Contributions of environmental signals and conserved residues to the functions of carbon storage regulator A of Borrelia burgdorferi

Abstract

Carbon storage regulator A of Borrelia burgdorferi (CsrABb) contributes to vertebrate host-specific adaptation by modulating activation of the Rrp2-RpoN-RpoS pathway and is critical for infectivity. We hypothesized that the functions of CsrABb are dependent on environmental signals and on select residues. We analyzed the phenotype of csrABb deletion and site-specific mutants to determine the conserved and pathogen-specific attributes of CsrABb. Levels of phosphate acetyltransferase (Pta) involved in conversion of acetyl phosphate to acetyl-coenzyme A (acetyl-CoA) and posttranscriptionally regulated by CsrABb in the csrABb mutant were reduced from or similar to those in the control strains under unfed- or fed-tick conditions, respectively. Increased levels of supplemental acetate restored vertebrate host-responsive determinants in the csrABb mutant to parental levels, indicating that both the levels of CsrABb and the acetyl phosphate and acetyl-CoA balance contribute to the activation of the Rrp2-RpoN-RpoS pathway. Site-specific replacement of 8 key residues of CsrABb (8S) with alanines resulted in increased levels of CsrABb and reduced levels of Pta and acetyl-CoA, while levels of RpoS, BosR, and other members of rpoS regulon were elevated. Truncation of 7 amino acids at the C terminus of CsrABb (7D) resulted in reduced csrABb transcripts and posttranscriptionally reduced levels of FliW located upstream of CsrABb. Electrophoretic mobility shift assays revealed increased binding of 8S mutant protein to the CsrA binding box upstream of pta compared to the parental and 7D truncated protein. Two CsrABb binding sites were also identified upstream of fliW within the flgK coding sequence. These observations reveal conserved and unique functions of CsrABb that regulate adaptive gene expression in B. burgdorferi.

Fig 1

Generation of csrA_Bb deletion and cis-complemented strains in B. burgdorferi strain B31-A3. (A) Schematic representation of the region of csrA_Bb in the chromosome of B. burgdorferi strain B31-A3 (wt). A csrA_Bb deletion strain was generated by replacing csrA_Bb with P_flgB-streptomycin (str^R; mt), and a cis-complemented strain was generated by restoring a functional copy of csrA_Bb in cis with downstream P_flgB-gentamicin (gent^R, ct). Arrows indicate orientations of primers S1 and S2 used to screen the mutant and cis-complemented strains. (B) Ethidium bromide-stained 1% agarose gel depicting amplicons generated using primers S1 and S2 specific to upstream and downstream regions of csrA_Bb. Total genomic DNAs from wild-type (wt), csrA_Bb mutant (mt), and cis-complemented (ct) strains were used as the template. Molecular mass markers in kilobases are indicated to the left. (C) Total protein profiles of wild-type (wt), csrA_Bb mutant (mt), and cis-complemented (ct) strains propagated under conditions mimicking the tick vector before (lanes 1; pH 7.6, 23°C) or after (lanes 2; pH 6.8, 37°C) a blood meal. Equivalent amounts of proteins were separated on an SDS–12.5% polyacrylamide gel and stained with Coomassie blue. Molecular mass markers in kilodaltons are indicated to the left. (D) Immunoblot analysis of the borrelial strains shown in panel C with antisera against CsrA_Bb, RpoS, BBA64, DbpA, BBK32, and OspC. Blots represent one of three independent experiments.

Fig 2

Immunoblot analysis of proteins involved in acetate metabolism in B. burgdorferi. Lysates of the wt, mt, and ct strains shown in Fig. 1C were analyzed using antisera against Pta, AckA, or P66. Horseradish peroxidase (HRPO)-conjugated secondary antibodies were used, and blots were developed with an enhanced chemiluminescence (ECL) system. Blots represent one of three independent experiments. Molecular masses in kilodaltons are indicated to the left.

Fig 3

Immunoblot analysis of RpoS and rpoS-regulated proteins in the csrA_Bb mutant grown with supplemental acetate. The wt, mt, and ct strains were propagated in BSK-II medium with 0 (lanes 1), 30 (lanes 2), or 90 (lanes 3) mM supplemental acetate at 37°C. Equivalent levels of proteins were loaded on SDS–12.5% PAGE and were either stained with Coomassie blue (A) or electrotransferred to PVDF membranes (B). The blots were probed with antisera against CsrA_Bb, RpoS, OppA5, BBA64, and DbpA and developed using an ECL system. Blots represent one of three independent experiments. Molecular masses in kilodaltons are indicated to the left.

Fig 4

Immunoblot analysis of csrA_Bb mutant for proteins involved in acetate metabolism. The wt, mt, and ct strains were propagated in BSK-II medium with 0 (lanes 1), 30 (lanes 2), or 90 (lanes 3) mM supplemental acetate at 23°C/pH 7.6 (A) or 37°C/pH 6.8 (B). Immunoblot analysis was done with antisera against Pta, AckA, OppA5, and P66 (loading control). Goat anti-mouse HRPO-conjugated antibodies were used as secondary antibodies and blots developed using an ECL system. Blots represent one of three independent experiments. Molecular masses in kilodaltons are indicated to the left.

Fig 5

Schematic representation of the mutations generated in CsrA_Bb. (A) The cis-complemented strain (ct) was generated to restore a functional native copy of csrA_Bb. The residues indicated in gray are the conserved amino acids that have been shown to interact with small RNA molecule CsrB in E. coli. (B) Schematic representation of the DNA construct lacking the sequence encoding the last seven amino acids of CsrA_Bb (7D). (C) Site-specific changes were made to change 8 critical residues (underlined) to alanines (8S). All three complemented strains were counterselected for resistance to gentamicin conferred by P_flgB-Gent^R.

Fig 6

Phenotypic analysis of csrA_Bb mutants with site-specific alterations or C-terminal deletion. (A) Proteins from cis-complemented strains carrying wild-type CsrA_Bb (ct), CsrA_Bb with a 7-amino-acid C-terminal deletion (7D), or CsrA_Bb with substitutions of 8 critical residues (8S) were propagated under conditions mimicking the tick vector conditions before (23°C/pH 7.6) or after (37°C/pH 6.8) a blood meal. Proteins were separated on an SDS–12.5% PAGE and stained with Coomassie blue. (B) Immunoblot analysis performed with antisera against CsrA_Bb, RpoS, BosR, BBA64, and DbpA. (C) Immunoblot analysis of ct, 7D, and 8S strains with antisera against Pta, AckA, and P66 (loading control). Molecular masses in kilodaltons are indicated to the left. (D) The CsrA_Bb binding box of the 5′UTR of bb0588 to bb0589 known to regulate levels of Pta was used to generate a biotinylated RNA probe (26). (E) Efficiency of binding of recombinant wt and mutant CsrA_Bb proteins to the 5′UTR of bb0588 to bb0589. A biotinylated RNA probe (20 fmol) was incubated with purified recombinant wt, 7D, and 8S fused to maltose binding protein (MBP) at the concentrations indicated above each lane. MBP alone was used at a 600-pmol concentration as a control (MBP lanes). Blots were developed using enhanced chemiluminescence (ECL) as described in Materials and Methods and represent one of three independent experiments.

Fig 7

Levels of acetyl-CoA accumulation in mutant csrA_Bb strains. Borrelial strains (ct, 7D, and 8S) were propagated at 23°C/pH 7.6 (black bars) and 37°C/pH 6.8 (open bars) to densities of approximately 5 × 10⁷ cells/ml and 1 × 10⁸/ml, respectively. Lysates were prepared from a total of 10⁹ spirochetes using perchloric acid, and levels of acetyl-CoA in the extracts were analyzed using a PicoProbe acetyl-CoA assay kit and Student's t test implemented in Prism software. Asterisks indicate levels of significance (***, P < 0.001; *, P < 0.05).

Fig 8

Real-time reverse transcription-PCR analysis of levels of select ORFs in the ct, 7D, and 8S strains. Total RNA was isolated from borrelial strains propagated at pH 6.8/37°C as described in Materials and Methods and subjected to reverse transcription-PCR. The values for all the samples were normalized relative to the value for recA, and the change in the C_T value for each transcript was obtained as an average of the values determined for each sample analyzed in triplicate. The 2^−ΔΔCT values for each transcript from the ct, 7D, and 8S strains are shown as fold difference on the y axis with corresponding error bars. The ΔC_T values obtained for all the ORFs analyzed from each borrelial strain were subjected to an unpaired Student's t test implemented in Prism software. The asterisks indicate levels of significance as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Fig 9

Immunoblot analysis of ct, 7D, and 8S mutant strains of B. burgdorferi grown with supplemental acetate. Total proteins from ct, 7D, and 8S strains were grown under unfed (23°C/pH 7.6) (A and B) or fed (37°C/pH 6.8) (C and D) tick conditions with 0 (lanes 1), 30 (lanes 2), and 90 (lanes 3) mm of supplemental acetate. Equivalent amounts of proteins were separated on an SDS–12.5% PAGE gel (A and B). Separated proteins were electrotransferred and subjected to immunoblot analysis (B and D) using antisera against Pta, AckA, OppA5, and P66 (loading control). Blots represent one of three independent experiments. Molecular masses in kilodaltons are indicated to the left.

Fig 10

Effect of CsrA_Bb on the members of the flgK motility operon. Separated proteins of strains wt, mt, and ct (A) and strains ct, 7D, and 8S (B) propagated under conditions that mimic the tick midgut before (23°C/pH 7.6; lanes 1) and after (37°C/pH 6.8; lanes 2) a blood meal were electrotransferred and subjected to immunoblot analysis using antisera against FlaB, BB0180 (FlbF), BB0181 (FlgK), BB0182 (FlgL, indicated by arrow), BB0183 (FliW), and P66 (loading control). Note the absence of any detectable levels of FliW in the csrA_Bb mutant and 7D strain. Levels of FliW were elevated in the 8S mutant compared to the ct strain under unfed-tick conditions. Levels of FlbF and FlgK were unchanged in the strains tested. Blots represent one of two independent experiments. Molecular masses in kilodaltons are indicated to the left.

Fig 11

CsrA_Bb binding sites upstream of FliW. (A) The sequence upstream of fliW was analyzed for potential CsrA_Bb binding domains, and two putative domains were identified within the coding sequence of flgK. (B) Sequenced borrelial strains were analyzed for the presence of CsrA_Bb binding domains, and B. burgdorferi strains B31 and ZS7 have identical sequences in these domains, while B. afzelii Pko and B. garinii PBi have one nucleotide difference in the second domain. Additional differences in the sequences in the relapsing fever borreliae have been observed in this analysis. (C) Mfold program was used to generate the secondary structure and free-energy values of the two binding sites of CsrA_Bb in the upstream region of fliW. Arrows indicate a conserved CsrA binding nucleotide at each site. (D) Efficiency of binding of recombinant CsrA_Bb to RNA probes corresponding to BS1 and BS2. A biotinylated RNA probe (20 fmol) was incubated with purified recombinant CsrA_Bb fused to maltose binding protein (MBP) at the concentrations indicated above each lane. MBP alone was used at a 600-pmol concentration as a control (MBP lane). Mutated probes with replacement of GGA>AAA at either or both of the two binding sites were used as indicated below the blots. Blots were developed using enhanced chemiluminescence (ECL) as described in Materials and Methods. Blots represent one of three independent experiments.