Convergence of Ser/Thr and two-component signaling to coordinate expression of the dormancy regulon in Mycobacterium tuberculosis

Abstract

Signal transduction in Mycobacterium tuberculosis is mediated primarily by the Ser/Thr protein kinases and the two-component systems. The Ser/Thr kinase PknH has been shown to regulate growth of M. tuberculosis in a mouse model and in response to NO stress in vitro. Comparison of a pknH deletion mutant (ΔpknH) with its parental M. tuberculosis H37Rv strain using iTRAQ enabled us to quantify >700 mycobacterial proteins. Among these, members of the hypoxia- and NO-inducible dormancy (DosR) regulon were disregulated in the ΔpknH mutant. Using kinase assays, protein-protein interactions, and mass spectrometry analysis, we demonstrated that the two-component response regulator DosR is a substrate of PknH. PknH phosphorylation of DosR mapped to Thr(198) and Thr(205) on the key regulatory helix α10 involved in activation and dimerization of DosR. PknH Thr phosphorylation and DosS Asp phosphorylation of DosR cooperatively enhanced DosR binding to cognate DNA sequences. Transcriptional analysis comparing ΔpknH and parental M. tuberculosis revealed that induction of the DosR regulon was subdued in the ΔpknH mutant in response to NO. Together, these results indicate that PknH phosphorylation of DosR is required for full induction of the DosR regulon and demonstrate convergence of the two major signal transduction systems for the first time in M. tuberculosis.

FIGURE 1.

Graphical analysis of iTRAQ ratios. A, distribution of iTRAQ ratios based on Rv accession number (TubercuList). Using arbitrary cutoff values of 1.25 and 0.8, 47 proteins were up-regulated and 21 were down-regulated in the pknH mutant in the absence of NO stimulus, whereas 20 were up-regulated and 17 were down-regulated after treatment with NO. Blue squares, untreated ΔpknH/WT ratios; red squares, acidified nitrite-treated ΔpknH/WT ratios. B, scatter plot of untreated/acidified nitrite-treated ratios of ΔpknH versus WT. Circled points represent proteins that clustered with the highest mean attribute value based on K-mean clustering of data points; eight of the nine points represent proteins encoded in the DosR regulon.

FIGURE 2.

PknH phosphorylation of DosR. A, in vitro kinase assay demonstrated phosphorylation of DosR by PknH using [γ-³²P]ATP. Upper, phosphorimage; lower, silver stain of DosR protein bands. B, one-dimensional phosphoamino acid analysis of PknH-phosphorylated DosR identified phosphorylation on Thr. Control phospho-Tyr (Y), phospho-Thr (T), and phospho-Ser (S) were visualized by spraying with ninhydrin, and radiolabeled DosR residues were visualized by phosphorimaging. Retention factors were calculated as follows: Tyr, 0.37; Thr, 0.31; Ser, 0.25; and DosR, 0.30. C, in vitro kinase assay confirmed that DosR(T198A) (A₁), DosR(T205A) (A₂), and the double mutant DosR(T198A/T205A) (AA) are defective for phosphorylation by PknH. Upper, phosphorimage; lower, silver stain. Arrowheads point to DosR.

FIGURE 3.

Identification of DosR phosphorylation sites. The MS/MS spectra represent peptide positions 198–209 with a monoisotopic mass of 1493.69 Da from DosR phosphorylated in a cell-based system showing diphosphorylation of Thr¹⁹⁸ and Thr²⁰⁵. Phosphorylation at Thr¹⁹⁸ is shown by the b N-terminal daughter ion series, where all b ions identified lose phosphoric acid (−98 Da). Phosphorylation at Thr²⁰⁵ is shown by the y C-terminal daughter ion series, where all y ions after Thr²⁰⁵ lose phosphoric acid. pT, phosphothreonine; amu, atomic mass units.

FIGURE 4.

PknH interaction with and dimerization of DosR in vivo. A, PknH and DosR protein-protein interaction facilitated the reassembly of the F1,2 and F3 domains of mDHFR, enabling growth of M. smegmatis strains coexpressing DosR-F1,2 and PknH-(1–401)-F3 fusion proteins in the presence of 20 μg/ml trimethoprim (TMP). Identical spots on control plates without trimethoprim revealed growth of all strains. Positive Control, Saccharomyces cerevisiae Gcn4 dimerization domains fused to F1,2 and F3, respectively; Negative Control, mDHFR fragments alone. The experiment is shown in duplicate. B, the specific interaction between PknH and the phosphorylation-defective DosR(T198A/T205A) (DosR_AA) mutant facilitated the reassembly of the N_Trp and C_Trp fragments required for Trp biosynthesis, thus enabling growth of M. smegmatis Trp⁻ strains coexpressing N_Trp-PknH-(1–401) with DosR(T198A/T205A)-C_Trp, but not with WT DosR-C_Trp (upper row). The positive control consisted of N_Trp-Cfp10 and Esat6-C_Trp. The negative control consisted of N_Trp and C_Trp alone. C, the growth of M. smegmatis Trp⁻ was dependent on the reassembly of N_Trp and C_Trp mediated by the dimerization of the C-terminal domains (amino acids 145–217) of the phosphomimetic DosR(EE) mutant (DosR_EE-C), but not of WT DosR (DosR_-C) or the WT DosR/DosR(EE) (DosR_-C/DosR_EE-C) combination (upper row). B and C, middle rows, Trp supplied exogenously; lower rows, no acetamide (Acet) induction of the fusion proteins. Data are representative of three separate experiments.

FIGURE 5.

Enhancement of DNA binding by PknH Thr-phosphorylated DosR. Shown are phosphorimages of EMSA comparing the effects of Thr and Asp phosphorylation of DosR on the ability of DosR to bind the D and CP DosR boxes of the hspX promoter. In vitro phosphorylation of DosR by PknH and/or DosS increased binding to the D box (A) and CP box (B). Cell-based phosphorylation of DosR by PknH increased binding and caused an addition shift to the D box (C) and CP box (D). DosR was incubated with radiolabeled DNA and run on a nondenaturing gel. Radiolabeled DNA was titrated with excess unlabeled probe by the dilution factor (DF) indicated to show specific binding. Arrowheads show unbound DNA. Data are representative of three separate experiments.

FIGURE 6.

Transcriptional analysis of DosR regulon genes in WT and ΔpknH. A, heat map of qRT-PCR results showing ΔpknH/WT ratios for various culture conditions as indicated. Each gene was normalized to sigA. Red and green spots indicate greater or lesser gene expression in the ΔpknH mutant relative to WT M. tuberculosis, respectively. The scale bar indicates the mean of the log₂ ratio. B, response of WT and the ΔpknH mutant to a 4-h treatment with 0.05 mm diethylenetriamine/NO (DETANO). Fold induction (±S.E.) was calculated by dividing gene expression levels in the treated cultures by the basal level expression in untreated cultures. *, p < 0.05; **, p < 0.01; ***, p < 0.001, significant difference compared with WT samples by Student's t test. Std, standing.