The diguanylate cyclase, Rrp1, regulates critical steps in the enzootic cycle of the Lyme disease spirochetes

Abstract

Rrp1 is the sole c-di-GMP-producing protein (diguanylate cyclase) of Borrelia burgdorferi. To test the hypothesis that Rrp1 regulates critical processes involved in the transmission of spirochetes between ticks and mammals, an rrp1 deletion mutant (B31-Δrrp1) and a strain that constitutively produces elevated levels of Rrp1 (B31-OV) were constructed. The strains were assessed for progression through the enzootic cycle using an Ixodes tick/C3H-HeJ mouse model and tick immersion feeding methods. B31-Δrrp1 infected mice as efficiently as wild type but had altered motility, decreased chemotactic responses to N-acetylglucosamine (NAG) and attenuated ability to disseminate or colonize distal organs. While this strain infected mice, it was not able to survive in ticks. In contrast, B31-OV displayed normal motility patterns and chemotactic responses but was non-infectious in mice. Using immersion feeding techniques, we demonstrate that B31-OV can establish a population in ticks and survive exposure to a natural bloodmeal. The results presented here indicate Rrp1, and by extension, c-di-GMP, are not strictly required for murine infection, but are required for the successful establishment of a productive population of B. burgdorferi in ticks. These analyses provide significant new insight into the genetic regulatory mechanisms of the Lyme disease spirochetes.

Figure 1. Generation and verification of rrp1 deletion, complementation, and overexpression strains and comparative analyses of their properties

Schematics in panel A and B represent the chromosome of the B31-Δrrp1 and B31-KI complement mutants after allelic replacement, respectively. Schematic C depicts the pBSV2-P_flaBrrp1 plasmid construct utilized in this study for the B31-OV mutant. Successful allelic exchange mutagenesis deleting and reinserting rrp1 was confirmed by PCR (Panel D), western blot (Panel E), and qRT-PCR analysis (Panel F). Verification of pBSV2-based overexpression of Rrp1 was likewise performed by PCR (Panel D), western blot (Panel E), and qRT-PCR (Panel F). Primers used for validation of proper integration are indicated by numbers above the schematic arrows and are listed to the left of the respective PCR panels. All primers used are listed in Table 1. The error bars in panel F indicate the standard deviation. Growth of each strain was assessed at 27, 33 and 37° over 20 days in BSK-H complete media. In panel H, HPLC chromatograms of nucleotide extracts from each strain are presented. The elution time of purified c-di-GMP is shown for reference. T. denticola nucleotide extracts were assessed as a positive control. All methods were as described in the text.

Figure 2. Rrp1 is not essential to mammalian infection, but overproduction inhibits disease establishment

C3H-HeJ mice were needle inoculated subcutaneously with 10⁴ spirochetes (5 mice per strain). Four weeks post-infection, mice were bled and sera collected. Seroconversion was evaluated using whole-cell ELISA (Panel A) and immunoblot (Panel B).

Figure 3. The diguanylate cyclase, Rrp1, is necessary for Ixodes infection

Evaluation of spirochete acquisition by Ixodes ticks was assessed by qPCR. DNA was isolated from naive ticks fed on infected mice till repletion (Panel A & B). Larval ticks were also immersed into spirochete culture, washed, fed, and assessed for spirochete uptake (Panel B).

Figure 4. Translational motion patterns requires Rrp1

Movement patterns of the B31-wt, B31-Δrrp1, B31-KI, and B31-OV strains were visualized using DIC microscopy. Images were captured at 5 sec intervals are shown. Motion tracks were manually recorded and overlaid on the images using motion tracking software. The tracks are colored according to motion achieved during each timelapse (blue - 0 sec, green - 5 sec, yellow - 10 sec, pink - 15 sec). Note that the patterns shown are representative of the entire population of cells.

Figure 5. Rrp1 positively regulates motility and chemotaxis

The B31-wt, B31-Δrrp1, B31-KI, and B31-OV strain motility and chemotaxis abilities were assessed by swarming assay and capillary chemotaxis assay. Borrelia were spotted into punched wells and diameters were measured at day 2, 4 and 6 (Panel B) from the respective swarms (Panel A). Mutant movement toward N-acetyl-D-glucosamine was analyzed by capillary assay. Spirochetes in capillary tubes with and without chemoattractant were counted after a 2h incubation at 33° (Panel C). The error bars indicate the standard deviation.