Figure 1.
Characterization of the ospB Mutant and the OspB Complemented Mutant
(A) Strategy for the inactivation of the ospB gene and for the generation of the OspB complemented mutant is shown. The top diagram represents the suicide vector pXLF11303 used for transformation into B. burgdorferi B31. Due to homologous recombination (double crossover) between the sequences of pXLF11303 and the native ospAB locus in B. burgdorferi B31, the ΔospB::kan fragment is inserted into the genome, resulting in the generation of the ospB mutant (ospB−). For the generation of the OspB complemented mutant, the PospAB and ospB gene were PCR-amplified from B. burgdorferi B31 total genomic DNA and cloned into the multiple cloning site (MCS) of the shuttle vector pKFSS1 [40], resulting in the construct pFGN1 (bottom diagram). The relevant structures of the plasmid and genome are shown. Arrows represent positions of the oligonucleotides used for the PCR analysis. Primer names are retained as shown in Table 1. Vertical lines represent the restriction enzyme sites. E, EcoRI; N, NotI; and P, PstI.
(B) PCR analyses of genomic DNA isolated from spirochetes were performed to confirm the inactivated ospB locus. wt, the wild-type isolate; ospB, the ospB mutant; ospB− , recovered-spirochetes recovered from mice infected with the ospB mutant; ospB−/pFGN1, the OspB complemented mutant; M, 1-kb DNA ladder; C in (B and E) refers to PCR reactions without template DNA.
(C) RT-PCR analysis of ospA, ospB, and flaB transcripts in spirochetes grown in BSK-H media. Shown is a gel image of RT-PCR reactions performed with (+RT) and without (−RT) reverse transcriptase. C refers to RT-PCR reactions without cDNA.
(D) SDS-PAGE (top) and immunoblot (bottom) analysis of whole-cell lysates of spirochetes. Arrows indicate the expression of OspA, OspB, and FlaB. Immunoblotting analysis performed with monoclonal antibodies directed against OspB (mAb B22J), OspA (C3.78), and FlaB (H9729) were reported previously [22,50].
(E) PCR analyses of genomic DNA to confirm the OspB complementation.
(F) Restriction digestion of pFGN1 plasmid recovered from the OspB complemented mutant (pFGN1-recovered) and plasmid controls pFGN1 and pKFSS1. Whole-cell lysate from the OspB complemented mutant was transformed into E. coli DH5α-competent cells and transformed clones were selected in the presence of spectinomycin (50 μg/ml). Plasmids isolated from E. coli cells were digested with NotI or EcoRI and PstI. Expected sizes of restrictive fragments are (i) 5,638 bp and 1,906 bp for NotI digestion of pFGN1; (ii) 5,638 bp and 453 bp for NotI digestion of pKFSS1; (iii) 3,840 bp, 2,413 bp, and 1,291 bp for EcoRI-PstI digestion of pFGN1; and (iv) 3,635 bp, 2,413 bp, and 43 bp for EcoRI-PstI digestion of pKFSS1.
Table 1.
Oligonucleotides Used in This Study
Figure 2.
Influence of OspB Deficiency on B. burgdorferi Infectivity for Mice
Spirochete burden (A) and joint swelling and inflammation (B) in C3H/HeN mice were quantified at 25 d after mice were needle-inoculated each with 105 wild-type (black bars) or ospB mutant (white bars) spirochetes. Spirochete burden in mice tissues (bladder, heart, joint, and skin) was quantified by Q-PCR and shown as pg of flaB DNA per μg of mouse β-actin. Both tibiotarsal and knee joints of each mouse were scored on a scale of 0–3 for joint swelling, exudation of fibrin and leukocytes into joint spaces, synovial thickening due to proliferation, and hypertrophy of synovial cells: absent (0), mild (1), moderate (2), or severe (3). Statistical analysis from Student t test is also shown. Error bars define standard deviation (+) from the average value (mean).
Figure 3.
Influence of OspB-Deficiency on the Ability of B. burgdorferi to Survive and Colonize in Ticks
(A) Results from the experiments with the ticks inoculated by feeding on infected mice are shown. The spirochete burden in nymphal and subsequently molted adult ticks is quantified by Q-RT-PCR. Value on Y-axis corresponds to pg of flaB/μg of tick β-actin in each cDNA sample. * indicates significant reduction (p < 0.05, Student t test) of the ospB mutant in ticks in comparison to the wild-type.
(B) RT-PCR analysis of the RNA samples extracted from nymphs is shown. RT-PCR of flaB and ospA transcripts were used to determine the presence of spirochetes; tick β-actin was used as an internal control for RT-PCR.
(C) Shown is the quantitative assessment of the number of spirochetes from 15 random microscopic field observations. Value on Y-axis corresponds to the number of spirochetes/microscopic field. Black and white bars in (A and C) indicate values for the wild-type and the ospB mutant spirochetes, respectively. Error bars define standard deviation (+) from the average value (mean).
(D) Shown are the representative confocal images of the I. scapularis nymphal ticks (fed on mice infected with the wild-type or the ospB mutant B. burgdorferi at the indicated time points) and molted adult ticks. Tick blood meal (in nymphs) and luminal content (in adult ticks) and gut tissues were stained for B. burgdorferi with FITC-labeled anti-Borrelia antibody (green) and for tick cell nucleic acid with propidium iodide (red). Arrow shown in the luminal content sample of the molted adult tick indicates the only ospB mutant spirochete seen in 15 random microscopic fields. Scale 20 μm.
Figure 4.
OspB Complementation Restores B. burgdorferi Ability to Survive and Colonize Ticks
Results from the experiments with the ticks inoculated by microinjection via rectal aperture are shown. Approximately 103 spirochetes were microinjected into each tick.
(A) Shown are representative confocal microscopic images of the blood meal and gut samples of microinjected nymphs at 48 h during feeding. Samples were stained with FITC-labeled anti-Borrelia antibody (green), and tick cell nucleic acid was stained with propidium iodide (red). Scale 20 μM.
(B) Average value of number of spirochetes from 15 random microscopic field observations is shown.
(C) Q-RT-PCR for the total RNA extracted from microinjected nymphs collected at 48 h during feeding is shown. Values on Y-axis represent pg flaB/μg tick β-actin cDNA.
(D) Shown are the readings from an in vitro binding assay of the wild-type, the ospB mutant, and the OspB complemented mutant B. burgdorferi to TGE- and FBS-coated wells (see Materials and Methods for details). Values on Y-axis are sample absorbance measured at 450 nm of wavelength. The values shown are the averages of four independent experiments. Black, open, and gray bars in (B, C, and D) indicate values for the wild-type, the ospB mutant, and the OspB complemented mutant spirochetes, respectively. Error bars define standard deviation (+) from the average value (mean). * indicates values that are statistically significant (p < 0.05, Student t test) in comparison to the ospB mutant.