c-di-GMP regulates activity of the PlzA RNA chaperone from the Lyme disease spirochete

Abstract

PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi during its enzootic life cycle. Unliganded apo-PlzA is important for vertebrate infection, while liganded holo-PlzA is important for survival in the tick; however, the biological function of PlzA has remained enigmatic. Here, we report that PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding. Holo- and apo-PlzA bind RNA and accelerate RNA annealing, while only apo-PlzA can strand displace and unwind double-stranded RNA. Guided by the crystal structure of PlzA, we identified several key aromatic amino acids protruding from the N- and C-terminal domains that are required for RNA-binding and unwinding activity. Our findings illuminate c-di-GMP as a switch controlling the RNA chaperone activity of PlzA, and we propose that complex RNA-mediated modulatory mechanisms allow PlzA to regulate gene expression during both the vector and host phases of the B. burgdorferi life cycle.

Keywords: Borrelia burgdorferi; Lyme disease; PlzA; RNA chaperone; c-di-GMP.

Figure 1.. Apo- and holo-PlzA have RNA annealing activity in vitro.

(A) Illustration of the RNA annealing assay measuring the annealing of two short unstructured complementary 21-mer RNAs labeled with Cy5 (red, J1) and Cy3 (green, M1) to form a dsRNA (yellow, J1M1). The reaction is initiated by adding 10 nM M1 RNA to 10 nM J1 RNA without or with 1.2 μM protein. Samples are withdrawn from the reaction at the indicated time points, mixed with stop buffer and analyzed by native polyacrylamide gel electrophoresis. The stop buffer includes excess unlabeled competitor M1 RNA, uM1, which has the identical sequence to the M1 RNA. The competitor RNA binds the unbound single-stranded J1 RNA forming a dsRNA that is only labeled with Cy5, J1uM1. The J1uM1 dsRNA is slightly smaller than the J1M1 dsRNA because it lacks the Cy3 label on M1. Representative images of RNA annealing gels are shown in Fig. S2B. (B) The Cy5 signal was quantified at each time point. Normalized linear regression of the ratio of J1M1 dsRNA to total RNA for each time point yielded annealing curves for each reaction: RNA only (red circle), StpA (purple square), PlzA (green triangle), PlzA and c-di-GMP (blue triangle), c-di-GMP (teal open square), and GrpE (black open circle). Error bars represent standard error of the means (SEM) of at least three replicates. (C) A bar graph illustrating the ratio of J1M1 dsRNA to total RNA at the endpoint of the RNA annealing assay. Annealing endpoint signals were quantified by ImageJ and analyzed by one-way ANOVA and Tukey’s multiple comparisons (GraphPad Prism): StpA vs RNA only (****, p<0.0001), PlzA vs PlzA plus c-di-GMP (ns, p>0.9999), PlzA vs RNA only (***, p=0.0002). (D) Illustration and representative image of the filter binding assay. 10 nM Cy3 M1 RNA and serial dilutions of PlzA proteins were incubated for 5 min, applied to a slot blot apparatus, and filtered through two membranes (nitrocellulose and positively charged nylon). Protein and RNA bound to protein bind to the nitrocellulose membrane, while unbound RNA flows through the nitrocellulose and binds to the nylon membrane. Membranes were imaged using an Azure Sapphire fluorescence imager and a representative image is shown. (E) RNA bound to each filter was quantified using ImageJ and the fraction of bound RNA was calculated as a function of protein concentration. Means and standard error of the means were calculated on GraphPad Prism and fit to the Hill equation for multiple binding sites (Table S5).

Figure 2.. PlzA RNA displacement activity is modulated by c-di-GMP.

(A) Illustration of RNA strand displacement assay measuring the amount of dsRNA that has one strand displaced and replaced with another strand. The dsRNA is formed with 10 nM two short unstructured complementary 21-mer RNAs J1 and M1 (described in Fig. 1) and purified. The reaction is initiated by adding 10 nM unlabeled M1 RNA (uM1) with or without 1.2 μM protein. Samples are withdrawn from the reaction at the indicated time points, mixed with stop buffer and analyzed by native polyacrylamide gel electrophoresis. If strand displacement occurs, then there will be a decrease in the preformed J1M1 dsRNA (yellow band) and an increase of both J1uM1 dsRNA (red band) and M1 RNA (green band) over time. Representative images of the RNA strand displacement gels are shown in Fig. S2C. The Cy5-signal was quantified. (C) Normalized non-linear regression of the ratio of dsRNA to total RNA for each time point yielded displacement curves for each reaction tested: RNA only (red circle), StpA (purple square), PlzA (green triangle), PlzA and c-di-GMP (blue triangle), PlzA and c-di-AMP (orange open triangle), c-di-GMP (teal open square), and GrpE (black open circle). Three replicates were performed for each protein. Error bars represent SEM. (C) A bar graph illustrating the ratio of J1M1 dsRNA to total RNA at the endpoint of the RNA strand displacement assay. Strand displacement endpoint signals were quantified by ImageJ and analyzed by one-way ANOVA and Tukey’s multiple comparisons(GraphPad Prism): StpA vs RNA only (****, p<0.0001), PlzA vs PlzA plus c-di-GMP (**, p=0.0006), PlzA vs RNA only (**, p=0.0020). (D) dsRNA PlzA filter binding assays. M1uJ1 RNA bound to each filter was quantified using ImageJ and the fraction of bound RNA was calculated as a function of protein concentration. Means and standard error of the means were calculated on GraphPad Prism and fit to the Hill equation for multiple binding sites (Table S5).

Figure 3.. c-di-GMP inhibits PlzA RNA unwinding activity in E. coli.

(A) Schematic of the trpL terminator assay. The trpL terminator stem loop is fused to the chloramphenicol (Cm) acetyltransferase (cat) gene. In the RL211 strain, the terminator stem loop is formed, transcription is terminated, and the cat gene is not expressed resulting in Cm susceptibility and no growth on Cm. However, an RNA chaperone expressed in RL211 that unwinds the terminator stem loop will allow transcription elongation to proceed and the cat gene to be expressed, resulting in Cm resistance and growth on Cm. (B) E. coli RL211 strains expressing wild-type PlzA, PlzA R145D (no c-di-GMP binding), PlzA R145K (increased c-di-GMP binding), or the positive control StpA were grown in LB overnight to an OD₆₀₀ of ~1. The overnight cultures were plated on LB (no antibiotic) or Cm and growth was assessed. Legend illustrates the apo-PlzA (PlzA), holo-PlzA (PlzA c-di-GMP) and StpA.

Figure 4.. PlzA structure reveals potential RNA binding sites.

(A) Structure of PlzA showing the positions of the mutated phenylalanines. Two views of the protein are shown, with the protein rotated approximately 180° between the top and bottom panels. On the left are ribbon views in which the mutated phenylalanines (F92, F95, F168, and F218) and arginine (R145) are colored with yellow bonds and the c-di-GMP molecules are colored with green bonds. On the right are the corresponding electrostatic surfaces, where electropositive regions are blue and electronegative regions are red. (B) Molar residue ellipticity (MRE) plotted versus wavelength for wild-type (WT) and mutant PlzA. Spectra were measured in 10 mM sodium phosphate buffer at pH 7 and 25°C using a 1-mm pathlength. Sample concentrations range from 4.2 μM to 5.5 μM.

Figure 5.. Aromatic amino acids protruding from the N- and C-terminal β-barrels are necessary for RNA binding and unwinding activity.

(A) E. coli RL211 strains expressing wild-type PlzA, PlzA R145D, PlzA R145D F92A F95A, PlzA R145D F168A F218A, or the positive control StpA were grown in LB overnight to an OD₆₀₀ of ~1 before plating on LB (no antibiotic) or Cm. (B) 10 nM Cy3 M1, M1uJ1 and trpL RNA and serial dilutions of PlzA proteins were incubated for 5 min, applied to a slot blot apparatus and filtered through two membranes (nitrocellulose and positively charged nylon). Protein and RNA bound to protein bind to the nitrocellulose membrane, while free RNA does not bind the nitrocellulose but instead binds to the nylon membrane. Membranes were imaged using an Azure Sapphire fluorescence imager. RNA bound to each filter was quantified using ImageJ and the fraction of bound RNA was calculated as a function of protein concentration. Means and standard error of the means were calculated on GraphPad Prism and fit to the Hill equation for multiple binding sites (Table S5).

Figure 6.. PlzA protein binds B. burgdorferi RNAs.

(A) Schematic of the His-tagged PlzA-RNA pulldown. 6×His-PlzA proteins were bound to a cobalt resin and washed several times. A cleared B. burgdorferi whole cell lysate was applied to the column. Complexes were washed several times and eluted in two fractions (E1 and E2) using an imidazole buffer. A control pulldown was performed where no PlzA was added. (B) Fractions E1 and E2 were analyzed by immunoblotting using anti-PlzA antibodies to assess the recovery from the columns. (C) RNA was isolated from E1 and run on a bioanalyzer.

Figure 7.. Model for the RNA chaperone activity of apo- and holo-PlzA throughout the enzootic cycle.

Holo-PlzA is not required for transmission to or survival in the mouse and constitutive production of c-di-GMP inhibits transmission and survival in the mouse, suggesting apo-PlzA plays an important role in the vertebrate phase of the enzootic cycle. We hypothesize that in the mouse, apo-PlzA (purple barbell) unwinds and anneals RNA, inducing vertebrate-phase-specific genes and repressing tick-phase-specific genes. Holo-PlzA (purple barbell with blue circle) and c-di-GMP (blue circle) are required for successful larval acquisition. Holo-PlzA anneals RNA but cannot strand displace or unwind RNA, suggesting that this activity may be needed in the repression of vertebrate-phase-specific genes and induction of tick-phase-specific genes. We propose that c-di-GMP acts as an ON/OFF switch for the RNA unwinding activity of PlzA: apo-PlzA (ON) and holo-PlzA (OFF). In contrast, PlzA RNA annealing activity is independent of c-di-GMP and is likely constitutive throughout the enzootic cycle. The role of PlzA and c-di-GMP in spirochete persistence through the molt and in the flat nymph has not been evaluated. Created with BioRender.com.