Association of Linear Plasmid 28-1 with an Arthritic Phenotype ofBorrelia burgdorferi

Abstract

Borrelia burgdorferi, the Lyme disease spirochete, has a genome comprised of a linear chromosome and up to 21 plasmids. Loss of plasmids is associated with decreased infectivity and pathogenicity. Sixteen transformants were generated by transforming the noninfectious clone 5A13 with the recombinant plasmid pBBE22. The transformants were classified into nine groups based on plasmid content analysis. An infectivity study revealed that all nine transformants examined, each of which represented one of the plasmid patterns, were infectious in mice with severe combined immunodeficiency (SCID) regardless of their genomic compositions. Tissue bacterial quantification revealed that the loss of plasmids significantly reduced the spirochete burden in the heart and joint tissues, not in the skin, suggesting virulence factors may be tissue specific. Four transformants containing lp28-1 induced severe arthritis in SCID mice, in contrast to the five transformants lacking lp28-1. These pathogenicity studies associated lp28-1 with an arthritic phenotype and further studies may identify factors that contribute to arthritic pathology.

Verification and dissection of the ospC operator by using flaB promoter as a reporter in Borrelia burgdorferi

“…Infected mice received a single subcutaneous injection of 100 μg of OspC MAb or purified mouse IgG as a control 3 weeks post-inoculation, as described previously [34]. Mice were sacrificed 1 week after passive immunization; heart, tibiotarsal joint, and skin specimens were aseptically collected for spirochete culture as previously described [42]. RNA was extracted from joint samples, and used for ospC expression analyses as described above.…”

“…Selected transformants were first surveyed for the presence of lp28-1 as this plasmid is essential for infection of an immunocompetent host [42,43]. Only clones that contained lp28-1 were further analyzed for plasmid content as described previously [42].…”

“…Constructs were electroporated into the ospC mutant, which was generated and characterized in our previous study [34]; resulting transformants were screened as described previously [42]. Selected transformants were first surveyed for the presence of lp28-1 as this plasmid is essential for infection of an immunocompetent host [42,43].…”

“…The copy numbers of the borrelial linear chromosome and the constructs were quantified by using the primers P5F and P5R or P6F and P6R (Table 4) specific for the genes flaB and kan, respectively. The recombinant plasmids pNCO1T-actin-flaB, which was constructed previously [42], and pBBE22 were used as DNA concentration standards for flaB and kan, respectively. Purified plasmids were determined for DNA concentrations by measuring the optical density at 260 nm wavelength, converted to copy numbers and 10-fold serially diluted within a range of 10 2 -10 7 DNA copies/ μl.…”

“…Selected transformants were first surveyed for the presence of lp28-1 as this plasmid is essential for infection of an immunocompetent host [42,43]. Only clones that contained lp28-1 were further analyzed for plasmid content as described previously [42]. Selected clones were grown to late-log phase in Barbour-Stoenner-Kelly H (BSK-H) complete medium at 33°C (Sigma Chemical Co., St. Louis, MO), and subjected to immunoblot analysis probed with a mixture of FlaB and OspC monoclonal antibodies (MAbs), as described previously [20].…”