Molecular dissection of RbpA-mediated regulation of fidaxomicin sensitivity in mycobacteria

Abstract

RNA polymerase (RNAP) binding protein A (RbpA) is essential for mycobacterial viability and regulates transcription initiation by increasing the stability of the RNAP-promoter open complex (RP_o). RbpA consists of four domains: an N-terminal tail (NTT), a core domain (CD), a basic linker, and a sigma interaction domain. We have previously shown that truncation of the RbpA NTT and CD increases RP_o stabilization by RbpA, implying that these domains inhibit this activity of RbpA. Previously published structural studies showed that the NTT and CD are positioned near multiple RNAP-σ^A holoenzyme functional domains and predict that the RbpA NTT contributes specific amino acids to the binding site of the antibiotic fidaxomicin (Fdx), which inhibits the formation of the RP_o complex. Furthermore, deletion of the NTT results in decreased Mycobacterium smegmatis sensitivity to Fdx, but whether this is caused by a loss in Fdx binding is unknown. We generated a panel of rbpA mutants and found that the RbpA NTT residues predicted to directly interact with Fdx are partially responsible for RbpA-dependent Fdx activity in vitro, while multiple additional RbpA domains contribute to Fdx activity in vivo. Specifically, our results suggest that the RP_o-stabilizing activity of RbpA decreases Fdx activity in vivo. In support of the association between RP_o stability and Fdx activity, we find that another factor that promotes RP_o stability in bacteria, CarD, also impacts to Fdx sensitivity. Our findings highlight how RbpA and other factors may influence RNAP dynamics to affect Fdx sensitivity.

Keywords: RNA polymerase; RbpA; antibiotic action; bacterial genetics; bacterial transcription; fidaxomicin; mycobacteria.

Figure 1

RbpA E17 and R10 synergize to promote Fdx activity against M. tuberculosis RNAP-σ^Ain vitro.A, schematic of RbpA’s four domain structure including the location of the substituted residues, R10, E17, R79, R88, and the two M. tuberculosis truncation mutants, RbpA 26–111 lacking the NTT, and RbpA 72–111 lacking the NTT and CD. B, representative gels showing Fdx (0 μM, 0.01 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM) inhibition of M. tuberculosis RNAP-σ^A production of three nucleotide transcripts alone or in complex with RbpA_Mtb^WT, RbpA_Mtb^R10A, RbpA_Mtb^E17A, RbpA_Mtb^R10A/E17A, RbpA_Mtb^R79A, RbpA_Mtb^R88A, RbpA_Mtb^26–111, or RbpA_Mtb^72–111 from a linear dsDNA template containing positions −80 to +70 of M. tuberculosis rrnAP3 (relative to the +1 transcription start site). C, dose–response curves of the experiments shown in (A). The curves are generated from at least four replicates from at least two different experiments. Percent inhibition at each Fdx concentration included in the plots compared to no drug is depicted as the mean ± SD. The IC50 for each replicate was calculated by nonlinear regression analysis with four-parameter (EC50, Hill Slope, top and bottom curve plateaus) fitting of log transformed Fdx concentration versus normalized response, with the mean IC50 and 95% confidence interval listed in the table. D, structural modeling of Fdx binding pocket on the RbpA-bound M. tuberculosis RNAP-σ^A from PDB structure 6BZO. Fdx and RNAP residues involved in the RNAP-Fdx binding interface are shown with PyMol stick representation while the rest of the structure is shown with PyMol cartoon representation. Polar interactions are indicated by red dashed lines, and potential van der Waals interactions are shown as gray lines. E, structural modeling of RbpA-bound M. tuberculosis RNAP-σ^A from PDB structure 6C04. RbpA R10 and RNAP σ^A D441 are shown with PyMol stick representation while the rest of the structure is shown with PyMol cartoon representation. The polar interaction between RbpA R10 and RNAP σ^A D441 is indicated by the red dashed line. CD, core domain; Fdx, fidaxomicin; NTT, N-terminal tail; RbpA, RNA polymerase binding protein A; RNAP, RNA polymerase.

Figure 2

Multiple RbpA domains impact Fdx activity in vivo.A, ratio of the doubling times of M. smegmatis strains expressing RbpA_Mtb^R10A, RbpA_Mtb^E17A, RbpA_Mtb^R10A/E17A, RbpA_Msm^28–114, or RbpA_Msm^72–114 as compared to the average doubling time for the strain expressing RbpA_Mtb^WT. The mean ± SD from at least two independent experiments with three replicates per experiment. B, zones of inhibition (ZOI) by Fdx on bacterial lawns of M. smegmatis expressing RbpA_Mtb^WT, RbpA_Mtb^R10A, RbpA_Mtb^E17A, RbpA_Mtb^R10A/E17A, RbpA_Mtb^R79A, RbpA_Mtb^R88A, RbpA_Msm^28–114, RbpA_Msm^72–114, or RbpA_Mtb^72–111 as the only copy of rbpA. C, mean radii of ZOI ± SD from at least two experiments with at least three replicates at 100 μM, 250 μM, and 500 μM Fdx is plotted. For A and C, statistical significance of differences was analyzed by ANOVA and Tukey’s multiple comparison test. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. All comparisons to RbpA_Mtb^WT were included in the analysis, but only statistically significant comparisons are indicated in the figure. Fdx, fidaxomicin; RbpA, RNA polymerase binding protein A; RNAP, RNA polymerase.

Figure 3

RP_ostability in associated with Fdx sensitivity in vivo.A, representative gels of three nucleotide transcripts produced by M. tuberculosis RNAP-σ^A alone or in complex with RbpA_Mtb^WT, RbpA_Mtb^72–111, RbpA_Mtb^R10/E17A, or RbpA_Mtb^R88A from a plasmid DNA template containing positions −39 to +4 of M. tuberculosis rrnAP3 relative to the +1 transcription start site. B, ratio of transcript produced as compared to the average of “No Factor” replicates included on the same gel. Results are plotted as individual values with the mean ± SD shown. Statistical significance of differences was determined by ANOVA and Tukey’s multiple comparison test. ‘ns’, not significant; ∗∗p < 0.01; ∗∗∗∗p < 0.0001. C, zones of inhibition (ZOI) by Fdx on bacterial lawns of M. smegmatis expressing CarD_Mtb^WT or CarD_Mtb^R25E as the only copy of carD. D, mean radii of ZOI ± SD from at least two experiments with at least three replicates at 100 μM, 250 μM, and 500 μM Fdx is plotted. Statistical significance was analyzed by two-tailed Welch’s t test. ∗p < 0.05; ∗∗p < 0.01. Fdx, fidaxomicin; RbpA, RNA polymerase binding protein A; RP_o, RNAP-promoter open complex.