Competitive advantage of Borrelia burgdorferi with outer surface protein BBA03 during tick-mediated infection of the mammalian host

Abstract

Linear plasmid lp54 is one of the most highly conserved and differentially expressed elements of the segmented genome of the Lyme disease spirochete Borrelia burgdorferi. We previously reported that deletion of a 4.1-kb region of lp54 (bba01 to bba07 [bba01-bba07]) led to a slight attenuation of tick-transmitted infection in mice following challenge with a large number of infected ticks. In the current study, we reduced the number of ticks in the challenge to more closely mimic the natural dose and found a profound defect in tick-transmitted infection of the bba01-bba07 mutant relative to wild-type B. burgdorferi. We next focused on deletion of bba03 as the most likely cause of this mutant phenotype, as previous studies have shown that expression of bba03 is increased by culture conditions that simulate tick feeding. Consistent with this hypothesis, we demonstrated increased expression of bba03 by spirochetes in fed relative to unfed ticks. We also observed that a bba03 deletion mutant, although fully competent by itself, did not efficiently infect mice when transmitted by ticks that were simultaneously coinfected with wild-type B. burgdorferi. These results suggest that BBA03 provides a competitive advantage to spirochetes carrying this protein during tick transmission to a mammalian host in the natural infectious cycle.

Fig 1

Expression of bba03 during tick blood meal. Wild-type B. burgdorferi-infected ticks were fed to repletion, pooled into groups of five ticks, and snap-frozen immediately upon drop-off or at 1 week postfeeding. RNAs were isolated from the frozen fed ticks and unfed ticks, converted to cDNAs, and used to quantify copies of the bba03 transcript relative to copies of the flaB transcript. *, the means of bba03 transcript relative to flaB transcript are significantly different between fed and unfed tick-derived spirochetes, as determined by the two-tailed unpaired t test (P = 0.0003).

Fig 2

Deletion and complementation of bba03. (A) Schematic diagram of the region of lp54 encompassing bba01 to bba05, illustrating the deletion of bba03 and introduction of the flaBp-driven streptomycin resistance cassette, aadA, by allelic exchange. (B) Schematic diagram of the pBSV2G-A03 shuttle vector that was used to complement the ΔA03 mutant. The region of lp54 encompassing bba02 and bba03 was amplified from wild-type B. burgdorferi and cloned into the pBSV2G shuttle vector.

Fig 3

Immunoblot confirming the absence and restoration of BBA03 production in the ΔA03 mutant and ΔA03/A03 complemented strains. Antisera to BBA03 and FlaB were used to probe lysates of approximately 10⁷ spirochetes. B. burgdorferi strains are identified above the lanes. Lysates from the A3ΔA1-7 mutant, which lacks the region of lp54 from bba01 to bba07, and strain B314, which lacks the entire lp54 plasmid, were used as negative controls for BBA03 production.

Fig 4

Infectivity of ΔA03 mutant in coinfected ticks. Five nymphs coinfected as larvae with WT and ΔA03 mutant spirochetes were fed on each mouse. Recovered fed ticks were crushed and plated to determine the ratio of ΔA03 mutant spirochetes to the total number of spirochetes per tick. Each point represents the ratio of ΔA03 to WT spirochetes from a single tick, with a value of 1.0 representing 100% ΔA03 mutant spirochetes and a value of 0.0 representing 100% WT spirochetes. The mean number (± standard deviation) of spirochetes within the coinfected ticks (ΔA03 mutant plus WT) was 1 × 10⁵ ± 2.7 × 10⁴. Mice were euthanized at 3 weeks postfeeding, and tissues were cultured in BSKII containing the appropriate antibiotics to select for ΔA03 mutant spirochetes (Kan^r Strep^r) versus WT spirochetes (Kan^r). The total number of ΔA03 mutant tissue reisolates (9/40 samples) was significantly different from the total number of WT tissue reisolates (38/40 samples) (P < 0.0001, as determined by Fisher's two-tailed exact probability test).

Fig 5

Surface exposure of BBA03 assessed by immunofluorescence assay. Wild-type B. burgdorferi was placed on slides and either heat fixed to prevent permeabilization of the cells or acetone fixed to permeabilize the cells. Cells were then probed with FlaB (periplasmic) and BBA03 antisera.

Fig 6

Proteinase K sensitivity of BBA03. Intact and disrupted cells from mid-log-phase wild-type B. burgdorferi cultures were incubated with various concentrations of proteinase K (PK), as indicated. Lysates of treated spirochetes were analyzed by immunoblotting with antibodies to FlaB (periplasmic protein), OspA (surface-exposed protein), and BBA03.