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. 2008 Jan 21;205(1):133–141. doi: 10.1084/jem.20070962

Figure 5.

Figure 5.

Complementation of bmpA/B− B. burgdorferi with bmpA and bmpB restores the ability of spirochetes to survive in joints and induces greater levels of arthritis. (A) The bmpA and B gene, including its native promoter region was amplified and cloned into the shuttle vector pKFSS1 and transformed into the bmpA/B− spirochetes. (B) Western blot demonstrates production of the BmpA and BmpB proteins by the complemented B. burgdorferi. Equal amounts of proteins from the mock-complemented bmpA/B− spirochetes (bmpA/B−), mock-complemented wild-type (WT), or complemented spirochetes (bmpAB Com) were separated on a SDS-PAGE gel, which was either stained with Coomassie blue (left) or transferred to nitrocellulose membrane. Individual Bmp proteins were probed with the affinity-purified BmpA (top right) or BmpB (bottom right) antibodies. (C) The B. burgdorferi burden in mice infected with mock-complemented wild-type (gray bar), mock complemented bmpA/B− mutant (black bar), and bmpA/B− mutant complemented with bmpA and bmpB (white bar). Groups of mice (10 animals/group) were infected with wild-type or genetically manipulated isolates of B. burgdorferi, and the spirochete burden was analyzed at day 14, 21, and 28 by measuring copies of the B. burgdorferi flaB gene. Amounts of mouse β-actin were determined in each sample and used to normalize the quantities of spirochete DNA. Mutant or complemented spirochete levels in the bladder and skin (ear) tissue were similar. n = 3. P > 0.5. Levels of bmpAB complemented isolates were significantly higher in the joints than bmpA/B− mutants. Error bars represent the mean ± the SEM of relative tissue levels of B. burgdorferi. n = 3. *, P < 0.05. (D) Severity of joint swelling in B. burgdorferi–infected mice. Groups of mice (10 animals/group) were separately infected with mock complemented wild-type B. burgdorferi (gray bar), mock complemented bmpA/B− mutant (black bar), and bmpA/B− spirochetes complemented with bmpA and bmpB gene (white bar). Arthritis was evaluated by the assessment of development of joint swelling after 14, 21, and 28 d of spirochete challenge using a digital caliper. Bars represent the mean measurements (± the SEM) from three independent experiments. Significant differences in the inflammation level were noticed between groups of mice infected with wild-type isolates with the bmpA/B− mutant at all time points. **, P < 0.001 - 0.04. Highly significant differences between joint inflammation was also noted in groups of mice infected with bmpA/B− spirochetes and bmpA/B− spirochetes complemented with bmpAB at day 21. **, P < 0.001.