Characterization of 6S RNA in the Lyme disease spirochete

Abstract

6S RNA binds to RNA polymerase and regulates gene expression, contributing to bacterial adaptation to environmental stresses. In this study, we examined the role of 6S RNA in murine infectivity and tick persistence of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi. B. burgdorferi 6S RNA (Bb6S RNA) binds to RNA polymerase, is expressed independent of growth phase or nutrient stress in culture, and is processed by RNase Y. We found that rny (bb0504), the gene encoding RNase Y, is essential for B. burgdorferi growth, while ssrS, the gene encoding 6S RNA, is not essential, indicating a broader role for RNase Y activity in the spirochete. Bb6S RNA regulates expression of the ospC and dbpA genes encoding outer surface protein C and decorin binding protein A, respectively, which are lipoproteins important for host infection. The highest levels of Bb6S RNA are found when the spirochete resides in unfed nymphs. ssrS mutants lacking Bb6S RNA were compromised for infectivity by needle inoculation, but injected mice seroconverted, indicating an ability to activate the adaptive immune response. ssrS mutants were successfully acquired by larval ticks and persisted through fed nymphs. Bb6S RNA is one of the first regulatory RNAs identified in B. burgdorferi that controls the expression of lipoproteins involved in host infectivity.

Keywords: Borrelia burgdorferi; 6S RNA; Lyme disease; RNA; bacterial; gene expression regulation; small untranslated; spirochete.

Figure 1.

Bb6S RNA in B. burgdorferi. (A) Genomic locus of ssrS encoding the B. burgdorferi homolog of 6S RNA (Bb6S RNA). (B) The predicted secondary structure of 6S RNA from B. burgdorferi and, for comparison, E. coli (Wassarman, 2018). The 5′ and 3′ ends of Bb6S RNA were experimentally determined by RACE. The majority of 5′ ends (5/6) mapped to a C five nucleotides downstream from the end of the bb0188 (rplT) ORF. The majority of 3′ ends (11/14) mapped to a U 24 nucleotides upstream from the start of the bb0187 ORF.

Figure 2.. Bb6S RNA binds to B. burgdorferi RNAP.

(A) Immunoblot of B. burgdorferi cell lysate one-half (1/2) or one-tenth (1/10) the equivalent used for immunoprecipitation (IP) of extracts with preimmune serum or anti-E. coli RNAP antiserum (clone WI 151). The blot was probed with the anti-E. coli RNAP antiserum (clone WI 151) to visualize B. burgdorferi RNAP core. Arrows denote proteins present in the cell extract and enriched in IP with anti-E. coli RNAP antiserum but not preimmune serum. (B) Northern blot of a B. burgdorferi total cell lysate and extracts immunoprecipitated with preimmune serum (preimmune IP) or anti-E. coli RNAP antiserum WI-151 (α-RNAP IP) separated on a urea gel and hybridized with a biotinylated RNA probe to Bb6S RNA (upper panel) or B. burgdorferi 5S rRNA (lower panel). The amount of cell lysate was 10% of the equivalent used for the immunoprecipitation.

Figure 3.. Bb6S RNA is processed by RNase Y.

RNase Y levels were depleted by fusing the artificially inducible promoter flacp to the rny gene, encoding RNase Y, and inserted into the rny locus on the B. burgdorferi chromosome. (A) flacp-rny spirochetes were grown in 0.5 mM IPTG to 10⁷ cells ml⁻¹. IPTG was removed from two-thirds of the cells, which were then placed in growth medium containing either 0 or 0.05 mM IPTG for 48 h. One-third of the flacp-rny cells remained in 0.5 mM IPTG for 48 h. Wild-type (WT) spirochetes were grown in the presence of 0.5 mM IPTG until late log phase. Levels of rny mRNA were quantified by TaqMan qRT-PCR and normalized to flaB mRNA levels relative to wild type. Values are the mean of four independent biological replicates and error bars represent the SE. * denotes a significant difference between WT and flacp-rny strains in all levels of IPTG, ** denotes a significant difference between flacp-rny in 0.5 mM IPTG and both 0.05 and 0 mM IPTG and † denotes a significant difference between 0.05 mM and 0 mM IPTG (P < 0.05 determined by one-way ANOVA with Tukey’s post-hoc test). (B) WT and flacp-rny spirochetes were grown in 0.5 mM IPTG for two days before IPTG was removed and cells were resuspended in growth medium containing 0, 0.05 or 1.0 mM IPTG. Cells were enumerated each day for the next eight days (days 2 to 9). Values are the means of three independent biological replicates and error bars represent the SE. Significance (P < 0.05) was determined by one-way ANOVA with Tukey’s post-hoc test. * denotes WT significantly different from 1.0, 0.05, and 0 mM IPTG-treated flacp-rny strains, ** denotes 0 mM IPTG-treated flacp-rny significantly different from all others and WT significantly different from 1.0 and 0.05 mM IPTG-treated flacp-rny strains, † denotes 0 mM IPTG-treated flacp-rny significantly different from all others and 0.05 mM IPTG-treated flacp-rny significantly different from WT and 1.0 mM IPTG flacp-rny strains, ‡ denotes 0 mM IPTG-treated flacp-rny significantly different from all others, # denotes 0 mM IPTG flacp-rny significantly different from WT and 1.0 mM IPTG flacp-rny and 0.05 mM IPTG flacp-rny significantly different from WT, ¶ denotes 0 mM IPTG-treated flacp-rny significantly different from WT and 0.05 mM IPTG-treated flacp-rny significantly different WT and 1.0 mM IPTG-treated flacp-rny. (C) Northern blot analyses of Bb6S RNA levels from the RNA isolated in (A) using a biotinylated RNA probe to Bb6S RNA and tmRNA as a control. The filled arrow marks the size of the mature Bb6S RNA and the open arrow indicates a large (>1000 nt) species present in the sample lacking mature Bb6S RNA, potentially representing an unprocessed Bb6S RNA transcript. (D) qRT-PCR analyses of RNA isolated from the same conditions in (A) using SYBR Green primers 6S 5F and 6S 114R (Table 1) to quantify Bb6S levels. (E) qRT-PCR analyses of the 5′ end of Bb6S using SYBR Green primers 6S U26F and 6S 24R (Table 1) expressed as a ratio of 5′ end to total Bb6S and normalized to the ratio in flacp-rny cells in 0.5 mM IPTG. * denotes significant difference between the 5′ end to Bb6S ratio in the flacp-rny strain in 0.5 mM and 0 mM IPTG (P < 0.05 by one-way ANOVA with Tukey’s post-hoc test). (F) qRT-PCR analyses of the 3′ end of Bb6S using SYBR Green primers 6S 95F and 6S D224R (Table 1) expressed as a ratio of 3′ end to total Bb6S and normalized to the ratio in flacp-rny cells in 0.5 mM IPTG. * denotes significant difference between the 3′ end to Bb6S ratio in the flacp-rny strain in 0 mM IPTG and all other values (P < 0.05 by one-way ANOVA with Tukey’s post-hoc test). Values are the mean of three independent biological replicates and error bars represent the SE in panels D, E and F.

Figure 4.. Bb6S RNA expression in vitro.

(A) Northern blot and (B) qRT-PCR analyses of Bb6S RNA and flaA mRNA levels from total RNA isolated from wild-type cultures grown at 35°C to different cell densities. (C) Northern blot and (D) qRT-PCR analyses of Bb6S RNA levels from wild-type cells grown to stationary phase (RPMI 0 h) and starved in RPMI for 2 h or 6 h. Values are the mean of three independent biological replicates and error bars represent the SE. P > 0.05 by one-way ANOVA with Tukey’s post-hoc test for comparison of Bb6S levels in both panels B and D.

Figure 5.. Mutation and complementation of ssrS encoding Bb6S RNA in B. burgdorferi.

(A) The ssrS mutant was constructed by replacing the ssrS gene encoding Bb6S RNA with a promoterless streptomycin resistance gene (aadA). The ssrS mutant was complemented in cis, using a gentamicin resistance gene (aacC1) fused to a B. burgdorferi promoter (flgBp) and a B. subtilis terminator (trpLt), to generate the ssrS complemented strain (ssrS reconstituted). (B) Bb6S RNA expression in the wild-type (WT), ssrS null mutant and ssrS complemented strains analyzed by Northern blot of total RNA. Samples were separated on a 6% TBE urea gel, blotted to membrane and hybridized with biotinylated flaA and Bb6S RNA single-stranded RNA probes.

Figure 6.. Bb6S RNA affects ospC and dbpA gene expression.

(A) Northern blot analyses of total RNA isolated from wild-type (WT), ssrS mutant (ssrS null) and ssrS complemented (ssrS comp) strains temperature-shifted from 23°C and grown at 35°C until mid-log phase. RNA was separated on an 0.8% agarose gel, transferred to membranes and hybridized with ³²P-labeled probes to ospC, ospA, dbpA and flaB mRNA (Table 1). (B) Total cell lysates from strains and conditions listed in (A) were separated by SDS-PAGE, transferred to PVDF membranes and analyzed by immunoblot using antibodies against OspC and FlaB.

Figure 7.. Serological response of mice injected with the ssrS null mutant.

Whole cell lysates from wild-type (WT), ssrS mutant (ssrS null) and ssrS complemented (ssrS comp) B. burgdorferi, as well as E. coli (Ec) as a negative control, were separated by SDS-PAGE, transferred to membranes and incubated with mouse serum collected five weeks post-injection of 1 × 10³ cells of the corresponding strains.

Figure 8.. Bb6S RNA expression in vivo in ticks.

Bb6S RNA levels in wild type-infected ticks as measured by qRT-PCR from RNA isolated from ticks: naive larvae one week or three weeks post-feeding to repletion on an infected mouse, unfed nymphs one month after molting into nymphs, and fed nymphs one week after feeding to repletion on a naive mouse. Values are the means of at least two independent groups of ticks and error bars represent the SE. * denotes P < 0.05 as determined by one-way ANOVA with a Tukey’s post hoc test.

Figure 9.. Persistence of the ssrS null mutant in ticks.

Quantification of spirochetes in ticks that had fed on mice infected with wild-type (black circles), ssrS null mutant (white circles) or ssrS complemented (gray circles) strains. Total DNA was isolated from larvae that had fed to repletion (fed larvae) or after larvae had molted to nymphs (unfed nymphs) or one week after nymphs had fed to repletion on uninfected mice (fed nymphs). The number of B. burgdorferi genome equivalents per tick was determined by qPCR using TaqMan primers/probe to flaB. Data were analyzed using one-way ANOVA with a Tukey’s post hoc test where * indicates P < 0.05.