Essential role of the response regulator Rrp2 in the infectious cycle of Borrelia burgdorferi

Abstract

Alteration of surface lipoprotein profiles is a key strategy that the Lyme disease pathogen, Borrelia burgdorferi, has evolved to be maintained within its enzootic cycle between arthropods and mammals. Accumulated evidence indicates that the central regulatory pathway controlling differential gene expression by B. burgdorferi is the RpoN-RpoS pathway (the sigma(54)-sigma(S) sigma factor cascade). It was previously shown that activation of the RpoN-RpoS pathway is controlled by Rrp2, a two-component response regulator and sigma(54)-dependent transcriptional activator. The role of Rrp2 in the infectious cycle of B. burgdorferi has not been determined heretofore. In this report, we demonstrate that an rrp2 mutant defective in activating sigma(54)-dependent transcription was unable to establish infection in mice, but the rrp2 mutant was capable of surviving within ticks during and after tick feeding. Because the rrp2 mutant was defective in the production of OspC, an outer surface lipoprotein essential for mammalian host infection, we further examined whether the loss of infectivity of the rrp2 mutant was solely due to the inability to produce OspC. While transformation with a shuttle vector carrying ospC under the control of a constitutive flaB promoter restored infection to an ospC mutant in immunodeficient SCID mice, it could not rescue the avirulent phenotype of the rrp2 mutant. These data indicate that, in addition to controlling OspC, Rrp2 controls another factor(s) essential for B. burgdorferi to establish infection in mammals. Furthermore, microarray analyses revealed that 125 and 19 genes were positively and negatively regulated, respectively, by Rrp2, which provides a foundation for future identification of additional Rrp2-dependent virulence determinants in B. burgdorferi.

FIG. 1.

Protein expression profiles of B. burgdorferi strains used for the infection and microarray analyses. Infectious clone 5A4NP1 [wild type (wt)], the isogenic rrp2 mutant [rrp2(G239C)], and the complemented rrp2 strain [rrp2(wt)] were cultivated in BSK-H medium and harvested at the late logarithmic phase of growth (5 × 10⁷ spirochetes/ml), and whole-cell lysates (5 × 10⁷ spirochetes/gel lane) were subjected to SDS-PAGE (Coomassie blue-stained gel). Numbers at the left denote protein molecular mass markers in kDa. The bands corresponding to OspC and OspA are indicated on the right.

FIG. 2.

Detection of wild-type or rrp2 mutant spirochetes in ticks by immunofluorescence assays. Unfed I. scapularis nymphs were microinjected with either wild-type 5A4NP1 or isogenic rrp2 mutant [rrp2(G239C)] spirochetes and allowed to feed to repletion on mice. After detachment, fed nymphs were dissected, and tick smear preparations were subjected to immunofluorescence assays with fluorescein isothiocyanate-labeled anti-B. burgdorferi antibody. Panels shown are representative images from three separate experiments.

FIG. 3.

Restoration of OspC expression with the pBBE22-flaB_p-ospC plasmid. SDS-PAGE (Coomassie blue-stained gel) (A) and immunoblot (B) analysis of whole-cell lysates of various strains of spirochetes harvested at the late logarithmic phase of growth (5 × 10⁷ spirochetes/ml). Lanes for both panels: 1, 13A (wild type); 2, 13A ospC::Gen^r; 3, 13A ospC::Gen^r/pBBE22-flaB_p-ospC; 4, 13A rrp2(G239C); 5, 13A rrp2(G239C)/pBBE22-flaB_p-ospC. For immunoblotting, a 1:10 dilution of the samples used for SDS-PAGE analysis was loaded and monoclonal antibodies directed against FlaB (loading control) and OspC were pooled. The bands corresponding to FlaB and OspC are indicated by arrows.

FIG. 4.

Summary of genes activated or repressed by Rrp2. Numbers represent the total numbers of genes activated (black bars) or repressed (gray bars) for each plasmid. Plasmid cp9 is absent from the parental strains and is not presented in the diagram. No genes on lp5 or lp21 were found to be activated by Rrp2. Only genes on the chromosome (chrom.), lp17, lp28-4, lp56, cp32-3, lp5, and lp21 were found to be repressed by Rrp2.

FIG. 5.

qRT-PCR analysis of representative genes regulated by Rrp2. mRNA levels of four plasmid-contained, Rrp2-activated genes (A), three chromosome-contained, Rrp2-activated, chemotaxis-related genes (B), and two Rrp2-repressed genes (C) in the 5A4NP1 rrp2(G239C) mutant (mut) relative to the levels in the 5A4NP1 wild type (wt) (value = 1). The level of flaB mRNA was used for normalization of the relative mRNA level of each gene. Data were calculated from three independent cultures, and the differences in mRNA levels between the wild type and the rrp2 mutant are statistically significant (P < 0.05).