initial gene filter created a threshold for each gene individually based on the local background of each spot (89). Only genes with raw data values ≥2 standard deviations (SD) above the local background were further analyzed (23). The raw data of each individual spot were divided by the total raw data of all spots to get a percent intensity. The data were analyzed in Excel workbooks. To determine which genes were differentially expressed, we used the criteria for significance developed by Conway et al. for Escherichia coli DNA array membranes (13, 23, 86; unpublished data [http://www.ou.edu/microarray ]). Differentially expressed genes were required to pass two sets of statistical criteria to be considered significant. The first criterion required that changes in each gene be ≥2 SD above or below the mean of the log ratio of the individual spot intensity of the CB2-treated replicate versus that of the untreated control. Log ratio plots were created based on the following formula: log [(percent spot intensity of the CB2-treated gene X)/(percent spot intensity of the untreated control gene X)]. For the second criterion, genes were required to pass an unpaired two-tailed Student t test with a P value of ≤0.01 (>99% probability).

Differentially expressed genes were further analyzed for paralogous family members and structural motifs, domains and regions by using TIGR and other protein databases (http://www.tigr.org , http://www.ch.embnet.org/software/TMPRED_form.html , http://smart.embl-heidelberg.de/ , and http://psort.nibb.ac.jp/form.html ).

Quantitative real-time PCR.This procedure was used for the validation of whole-genome DNA array experiments comparing untreated control versus CB2-treated samples. For cDNA synthesis, 5 μg of total RNA was used as described previously, with genome-directed primers (2, 62, 84). Genome-directed primers were used to generate cDNA from untreated control RNA samples and from RNA samples treated with 20 μg of CB2/ml at the designated time points. The resulting cDNA sample was diluted 1:100 for use in the real-time PCR. LightCycler-FastStart DNA Master SYBR I Green (Roche) reaction mix was used in the experimental procedure with the LightCycler (Roche) based on the manufacturer's protocol. Cycle parameters were as follows: preincubation, 1 cycle at 95°C for 5 min; amplification (quantification), 40 cycles at 95°C for 15 s, 52°C for 5 s; and 72°C for 6 s; melting curve, 95°C for 0 s, 65°C for 15 s, and 72°C for 0 s; and cooling, 1 cycle at 40°C for 30 s. A region of the 16S rRNA was used as an internal control for all data as shown previously by Revel et al. (62). GAPDH (glyceraldehyde-3-phosphate dehydrogenase; BB0057) and flaB were also used as secondary controls for some genes. Oligonucleotide primers for real-time PCR were designed by using MacVector software (Kodak International Biotechnologies, Inc., New Haven, Conn.) (Table 1).

SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting.Borreliae were harvested and prepared as described above for RNA isolation. Hanks balanced salt solution (Gibco, Carlsbad, Calif.) was used for washes and final resuspension. Eight 400-μl replicates of both untreated control samples and samples treated with 20 μg of CB2/ml were prepared for each 5-, 27-, 35-, and 60-min time point. After each time point, samples were centrifuged at 10,000 × g for 45 s. The supernatant was removed and pooled for concentration to a volume of 0.2 ml by using a Centricon YM-3, with a nominal molecular mass limit of 3 kDa, according to the manufacturer's protocol (Millipore, Bedford, Mass.). Each pellet was resuspended in 100 μl of Hanks balanced salt solution and pooled. The total protein concentration of both supernatant and pellet fractions was determined with the BCA protein assay kit (Pierce, Rockford, Ill.). In addition, gels were also run with equivalent volume ratios. Minislab 16.5% acrylamide gels (8 by 9 by 0.1 cm) were cast with a 1.5-mm 10-well comb, and samples (250 ng/well) were applied and run for 1.5 h at 20 mA per gel (52). E. coli strain MM294 harboring pTG3, containing the blyAB operon, was used as a marker, as described previously (26, 42). Gels were stained by using Silver Stain-Plus based on the manufacturer's protocol (Bio-Rad, Hercules, Calif.). Some gels were transferred overnight to Immobilon-P^SQ (Millipore) in CAPS transfer buffer (10% methanol, 20% 0.05 M 3-cyclohexyl-amino-1-propane sulfonic acid [CAPS] [pH 11]) at 30 mA and blocked in 2% casein blocking solution. The membranes were probed with rabbit anti-BlyA peptide antiserum in casein blocking solution. The secondary antibody was alkaline phosphatase-conjugated goat anti-rabbit whole-molecule immunoglobulin G (IgG) (Sigma). Bands were visualized with nitroblue tetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate; KPL, Gaithersburg, Md.). Both BlyA antibody and MM294-pTG3 were a generous gift of Donald Oliver, Wesleyan University (42).

B. burgdorferi strain B31 treated with 20 μg of CB2/ml proves to be the optimal, sublethal concentration for assessing differential gene expression.Prior to utilizing the whole-genome DNA arrays for the effect of CB2 on Borrelia gene expression, the desired antibody concentration used for treating the spirochetes was determined. To examine the feasibility of harvesting RNA under lethal or near-lethal conditions, B. burgdorferi were subjected to either HS or FT conditions, followed by RNA isolation. Both HS and FT treatments had marked effects on the numbers and morphology of spirochetes compared to the untreated control (data not shown). In line with these effects, RNA isolated from both HS and FT samples was 33% less concentrated than that obtained from untreated samples (Fig. 1A). These data served as a reference to assess the effects of CB2 on our ability to obtain RNA from the treated organisms.

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FIG. 1.

Verification of 20 μg of CB2/ml as the optimal sublethal concentration. (A) Borrelia RNA concentration after HS or FT conditions. Error bars represent the mean of triplicate values ± the standard error. (B) Borrelia RNA concentration after treatment with various CB2 concentrations for 60 min. (C) 1% agarose gel electrophoresis of RNA extracts shown in panel B. Lane 1, RNA ladder; lanes 2 and 3, untreated; lanes 4 and 5, 2 μg of CB2/ml; lanes 6 and 7, 20 μg of CB2/ml; lanes 8 and 9, treatment with 200 μg of CB2/ml. Arrows indicate rRNA subunits. (D) Recovery of Borrelia after treatment with 2 (□), 20 (▴), or 200 (×) μg of CB2/ml or no treatment (♦).

Preliminary experiments were done to determine the antibody concentration and time needed for gene expression. Response of B. burgdorferi to CB2 was analyzed with increasing 10-fold CB2 concentrations (2, 20, and 200 μg/ml) at a 60-min time point to determine the concentration that affected but did not obliterate the organisms. The RNA yield showed a slight decrease as CB2 concentration increased in comparison to the untreated control (Fig. 1B). However, agarose gel electrophoresis of representative samples verified RNA quality and showed that the harvested RNA was not degraded under these conditions (Fig. 1C). In fact, RNA isolated from B. burgdorferi treated with the various CB2 concentrations did not yield concentrations of RNA lower than that observed with the HS and FT conditions. Spirochete recovery experiments were performed in which 400 μl of each sample (untreated or 2, 20, or 200 μg of CB2/ml) was added to fresh BSK-H. The spirochetes were then counted for 5 days to track growth patterns (Fig. 1D). The 2-μg/ml CB2 concentration had little or no effect on the growth of the bacteria. The sample treated with 200 μg of CB2/ml showed considerable delayed recovery, whereas the 20-μg/ml concentration of CB2 displayed a balance between having an effect on the organism and their potential for recovery. For this reason, we chose the CB2 concentration of 20 μg/ml. Spirochete viability was determined through direct enumeration after treatment of 20 μg of CB2/ml at 20 min under dark-field microscopy. The mean percentage of motile organisms after this treatment was 81.3 (±4.26 [standard error]) from three separate enumerations. To further verify the 20 μg of CB2/ml as the optimal sublethal concentration, flaB slot blot analysis of each cDNA sample was carried out. flaB was chosen as the optimal gene to analyze because its mRNA transcript should be unaffected by CB2. Each RNA sample (untreated or 2, 20, or 200 μg of CB2/ml) was used to create radiolabeled cDNA, which was then hybridized to flaB spotted on membrane strips. At each CB2 concentration, the density of the spotted flaB transcript remained unaffected compared to the untreated control (data not shown). Slot blot data of flaB proved that transcript was present and constant at all tested CB2 concentrations. This experiment also provided evidence for stable mRNA despite the concentration of CB2 and its effect on the organisms. All subsequent experiments were carried out by using a final CB2 concentration of 20 μg/ml.

DNA array experiments with borreliae incubated with CB2.DNA array membranes were probed with radiolabeled cDNA from separate RNA isolations of untreated spirochetes and spirochetes treated with 20 μg of CB2/ml at various times ranging from 5 min to 1 h. Spots that did not pass the initial background filter (see methods) were not further analyzed but cannot be excluded from potential involvement given their low expression level.

DNA array data at various time points provided a preliminary assessment of gene activity. These data served as a basis for subsequent experiments at a relevant time point.

Transcriptome analysis of cultures (5 × 10⁹ spirochetes) grown in 50-ml volumes were subsequently treated with 20 μg of CB2/ml for 25 min. This DNA array experiment was carried out twice with the same RNA sample to create cDNA for both experiments (Fig. 2). This method allowed a “snapshot” of gene activity to be created because the same RNA sample was used in creating cDNA for both membrane hybridizations. A constant pH and temperature of 33°C were maintained throughout the experiment. Figure 2 illustrates the log ratio plots of all of the genes analyzed, and Table 2 lists the respective differentially expressed genes that met the statistical requirements (≥2 SD from the mean of the log ratios and of P value of ≤0.01).

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FIG. 2.

Spot graph log ratio plot analysis of Borrelia gene expression from array raw data of untreated versus CB2-treated (20 μg/ml) condition at 25 min. Dashed horizontal lines represent 2 SD above or below the mean of the log ratio plot. Solid vertical lines separate chromosomal (left) from plasmid (right) genes. Highlighted spots (○) represent genes that are significant by both criteria (see also Table 2).

Four chromosomal genes were downregulated in response to CB2 at 25 min. BB0055 and BB0056, triosephosphate isomerase and phosphoglycerate kinase, respectively, are located immediately upstream of BB0057 (GAPDH). All three genes are glycolytic pathway enzymes involved in energy metabolism and are in an operon (39). BB0354 is characterized by The Institute for Genomic Research (www.tigr.org ) as a hypothetical protein but shares 31% identity and 58% similarity to a Fusobacterium nucleatum integral membrane protein (FN1300). The membrane-associated protein BB0603, p66 or Oms66, was also downregulated in this experiment. This protein has been characterized as a porin and as a protective immunogen (8, 17, 18, 28, 35, 56, 76). Sixteen (80%) of the differentially expressed genes were plasmid encoded. Analysis of the results obtained with this array (Table 2) again showed a marked activity of genes encoded by cp32. Cluster analysis of these 20 genes, many of which are hypothetical proteins, would not yield significant data when separated based on functional category. These genes were analyzed for unique motifs, regions, and/or domains by using TIGR, TMPRED, PSORT, and SMART protein databases. No obvious trends were observed with these analyses. However, paralogous family groupings showed three paralogs from both the cp32-encoded family 112 and 143 genes that are upregulated in response to CB2 (Table 2). BBA39, BBA40, and BBA41, in a possible operon with BBA38 on lp54, were all upregulated in response to CB2. There is a perfect −10 consensus and a partially conserved −35 consensus sequence upstream of BBA38. In addition, blyA homologs from the cp32 plasmids were associated with CB2 treatment in both experimental procedures, as BBO23, BBP23, and BBR23 were upregulated.

blyA, a holin system gene, was differentially expressed in two independent experiments at two different time points (20 and 25 min) and therefore was chosen for further analysis. blyA homologs and genes in the operon (BBP23 to BBP26) were differentially expressed along with cp32 plasmid activity at 25 min, and we attribute this effect to the action of CB2.

Quantitative real-time PCR analysis of blyA and blyB and other genes validate array results.Both blyA and blyB displayed similar trends of gene expression in untreated versus CB2-treated samples, as would be expected from genes encoded in an operon. Because neither blyA nor blyB displayed a change in gene expression at 5 or 60 min in preliminary arrays, we performed real-time PCR analysis at these time points and confirmed the earlier results with a −1.39-fold and −1.03-fold change in blyA expression at 5 and 60 min, respectively, when normalized to the 16S ribosomal subunit. In similar experiments, blyB had a −1.4-fold and −1.06-fold change in gene expression at 5 and 60 min, respectively. However, at 20 min blyA was found to be upregulated, on average 76-fold, whereas blyB was upregulated 56-fold, relative to either 16S or GAPDH as internal controls. Two separate time courses were further analyzed to see exactly when blyA and blyB gene homologs were active with four separate RNA samples. The first time course of 20, 21, 23, and 25 min shows both blyA and blyB to be upregulated significantly at 25 min in the CB2-treated sample compared to the untreated control (Fig. 3A and D). The second time course of 18, 25, 30, and 35 min again illustrates 25 min as the prominent time point for blyA and blyB gene activity (Fig. 3B and E). For the second time course, blyA and blyB were also normalized to flaB, and a similar trend in gene expression was observed (Fig. 3C and F). It is clear that both blyA and blyB are upregulated significantly, with the 25-min time point as the critical peak time. Evidence for this upregulation comes from significant concordance between array and quantitative real-time PCR data.

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FIG. 3.

Real-time PCR analysis of blyA (A to C) and blyB (D to F) in two different time courses. A, B, D, and E are ratios relative to the 16S ribosomal subunit; C and F are ratios relative to flaB. Black bars (U) represent untreated samples; gray bars (C) represent samples treated with 20 μg of CB2/ml. Error bars represent the mean of three replicates ± the standard error.

To validate the analyses of the array, 16 genes at various time points for a total of 28 triplicate quantitative real-time PCR reactions were completed. Unchanged genes in untreated control versus CB2-treated samples, as determined by previous array analysis, were randomly selected as negative controls for real-time PCR. Selected genes included BB0011, BB0057, BB0147, BB0377, BB0516, BB0615, BB0663, BBA30, BBB19, BBP10, and BBM35. Real-time PCR investigation of these genes, along with genes analyzed over time, and several differentially expressed genes was significantly consistent with array predictions with a correlation coefficient value of 0.90 (r² = 0.81) (Fig. 4).

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FIG. 4.

Concordance of differential gene expression between DNA array and quantitative real time-PCR Analysis. Fold change values for negative controls and differentially expressed genes were generated by comparing untreated to CB2-treated samples. In all cases, 16S ribosomal subunit was used as the internal control for real-time PCR (RT-PCR). Correlation coefficient = 0.90; trend line was generated with linear regression analysis.

BlyA protein expression in untreated Borrelia samples and in Borrelia samples treated with 20 μg of CB2/ml. blyA and blyB were transcriptionally active and differentially expressed in response to CB2 at a critical 25-min time point. The translation of these genes was next analyzed via SDS-PAGE silver stain and Western blot with anti-BlyA peptide antiserum (Fig. 5). Untreated and CB2-treated samples were harvested at 5, 27, 35, and 60 min. Each time point showed the same trend in BlyA expression. Both pellet fractions contained similar amounts of BlyA, whereas there was clearly an absence of BlyA in the CB2 supernatant compared to the untreated control (Fig. 5B).

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FIG. 5.

Representative SDS-PAGE gels of samples were collected at 5, 27, 35, or 60 min; the 35-min time point is shown. Silver stain (A) and anti-BlyA Western blot (B) of untreated samples and samples treated with 20 μg of CB2/ml. S, supernatant; P, pellet. Lane 1, MM294-pTG3, positive BlyA control; lanes 2 and 3, untreated Borrelia; lanes 4 and 5, CB2-treated Borrelia samples.