Role of outer surface protein D in the Borrelia burgdorferi life cycle

Abstract

Borrelia burgdorferi preferentially induces selected genes in mice or ticks, and studies suggest that ospD is down-regulated in response to host-specific signals. We now directly show that ospD expression is generally elevated within Ixodes scapularis compared with mice. We then assessed the importance of OspD throughout the spirochete life cycle by generating OspD-deficient B. burgdorferi and examining the mutant in the murine model of tick-transmitted Lyme borreliosis. The lack of OspD did not influence B. burgdorferi infectivity in mice or the acquisition of spirochetes by I. scapularis. OspD adhered to tick gut extracts in vitro, and the OspD-deficient B. burgdorferi strain had a threefold decrease in colonization of the tick gut in vivo. This decrease, however, did not alter subsequent spirochete transmission during a second blood meal. These data suggest that B. burgdorferi can compensate for the lack of OspD in both ticks and mice and that OspD may have a nonessential, secondary, role in B. burgdorferi persistence within I. scapularis.

FIG. 1.

Differential ospD expression in B. burgdorferi during its life cycle in mice and ticks. The relative ospD expression level in each cDNA sample was calculated as the number of copies of the ospD transcript per 1,000 copies of the flaB transcript. In the left panel, the open circles represent values of individual mouse tissue samples and a black bar represent the mean value of each column. Shown in the right panel are the means and standard deviations of duplicate measurement of pooled tick samples (five ticks per time point), and the shaded area represents the time window during which the majority (>95%) of the ticks feed to repletion.

FIG. 2.

Construction and characterization of the ospD mutant. (A) Schematic drawings of the wild-type B. burgdorferi strain (w) and the ospD mutant (m) near the ospD locus. The ospD (BBJ09) gene and its surrounding open reading frames, BBJ08 (8), BBJ10 (10), and BBJ11 (11), are illustrated as box arrows, the lengths and directions of which reflect the lengths and directions of the open reading frames. In the mutant, the ospD gene is replaced with the kanAn cassette. Primers P1 to P9 are illustrated as arrows with their positions and directions representing those of the primers. The positions of the two DNA fragments mediating the homologous recombination event (the 5′ and 3′ arms) are shown. (B) PCR analysis of the wild-type B. burgdorferi strain (w) and the ospD mutant (m). The primers used in each PCR are indicated above the gel. The molecular masses of the DNA standards are indicated on the right in base pairs. (C) Protein analysis of whole-cell lysates of the wild-type B. burgdorferi strain (w) and the ospD mutant (m). Borrelia lysates were separated by SDS-PAGE and then stained with Coomassie brilliant blue (left panel) or transferred to a membrane and probed with antibodies against OspD (right panel). An arrow points to the OspD protein. The molecular masses of the protein standards are shown on the right in kilodaltons.

FIG. 3.

Quantification of spirochete burdens in mice and ticks. DNA samples from mice and ticks infected with the wild-type B. burgdorferi strain (w) or the ospD mutant (m) were subjected to Q-PCR analyses to determine the copy number of the B. burgdorferi flaB gene and that of the mouse or tick actin gene. The relative spirochete burden is shown as the number of copies of the B. burgdorferi flaB gene per 10⁶ copies of the mouse β-actin gene or per 10³ copies of the tick actin gene. (A) Spirochete burdens in bladder, heart, joint, and skin samples of mice at 21 days after syringe inoculation with spirochetes at a dose of 10⁵ spirochetes per mouse. (B) Spirochete burdens in larvae and nymphs after feeding on infected mice. (C) Spirochete burdens in infected nymphs before feeding (unfed) and during and after feeding (at 48, 72, and 96 h after tick attachment). (D) Spirochete burdens in bladder, heart, joint, and skin samples of mice at 21 days after being fed upon by infected nymphs. Individual circles indicate spirochete burdens in individual mouse tissue or tick samples, and a black bar represents the median of each column. The statistical significance of comparison of data is indicated by * (P < 0.005) or ** (P < 0.01).

FIG. 4.

Immunofluorescence microscopy analysis of ticks. Naive nymphs were fed on mice infected with the wild-type B. burgdorferi strain (w) or the ospD mutant (m). Shortly after tick feeding (at 96 h after tick attachment), ticks were dissected and the blood meal and tick midgut samples were separately isolated and processed for immunofluorescence microscopy as described in Materials and Methods. (A) Representative confocal images of gut and blood meal samples. Both samples were stained for B. burgdorferi with FITC-labeled anti-Borrelia antibody (green) and for tick cell nucleic acid with propidium iodide (red). Scale bar, 20 μm. (B) Quantitative assessment of the number of spirochetes from 15 random microscopic field observations. Values on the y axis are the numbers of spirochetes per microscopic field. Error bars define standard deviations from the mean. The statistical significance of comparison of data is indicated by *** (P < 0.001).

FIG. 5.

In vitro TGE binding assay. The binding of FITC-labeled OspD, OspA, OspB, OspC, and Dps to TGE prepared from unfed (gray bar) and fed (black bar) ticks and to a mouse cell line extract (open bar) was assayed in triplicate or quadruplicate. The bars and error bars represent the means and standard deviations of three or four readings of optical density at a 450-nm wavelength.