Borrelia host adaptation Regulator (BadR) regulates rpoS to modulate host adaptation and virulence factors in Borrelia burgdorferi

Abstract

The RpoS transcription factor of Borrelia burgdorferi is a 'gatekeeper' because it activates genes required for spirochaetes to transition from tick to vertebrate hosts. However, it remains unknown how RpoS becomes repressed to allow the spirochaetes to transition back from the vertebrate host to the tick vector. Here we show that a putative carbohydrate-responsive regulatory protein, designated BadR (Borrelia host adaptation Regulator), is a transcriptional repressor of rpoS. BadR levels are elevated in B. burgdorferi cultures grown under in vitro conditions mimicking unfed-ticks and badR-deficient strains are defective for growth under these same conditions. Microarray and immunoblot analyses of badR-deficient strains showed upregulation of rpoS and other factors important for virulence in vertebrate hosts, as well as downregulation of putative tick-specific determinants (e.g. linear plasmid 28-4 genes). DNA-binding assays revealed BadR binds to upstream regions of rpoS. Site-directed mutations in BadR and the presence of phosphorylated sugars affected BadR's binding to the rpoS promoters. badR-deficient B. burgdorferi were unable to colonize mice. Several putative tick-specific targets have been identified. Our study identified a novel regulator, BadR, and provides a link between nutritional environmental cues utilized by spirochaetes to adaptation to disparate conditions found in the tick and vertebrate hosts.

Fig. 1

Analysis of xylose utilization of B. burgdorferi and ROK DNA-binding domains. BLAST analysis in conjunction with the borrelial genome annotations (Fraser et al., 1997) demonstrated xylulokinase (BB0545) as the only enzyme for xylose utilization in B. burgdorferi suggesting ORFs annotated as xylose operon regulatory proteins (XylR1 and XylR2) are misannotated and have alternative functions (A). Comparison of ROK DNA-binding motifs. Alignment of the HTH motifs in the N-terminal regions of NagC and Mlc from E. coli, XylR repressor from Bacillus subtilis, and XylR1 from B. burgdorferi. The locations of the motifs relative to the N-terminus of the proteins are given. Conservation stringency of residues is depicted using symbols below (B).

Fig. 2

Immunoblot analysis determines regulatory contributions of BadR. Coomassie blue stained SDS-PAGE of B. burgdorferi B31-A3 lysates grown under host-mimicking conditions (A). Immunoblot analysis using depicted antibodies (B). Lanes: MW: molecular weight marker, 1: grown at pH7.6/32°C; 2: grown at pH 7.6/23°C (mimicking unfed-tick); 3: grown at pH 6.8/37°C (mimicking fed-tick).

Fig. 3

Genetic analyses of badR-deficient B. burgdorferi strains. Total genomic DNA from parental B31-A3 (WT) and badR-deficient strain (badR⁻) was digested with HindIII (lanes 1 and 3) or EcoRI and NdeI (lanes 2 and 4), separated on a 1% agarose gel, and transferred to nylon membranes (A, B, C). Membranes were hybridized with PCR amplified probes corresponding to the aadA gene (Str^R marker) (B) or to a region upstream of the badR gene (C). Schematic representation of the badR region of the chromosome for both WT and badR⁻. Probes used are indicated with brackets. HIII-HindIII, ERI-EcoRI, ND1-NdeI (D). PCR confirmation to assess plasmid profile was performed on parental and badR-deficient strains. Strains and molecular weight (in base pairs) are indicated on the left (E).

Fig. 4

Growth analysis and protein profile of badR-deficient strains. Growth analysis of WT and badR-deficient strains grown under laboratory (pH7.6/32°C) (A) and unfed-tick (pH7.6/23°C) (B) conditions. Bacteria were diluted from stationary phase (1×10⁸ bacteria ml⁻¹), seeded at 5×10⁵ bacteria ml⁻¹, and enumerated every 24 hours using dark field microscopy. The cultures were grown in triplicate, with three independent trials. Error bars indicate standard error. Levels of significance were determined using two-way ANOVA with α=95% and there were statistical differences in growth in the badR-deficient strains from day 11 on with a P<0.01 (A, B). Protein profile of badR-deficient B. burgdorferi. Coomassie blue stained SDS-PAGE of B. burgdorferi lysates from WT (B31-A3) and badR-deficient strains grown under laboratory growth conditions (pH7.6/32°C) (C). Immunoblot analysis using depicted antibodies. Lanes: MW: molecular weight marker in kDa; 1: parental B31-A3 (wt); 2: badR⁻ (mt1); 3: badR2⁻ (mt2) (D).

Fig. 5

BadR binds rpoS promoters. Schematic diagram illustrating the two regions upstream of rpoS used for the EMSAs. RpoS 1 promoter (PrpoS 1_{(813088-813282)}) includes the RpoS start codon (boxed) and BosR binding site 3 (BS3 -Underlined). RpoS2 promoter (PrpoS 2_{(813258-813471)}) includes the RpoN binding site (bold), both the BosR binding sites 1 and 2 (BS1, BS2 -Underlined), and the rpoS transcriptional start site (bolded with asterisk) (A). 5′ biotin-labeled promoters (2 nmols) were mixed with various amounts of purified BadR_{(734081-735289)} (130, 200, 270 pmols) in a 20μl binding reaction. Some reactions were incubated with unlabeled promoters (200-fold molar excess) for competition reactions or a N′ terminal HTH deficient BadR_{(734225-735289)} (BadR Δ HTH) (B). The binding reactions were incubated at room temperature for 20 minutes, run on a 6% polyacrylamide gel, and transferred onto a positively charged Nylon membrane. After transfer the membrane was cross-linked by UV irradiation, blocked, incubated with streptavidin-horseradish peroxidase conjugate and luminol enhancer substrate solution allowed visualization of labeled DNA by exposure to X-ray film.

Fig. 6

Phosphorylated sugars alleviate BadR binding to rpoS promoters. Two nmols of 5′ biotinylated rpoS promoters (PrpoS 1(A), PrpoS 2 (B)) were mixed with 130 pmols of WT BadR. The binding reactions were performed in the absence or presence of various sugars (GlcNAc-6P, glucose-6P (Glc-6P), xylulose-5P, ribose-5P, xylose, or chitobiose). Binding reactions with various sugar concentrations (10, 30, 50,100, or 200mM) determined the influence of each sugar on BadR binding to the rpoS promoters.

Fig. 7

Characterization of BadR: Site-directed mutations and domain-deficient BadRs. Schematic representation of BadR domains (A) and comparisons of residues in ROK inducer-binding domains (IBD). Mutated residues are indicated (star) (B). 5′ biotinylated rpoS promoter 1 (2 nmols) was incubated with WT or mutant BadR proteins (130pmols) as previously described. Lanes: binding reaction absent of BadR (1), WT BadR_{(734081-735289)} (2), BadR Δ HTH (BadR_{(734225-735289)}) (3), BadR deficient of inducer binding domain BadR_{(734081-734659)} (BadRΔIBD) (4), BadR with a site directed mutation in the equivalent residue of the ROK conserved residue, H247, (H243A) (5), BadR with site-directed mutations in the putative ROK CxxC metal binding/dimerization residues, (C254A/C257A) (6), BadR with site directed mutations in unique residues in BadR’s putative inducer binding domain (N253A/P255A) (7) (C).

Fig. 8

BadR represses rpoS to regulate host-specific adaptation in B. burgdorferi. The rpoS gene is upregulated in a badR-deficient B. burgdorferi strain. Log2 fold change of rpoS derived from two microarrays from (pH7.6/32°C) grown cultures. P values from pooled (overall) and individual arrays (Rep.1, Rep.2) are indicated using n=4 for both A3 (WT) and badR⁻ (A). Quantitative RT-PCR fold changes of rpoS are indicated with P≤0.01. Error bars indicate s.e.m. (B). Predicted BadR regulatory network. Many activators of rpoS have been identified (e.g. DsrA_Bb, BosR, RpoN, RpoD, CsrA_Bb). Our study is the first to demonstrate BadR as a repressor of rpoS and thus may facilitate the spirochete’s transition back into ticks. A model for BadR regulation entails the following: 1.) BadR binds upstream of rpoS to repress rpoS in unfed ticks; 2.) Since ROK regulators are nutrient-responsive, BadR regulation may be modulated directly or indirectly by nutrient inducers; 3.) Nutrient surges from the blood meal may provide the inducer to allow BadR’s derepression of rpoS; and 4.) BadR regulates chitobiose utilization genes and may activate genes to enhance the spirochetes maintenance in ticks (lp28-4 genes). square= activation, oval=repression by BadR, respectively (C).