Cyclic di-GMP is essential for the survival of the lyme disease spirochete in ticks

Abstract

Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogen's life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol). To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogen's survival in its vector host.

Figure 1. Construction of the rrp1 mutant and the repaired strain.

(A) Strategy for construction of the rrp1 mutant and the repaired strain (rrp1^com). Arrows indicate the approximate positions of the oligonucleotide primers used for PCR analysis. (B) PCR analysis of strains. The specific primer pairs used in PCR are indicated above lanes. Lane W, wild-type (5A4NP1); lane M, the rrp1 mutant; lane C, rrp1^com. (C) Western blot analysis of whole-cell lysates of WT, rrp1, and rrp1^com spirochetes probed with α-Rrp1 and α-FlaB monoclonal antibodies.

Figure 2. The rrp1 mutant failed to survive in ticks upon acquisition.

(A) IFA analysis of spirochetes from fed larvae. I. scapularis larvae were fed on needle infected C3H/SCID mice harboring wild-type (5A4NP1), rrp1, or rrp1^com spirochetes. Engorged larvae were collected after repletion and subjected to IFA analysis using fluorescein isothiocyanate-labeled anti-B. burgdorferi antibody. Twelve ticks were examined in each group and a representative image for each group of ticks is shown. (B) qPCR analyses of spirochete burden in fed larvae. Quantitative PCR for the B. burgdorferi flaB gene was performed with DNA extracted from fed larvae. Each dot represents one data point from one three larval tick (a total of 20 samples with 60 ticks examined for each group from two independent experiments). The horizontal bar represents the mean value of each group.

Figure 3. The rrp1 mutant could not survive in artificially infected ticks upon feeding.

Unfed I. scapularis nymphs were microinjected with wild-type (5A4NP1), rrp1, or rrp1^com spirochetes and then fed on naïve mice. The engorged nymphs were collected after repletion and subjected to IFA analysis using fluorescein isothiocyanate-labeled anti-B. burgdorferi antibody. Ten ticks were examined in each group and a representative image for each group of ticks is shown in this figure.

Figure 4. Rrp1 controls expression of the glycerol gene operon (bb0240-0243).

(A) Relative transcript levels of the glycerol operon glp (bb0240-bb0243) in wild-type, rrp1, and rrp1^com by real-time RT-PCR. RNA was isolated from late logarithmic phase cultures grown at 35°C in standard BSK-II medium. Values represent the average copy number for each gene (± standard deviation) normalized per 1000 copies of flaB. (B–E) growth curves of wild-type, rrp1, rrp1^com or rrp1/flaBp-glp at 23°C (D, E) or 35°C (B, C) in standard BSK-II medium (B, D) or BSK-glycerol medium (C, E). The initial cell density was 3×10⁵ cells/ml for each strain. Spirochetes were enumerated under dark-field microscopy. Data presented here is from one representative experiment with three independent cultures. Each data point was the average of data from three independent cultures. *, P<0.05.

Figure 5. Glycerol induces expression of rrp1 and bb0240-bb0243.

Wild-type B. burgdorferi strain B31 5A4NP1 was grown at 35°C in either standard BSKII or BSK-glycerol medium. Cells were harvested at late logarithmic phase. (A) qRT-PCR. RNAs were extracted and subjected to real-time RT-PCR analyses for rrp1, bb0240, bb0241, bb0242, bb0243, rpoS, ospC, and flaB. Levels of expression of each gene were normalized with the level of flaB expression in each sample. Relative fold change of gene expression between the two growth conditions were reported (with levels of expression of each gene in standard BSKII media as values of 1). **, p<0.01. (B) immunoblot against Rrp1, FlaB, or OspC. Lane 1, spirochetes cultivated in BSK-II medium. Lane 2, spirochetes cultivated in BSK-glycerol medium. Upon normalization against FlaB, Rrp1 level is 1.7 fold higher in BSK-glycerol medium than that in standard medium.

Figure 6. Construction of the glp mutant and the repaired strain.

(A) Strategy for construction of the glp mutant and the repaired strain (glp^com). (B) RT-PCR analysis of expression of bb0240-bb0243. RNA was isolated from late logarithmic phase cultures grown at 35°C in the BSK-II medium (pH 7.5). W: wild-type strain 5A4NP1; M: the glp mutant; C: the repaired strain (glp^com). (C) and (D) Growth curve of wild-type, glp and glp^com at 35°C in standard BSK-H medium (C) or BSK-glycerol medium (D). The initial cell density was 3×10⁵ cells/ml for each strain. Spirochetes were enumerated under dark-field microscopy. Data presented here is from one representative experiment with three independent cultures. Each data point was the average of data from three independent cultures. *, p< 0.05.

Figure 7. Glycerol transport/metabolism is important during tick residence.

Naïve larvae fed on needle inoculated C3H/HeN mice infected with wild-type, glp, or glp^com. Fed larvae were allowed to molt to nymphs. Infected unfed nymphs were then fed on naïve mice to produce fed nymphs. Fed nymphs were then subjected to IFA analysis (A) and qPCR analysis (B). For qPCR, copies of the B. burgdorferi flaB genes were chosen to represent spirochete numbers and the values were reported relative to 100 copies of the tick actin gene. Each data point is from one tick.

Figure 8. Constitutive expression of glycerol metabolism genes in the rrp1 mutant partially restored spirochetal survival in ticks.

(A) Strategy for construction of an rrp1 mutant with constitutive expression of bb0240-bb0243, by replacing the promoter region of bb0240 with the flaB promoter. (B) Real-time RT-PCR analysis of expression of b0240, bb0241, and bb0243. RNA was isolated from late logarithmic phase cultures of wild-type (WT), the rrp1 mutant (rrp1), or rrp1/flaBp-glp that had been grown at 35°C in the BSK-II medium (pH 7.5). Levels of gene expression were normalized with the level of flaB gene expression. Fold changes of gene expression relative to wild-type strain (set as 1) were reported. (C) and (D), qPCR analysis of spirochetal burden in fed larvae one day (C) or fourteen days (D) after repletion. Copies of the B. burgdorferi flaB genes were chosen to represent spirochete numbers and are normalized with 100 copies of the tick actin gene in each sample. Each data point is from three fed larval ticks. **, p<0.01.