The Borrelia burgdorferi RelA/SpoT Homolog and Stringent Response Regulate Survival in the Tick Vector and Global Gene Expression during Starvation

Abstract

As the Lyme disease bacterium Borrelia burgdorferi traverses its enzootic cycle, alternating between a tick vector and a vertebrate host, the spirochete must adapt and persist in the tick midgut under prolonged nutrient stress between blood meals. In this study, we examined the role of the stringent response in tick persistence and in regulation of gene expression during nutrient limitation. Nutritionally starving B. burgdorferi in vitro increased the levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp, products of the bifunctional synthetase/hydrolase RelBbu (RelA/SpoT homolog). Conversely, returning B. burgdorferi to a nutrient-rich medium decreased (p)ppGpp levels. B. burgdorferi survival in ticks between the larval and nymph blood meals, and during starvation in vitro, was dependent on RelBbu. Furthermore, normal morphological conversion from a flat-wave shape to a condensed round body (RB) form during starvation was dependent on RelBbu; relBbu mutants more frequently formed RBs, but their membranes were compromised. By differential RNA sequencing analyses, we found that RelBbu regulates an extensive transcriptome, both dependent and independent of nutrient stress. The RelBbu regulon includes the glp operon, which is important for glycerol utilization and persistence in the tick, virulence factors and the late phage operon of the 32-kb circular plasmid (cp32) family. In summary, our data suggest that RelBbu globally modulates transcription in response to nutrient stress by increasing (p)ppGpp levels to facilitate B. burgdorferi persistence in the tick.

Fig 1. Starvation of B. burgdorferi in RPMI increases (p)ppGpp levels.

(A) Analysis of radiolabeled nucleotides in wild-type strain B31-5A4 grown in ³²P-orthophosphate. Cultures were grown to late log phase in BSK + RS (lane 1) and then starved in RPMI culture medium for 30 min (lane 2) or 6 h (lane 3) and then shifted back to BSK + RS for 10 min (lane 4) or 2 h (lane 5). At each time point, nucleotides were extracted and separated by TLC. TLC plates were dried, exposed to a phosphor screen and visualized using a phosphorimager. (B) Quantification of (p)ppGpp levels by densitometry. Values represent the mean of three independent experiments, performed as described in A (lanes 1–5), expressed as (p)ppGpp normalized to (p)ppGpp + GTP. Error bars represent SEM. Asterisks indicate P < 0.05 as determined by one-way ANOVA and a Tukey’s post-hoc test.

Fig 2. rel _Bbu transcript levels change in response to nutrient levels.

(A) Temperature, growth phase and pH do not affect rel _Bbu transcript levels. B. burgdorferi strain B31-5A4 was grown in BSK + RS at 23°C or 35°C to log or stationary (stat) phase at pH 7.4 or 6.8 before RNA was isolated, cDNA synthesized and rel _Bbu transcript levels measured by qRT-PCR. (B) Starvation conditions reduce rel _Bbu transcript levels. B. burgdorferi strain B31-5A4 was grown in BSK + RS at 35°C to late log phase before cultures were collected and shifted to RPMI, RPMI without amino acids (aa) and glucose, RPMI without amino acids (aa) and phosphate (PO₄), or returned to BSK + RS at 35°C. After 30 min, RNA was isolated, cDNA synthesized and rel _Bbu transcript levels measured by qRT-PCR. Values are normalized to rel _Bbu transcript levels at the beginning of each experiment (0 h). (C) B. burgdorferi strain B31-5A4 was grown in BSK + RS at 35°C (0 h), starved in RPMI for 6 h or starved in RPMI for 6 h, and then shifted back to BSK + RS for 2 h. At each time point, rel _Bbu transcript levels were measured as described above. Each value is the mean of three independent experiments and error bars represent the SEM.

Fig 3. Mutation and complementation of rel _Bbu in B. burgdorferi.

(A) The rel _Bbu mutant (rel _Bbu ^-) was constructed by replacing the rel _Bbu gene in strain B31-5A4 with the streptomycin resistance gene aadA fused to the B. burgdorferi flgB promoter and the B. subtilis trpL terminator. The rel _Bbu mutant was complemented in trans by transformation with the Borrelia shuttle vector pBSV2 containing the rel _Bbu gene fused to either the flacp inducible promoter (pBS-flacp-rel _Bbu) or its native promoter (pBS-rel _Bbu). (B) RT-PCR analysis of RNA isolated from wild-type (lane 1), rel _Bbu ^- (lane 2), rel _Bbu ^- pBS-flacp-rel _Bbu (lane 3), or rel _Bbu ^- pBS-rel _Bbu (lane 4) strains. Samples were incubated with (+RT) or without (-RT) reverse transcriptase, and rel _Bbu and flaB transcripts were detected by PCR using primer pairs rsh 981F/rsh 1984R and flaB 423F/flaB 542R, respectively. Products were separated on 1% (rel _Bbu) or 2% (flaB) agarose gels and stained with ethidium bromide. ntc = no template control. (C) Production of (p)ppGpp in the wild-type (WT), rel _Bbu ^- and rel _Bbu ^- pBS-rel _Bbu (rel _Bbu ^- comp) strains. ³²P-labeled cultures were grown to log phase, shifted to RPMI for 6 h and nucleotides were extracted and analyzed by TLC.

Fig 4. Survival of B. burgdorferi during starvation conditions in vitro depends on rel _Bbu.

Strains were grown to late log phase in BSK + RS (0 h) before shifting to RPMI for 24 h or 72 h. Wild-type (WT), rel _Bbu mutant (rel _Bbu ^-), and rel _Bbu ^- pBS-flacp-rel _Bbu cells were stained with PI and visualized by DIC and fluorescence microscopy. Images are overlays of DIC and fluorescence images with PI shown in blue. Arrowheads are RBs that did not stain with PI and arrows point to PI-stained RBs. Scale bar = 10 μm. (B) The percentage of live spirochetes as quantified by PI staining following incubation in RPMI for various times (24 h to 96 h). Black squares represent WT in BSK + RS, black circles represent WT in RPMI, white squares represent rel _Bbu ^- in BSK + RS, white circles represent rel _Bbu ^- in RPMI, gray squares represent rel _Bbu ^- pBS-flacp-rel _Bbu in BSK + RS, and gray circles represent rel _Bbu ^- pBS-flacp-rel _Bbu in RPMI. Values are the means of three independent experiments with at least 100 spirochetes counted for each time point and error bars represent SEM. (C) Wild-type (black bars), rel _Bbu ^- (hatched bars) and rel _Bbu ^- pBS-rel _Bbu (gray bars) strains were grown in BSK + RS to late log phase before shifting to RPMI. To quantify the number of live B. burgdorferi at the time points indicated, samples of each culture were plated in semi-solid BSK, allowed to grow for two weeks and colonies enumerated to yield colony forming units (CFUs). Each value represents the mean of three independent experiments normalized to the initial number of colonies before shifting to RPMI (0 h). Error bars represent SEM.

Fig 5. rel _Bbu regulates round body formation under starvation conditions in vitro.

Fluorescence microscopy of live B. burgdorferi wild-type (WT), rel _Bbu mutant (rel _Bbu ^-), and rel _Bbu ^- pBS-rel _Bbu strains grown in BSK + RS to late log phase (A-C) or shifted to RPMI for 2 days (D-F) and stained with WGA-Alexa Fluor 594. Scale bar = 10 μm. Scanning electron microscopy of rel _Bbu ^- strains after shifting to RPMI for 2 days (G-J). Arrows indicate folded and disrupted membranes and arrowheads indicate membrane beading. Scale bar = 600 nm.

Fig 6. rel _Bbu affects B. burgdorferi morphology in stationary phase.

(A) Live cell microscopy of wild-type (WT), rel _Bbu mutant (rel _Bbu ^-) and rel _Bbu ^- pBS-flacp-rel _Bbu (rel _Bbu ^- comp) strains grown to late log phase or late stationary phase in BSK + RS before staining with WGA-Alexa Fluor 594. Scale bar = 10 μm. (B) RB quantification in log (gray bars) and stationary (black bars) phase. Each value represents the mean of three independent experiments; error bars represent SEM.

Fig 7. Tick persistence depends on rel _Bbu.

(A) Immunofluorescence microscopy of ticks allowed to feed on mice infected with wild-type (WT), rel _Bbu mutant (rel _Bbu ^-) or rel _Bbu ^- pBS-rel _Bbu strains. Ticks were dissected and fixed on slides one week after naïve larvae had fed to repletion (fed larvae) or after larvae had molted to nymphs (unfed nymph) or one week after nymphs had fed to repletion on uninfected mice (fed nymph). Samples were processed for IF microscopy using rabbit polyclonal anti-B. burgdorferi antibodies followed by goat anti-rabbit Alexa Fluor 488 antibodies to visualize spirochetes (green). Tick cells were visualized by staining with WGA-Alexa Fluor 594 (red). Scale bar = 10 μm. (B) Quantification of Borrelia in ticks that had fed on mice infected with wild-type (black circles), rel _Bbu ^- (white circles) or rel _Bbu ^- pBS-rel _Bbu (gray circles) strains. Total DNA was isolated from larvae that had fed to repletion (fed larvae) or after larvae had molted to nymphs (unfed nymph) or one week after nymphs had fed to repletion on uninfected mice (fed nymph). The number of B. burgdorferi genome equivalents per tick was determined by qPCR using TaqMan primers/probe to flaB. The difference between the number of WT and rel _Bbu ^- in fed nymphs was statistically significant (P = 0.018) by a one-way ANOVA with a Tukey’s post hoc test.

Fig 8. Changes in transcript levels in wild-type B. burgdorferi during nutrient stress.

Genes significantly differentially expressed more than twofold as determined by RNA-seq of wild-type B31-5A4 (A) shifted from stationary phase to starvation (6 h RPMI) and (B) in recovery (2 h BSK + RS) from starvation. The number of genes upregulated (black bars) and downregulated (gray bars) are divided by functional category using the following abbreviations: BP, bacteriophage; CD, cell division; CE, cell envelope; CM, chemotaxis and motility; MT, metabolism; PD, protein degradation; RR, DNA replication and repair; SR, stress response; TL, translation; TP, transporter proteins; TR, transcription and transcriptional regulation; and U, unknown.

Fig 9. Rel_Bbu-upregulated genes during nutrient stress.

The number of genes significantly Rel_Bbu-upregulated (higher in wild type than rel _Bbu ^-) greater than twofold as determined by RNA-seq in (A) stationary phase, (B) starvation and (C) recovery from starvation. Bars are color-coded based on the color scheme assigned in (D) for unique and overlapping conditions that upregulate genes. (D) Venn diagram of the number of genes Rel_Bbu-upregulated in stationary phase, starvation and recovery from starvation. Functional gene category abbreviations are the same as in Fig 8.

Fig 10. Rel_Bbu-downregulated genes during nutrient stress.

The number of genes significantly Rel_Bbu-downregulated (lower in wild type than rel _Bbu ^-) greater than twofold as determined by RNA-seq in (A) stationary phase, (B) starvation and (C) recovery from starvation. Bars are color-coded based on the color scheme assigned in (D) for unique and overlapping conditions that downregulate genes. (D) Venn diagram of the number of genes Rel_Bbu-downregulated in stationary phase, starvation and recovery from starvation. Functional gene category abbreviations are the same as in Fig 8.