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. 2026 Feb 20;11(9):14707–14718. doi: 10.1021/acsomega.5c10658

Regulation of Dormancy-Associated Genes dosR by TetR Family Regulator MRA_0776 in Mycobacterium Tuberculosis H37Ra

Shuxian Wang †, Luyue Zhang †, Ruiying Wu †, Hui Wang †, Mingrui Sun †, Yang Zhang †, Jiayin Xing †, Ren Fang †, Xiaotian Li †, Yonghao Wang †, Zhaoli Li ‡,§,*, Ningning Song †,*
PMCID: PMC12980445  PMID: 41835575

Abstract

Background: The Dormancy survival regulator (DosR), Rv3133c, is one of the key transcriptional proteins that regulate the dormancy of Mycobacterium tuberculosis (MTB), participates in various metabolic processes, and is essential for the survival of MTB in the host. MRA_0776 (homologous with Rv0767c and MSMEG_5860) belongs to the TetR family of regulators; we previously found that MRA_0776 can directly regulate the expression of dosR, although the mechanism by which MRA_0776 acts directly on dosR and other small molecules affecting the interaction of MRA_0776 with dosR has not been elucidated. Results: In the present study, primary transcriptomic analysis of a MRA_0776 overexpression strain revealed that MRA_0776 is a broad-spectrum regulatory protein and that MRA_0776 specifically binds to the dosR promoter both in vitro and in vivo. In addition, we found that a range of ligands, including lysine, arginine, tyrosine, Cu2+, Fe3+ and Pb2+, inhibited the interaction of MRA_0776 with the dosR promoter. Furthermore, molecular docking predicted that 125-Thr is likely to be a key amino acid site for the interaction of MRA_0776 with ligands. Finally, MRA_0776 promoted bacteria colonization and induced pathological damage in livers, spleens, lungs, and kidneys in mice. Conclusions: In conclusion, our analyses suggested that MRA_0776 is a pleiotropic regulator, and its overexpression can promote colonization and pathogenicity of MTB.

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Introduction

The Mycobacterium tuberculosis complex (MTBC) is a complex of pathogens causing tuberculosis (TB), with M. tuberculosis (MTB) and Mycobacterium bovis being the main pathogens of human TB. The latest report from the World Health Organization reported that in 2023, approximately 10.8 million people suffered from TB and that 1.25 million people died from TB. An important factor underlying the recurrent attacks of TB and the difficulty in eradicating TB is that MTB has the ability to adapt to the harsh environments in hosts, including low-oxygen, acidic, and nutrient-limited conditions. Furthermore, this level of adaptation is highly dependent on the complex transcriptional regulatory system, which can help MTB to survive and maintain its latent state by regulating target genes. The TetR family of transcriptional regulators is the most abundant family of regulators in Mycobacteria. There are 51 transcriptional regulators in the TetR family and more than 200 transcriptional regulatory proteins in MTB. , In MTB, TetR regulators participate in multiple physiological functions. For instance, the family of TetR proteins can regulate genes required for carbon utilization (kstR, kstR2, and mce3R); − bkaR, which assists in the catabolism of branched-chain amino acids, the antibiotic resistance genes rv3066 and ethR, , and play multiple roles in intracellular signaling, cell division, and the metabolism of carbon, nitrogen, and cofactors.

Rv0767c (HpoR) belongs to the TetR family and is highly conserved in MTB, Mycolicibacterium smegmatis (Ms), and M. bovis Bacillus Calmette–Guerin vaccine (BCG). Bacterial genome sequencing identified a nonsynonymous mutation frequency significantly in rv0767c in drug-resistant MTB clinical isolates. HpoR (MSMEG_5860), a homologue of Rv0767c in MTB, is known to act as an effector protein of the second messenger cyclic diguanosine monophosphate (c-di-GMP) and links the regulatory function of the second messenger to the antioxidant defense of Mycobacterium by acting as a c-di-GMP receptor to negatively regulate resistance to H2O2. In addition, c-di-GMP is known to integrate and interact with two functionally opposing receptor transcription factors, LtmA and Rv0767c (HpoR). However, little is known about the precise physiological function of Rv0767c and its regulons.

Rv0767c was predicted as a potential regulator of dosR, and in our study, MRA_0776 overexpression induced high expression of dosR and EMSA combining with ChIP-PCR assay demonstrated that dosR is a target of MRA_0776. DosR is known to be critical for M. bovis infection and persistence in a host under hypoxia. Transcriptomic analysis previously revealed that dosR as well as a cluster of genes regulated by DosR were induced during the early stages of hypoxia. Expression of the dosR gene was found to be induced in microenvironments containing elevated levels of nitric oxide (NO), carbon monoxide (CO), reactive oxygen species (ROS) and in a hypoxia and vitamin C model. , Several animal studies, including guinea pigs and rabbits, confirmed that the upregulation of dosR favors the survival of MTB in vivo. Under hypoxia, phosphorylated DosR interacts with multiple binding sites within the promoter regions of target genes to regulate the expression of approximately 48 genes in MTB. , The deletion of dosR has been found to significantly influence the transcription of 104 genes and the translation of 179 proteins; metabolomics analysis also showed that the knockdown of dosR significantly up-regulated the synthesis of l-aspartic acid and serine, but down-regulated the synthesis of seven other amino acids, including l-histidine and lysine. Thus, the regulatory role of DosR in MTB is more complex and extensive than previously thought for the complexity of hypoxia in MTB physiology. DosR regulates amino acid synthesis and metabolism, maintains ribosome stability, induces TLR2 pathway-dependent macrophage activation during hypoxia, , participates in nitrate metabolism and deoxyribonucleotide synthesis processes, and is involved in the adaptation to oxidative stress and maintenance of redox homeostasis. ,

In the present study, we aimed to gain further insights into the regulatory role of MRA_0776 and its precise physiological role and used electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation PCR (ChIP-PCR) assays to confirm that MRA_0776 directly regulates dosR, both in vitro and in vivo. Furthermore, we identified and validated additional MRA_0776 binding sites in the genome of MTB by in vitro DNA-binding assays for a subset of selected targets. We also found that MRA_0776 binds with a broad range of ligands to influence the DNA-binding capacity. Finally, we found that MRA_0776 could promote MTB colonization and organ damage in mice. Collectively, our findings demonstrate the novel regulons and ligands of MRA_0776 and identify its precise physiological role in MTB. These findings will benefit the identification of drug candidates and enhance our future ability to treat TB.

Materials and Methods

Bacterial Strains, Plasmids, and Culture Conditions

Escherichia coli BL21 (DE3) was grown in flasks containing LB broth. Mycobacterium BCG Tokyo 172, M. tuberculosis H37Ra, and Mycolicibacterium smegmatis MC2 155 were cultured in Middlebrook 7H9 broth medium supplemented with 10% OADC (v/v) (BD) and 0.05% Tween 80 (v/v). The pET22b-MRA_0776-6xHistag was used to express recombinant protein MRA_0776, while the pMV262-MRA_0776-Ms and pMV262-Ms strains were used for the physiological study. The pMV262-MRA_0776-H37Ra and pMV262-H37Ra strains were used for overexpression transcriptomics analysis.

Animals and ethics

Wild-type (WT) C57BL/6 mice (6 weeks, female) and New Zealand rabbits were purchased from Jinan Pengyue Laboratory Animal Breeding Ltd. (Jinan, China). All mice were housed in a specific pathogen-free (SPF) environment in accordance with standard humane animal husbandry protocols, which were approved by the Ethical Committee of Animal Experiments of Shandong Second Medical University.

Purification of the Recombinant MRA_0776 Protein

The genome of MTB H37Ra was used as a template, and the MRA_0776 gene was amplified by specific primers (Table S1) and cloned into the pET22b vector to construct a recombinant plasmid (pET22b-MRA_0776-6xHistag). This plasmid was transformed into BL21 cells to generate a pET22b-MRA_0776-BL21 strain in LB broth. Protein expression was induced by adding a final concentration of 0.75 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the OD600 nm reached 0.9, and then 150 rpm, 30 °C, induced for 9 h. Following induction, the culture was resuspended in lysis buffer (20 mM Tris-HCl, 150 mM NaCl and 10% glycerol, pH 8.0) and then sonicated. The recombinant MRA_0776 protein was purified by affinity chromatography, and protein concentration was determined by the BCA protein assay. The recombinant protein was subsequently analyzed by SDS-PAGE and western-blotting.

Transcriptomic Analysis of the pMV262-MRA_0776-H37Ra Overexpression Strain

RNA-seq was performed as described previously to analyze the transcriptome of the pMV262-MRA_0776-H37Ra overexpression strain. In brief, when the OD600 nm reached 0.8, pMV262-MRA_0776-H37Ra was overexpressed at 45 °C for 2 h. The regulatory sequences of the BCG hsp60 genes were chosen to drive the expression of foreign antigen genes in MTB H37Ra, which requires 45 °C heat excitation for overexpression. Then, the cells were harvested by centrifugation, stirred, and homogenized at a high speed in a bead mill in the presence of glass beads and lysis buffer. Total RNA was then extracted using an RNeasy mini kit and subjected to on-column DNase I treatment to remove any contaminating genomic DNA. Ribosomal RNA was removed using a Ribo-Zero Magnetic kit, in accordance with the manufacturer’s instructions. Following cDNA synthesis, mRNA was fragmented with a TruSeq stranded RNA Sample Preparation Kit, in accordance with the manufacturer’s instructions. Deoxythymidine triphosphate was replaced with deoxyuridine triphosphate during second-strand cDNA synthesis, followed by digestion with uracil-DNA glycosylase. The resulting cDNA library was enriched with PCR and purified. Subsequently, libraries were quantified byTBS380 minifluorometer systems using PicoGreen and sequenced on the HIseq platform. Raw sequencing data were analyzed by Base Calling and Trimmomatic software for the quality assessment and base distribution analysis. After comparison with the Rfam database, UMI redundancies were removed, and reads matching rRNA (rRNA) were excluded. Subsequently, the sequencing data of each sample were mapped to the reference genome using Rockhopper, a specialized software for prokaryotic transcriptome analysis. Gene expression levels were determined by a Rockhopper. Local regression was used to determine gene expression patterns, and statistical comparisons provided P-values using a negative binomial distribution model. Finally, these P-values were corrected for Benjamin-Hochberg multiple testing to obtain Q-values for significant gene comparisons. To assess the potential functions of differentially expressed genes, we assigned gene ontology (GO) categories. The significant transcript enrichment analysis of pMV262-H37Ra and pMV262-MRA_0776-H37Ra overexpression strains were carried out using the Goatools program (https://github.com/tanghaibao/GOatools).

cDNA Synthesis and Quantitative RT-PCR

First-strand cDNA was synthesized by using a SuperScript III First-Strand Synthesis SuperMix Kit. Quantitative real-time PCR (qRT-PCR) was carried out in a LightCycler480 II (Roche, Basel, Switzerland) RT-PCR machine with SYBR Green Supermix, as described previously. The PCR conditions were as follows: 95 °C for 3 min, 40 cycles at 95 °C for 10 s, 55 °C for 30 s, 72 °C for 10 s, and one cycle at 50 °C for 3 min. Reactions were performed with 12.5 μL of SYBR Green Supermix and cDNA (diluted by 20-fold) in a total volume of 25 μL. All qRT-PCR assays were carried out in technical duplicates for three biological replicates with no-template and no-RT controls. MRA_2731, encoding sigA, was used as a housekeeping gene for normalization. The efficiencies of gene-specific primer pairs were calculated by measuring the slope of a linear regression curve arising from the Cq values of a 10-fold dilution series with g-DNA as the template. Relative expression values were then calculated using the 2–ΔΔCt method.

Electrophoretic Mobility Shift Assays

Electrophoretic mobility shift assays (EMSAs) were performed to verify the binding ability of MRA_0776 with target promoters, and the EMSA reaction volume was 20 μL. Specific primers were labeled with Cy-5 and synthesized by TONGYONG and then amplified by PCR. Primer sequences are shown in Table S1. For EMSA assays, a mixture of MRA_0776 and DNA fragments was incubated in a binding buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM DTT, and 5% glycerol) at 37 °C for 20 min and then loaded on to an 8% nondenaturing acrylamide gel in Tris-Borate-EDTA buffer at 200 v for 4 h. Subsequently, the gel was analyzed and imaged using a gel imaging analysis system (Junyi, Beijing, China). For binding specificity tests, we added high concentrations of cold probes to the binding buffer to perform competition inhibition experiments, the Cy5-labeled probe was at 8 nM, and the concentrations of the cold probe were 800 nM, 1600 nM, and 3200 nM (corresponding to 100×, 200×, and 400×, respectively). For ligand tests, ligands with various concentration gradients were added to the reaction to evaluate the ligand specificity of MRA_0776.

Biofilm Layer Interference (BLI)

The binding affinity of MRA_0776 for the dosR promoter was investigated by biofilm layer interference (BLI). BLI was performed by using streptavidin (SA)-labeled sensors in combination with biotin-labeled SN441 nucleic acid probes (Table S1). Sensor tips were preincubated for 10 min in BLI buffer (PBST, 0.02% (v/v) Tween-20). Next, we immobilized 5′-biotinylated PCR products for 60 s. MRA_0776 protein was added to reactions in a gradient series of concentrations (313.5 nM, 625 nM, 1250 nM, 2500 nM, 5000 nM, and 10000 nM). Association was recorded for 90 s, followed by a 90 s dissociation step. The reaction volume of all sample wells in the experiment was 200 μL at 30 °C. All binding sensor grams were recorded on a forteBIO OctetRED96e instrument. Data were normalized by Octet data analysis software and then plotted using PRISM.

Animal Immunization and Polyclonal Antibody Purification

The MRA_0776 protein was emulsified with Fuchs’ incomplete adjuvant at a 1:1 ratio. Rabbits were then immunized with 400 μg of MRA_0776 protein by multiple subcutaneous injections at intervals of 14 d. One week after the final immunization, the rabbits were bled, and an enzyme-linked immunosorbent assay (ELISA) was used to determine the titer of anti-His-MRA_0776. The anti-MRA_0776 rabbit serum was purified by passaging the protein A + G agarose column. The specificity of the purified antibody was determined with antigen MRA_0776 by Western blotting.

Chromatin Immunoprecipitation-PCR (ChIP)

Next, ChIP was performed in vivo to confirm the binding of MRA_0776 to the promoter of dosR in accordance with a previous study. M. tuberculosis H37Ra was harvested during the midexponential growth phase and were fixed with a final concentration of 1% formaldehyde for 10 min. The reaction was quenched by adding glycine to a final concentration of 125 mM for 5 min. The precipitate was then resuspended with IP buffer and used ultrasonic cell disruptor sonicated for 5 min at 20% amplitude. Purified MRA_0776 polyclonal antibody was coupled with M-280 goat antirabbit antibody to prepare coated magnetic beads at 4 °C overnight. After the magnetic beads were washed, the MRA_0776-DNA complex was collected and purified with an iPure DNA extraction kit, and the collected DNA was used as a template for PCR amplification with primers SN441-F/SN441-R (Table S1).

Tertiary Structure Prediction and Molecular Docking

The modeled tertiary structure of MRA_0776 protein was predicted using Alphafold2 for molecular docking. The three-dimensional (3D) structure of each small molecule was downloaded from the PUBCHEM database, and energy was minimized using Chem3D 20.0 under the MMFF94 force field.

AutoDock Vina 1.1.2 software was used for molecular docking analysis, and before docking started, the receptor proteins were processed using PyMol 2.5.2, including the removal of water molecules, salt ions, and small molecules. Subsequently, the docking box was set up so that it wrapped around the entire protein structure. In addition, all processed small molecules as well as receptor proteins were converted to a PDBQT format for AutoDock Vina 1.1.2 docking using ADFRsuite 1.0. For docking, the exhaustiveness of the global search was set to 32 and the rest of the parameters were kept at the default settings. Output docking conformations with the highest scores were considered as binding conformations, and finally, the docking results were visualized and analyzed by PyMol 2.5.2.

Mouse Infection Assays, Colony-Forming Unit (CFU) Counting, and Histopathological Analysis

Ms has the advantages of fast growth, low pathogenicity, a high homology with MTB, and has been used as a model strain for mycobacterial research in the laboratory. − Therefore, we constructed a pMV262-MRA_0776-Ms strain to study the physiological functions of MRA_0776 of Mycobacterium. Sixty 6–8 week-old C57BL/6 mice were divided into three groups: PBST control group (n = 20), pMV262-Ms group (n = 20), pMV262-MRA_0776-Ms group (n = 20). Mice were infected with 5.0 × 106 CFU bacteria suspended in 100 μL of PBST via tail vein injection. The mice of each group were humanely euthanized at each designated time point (4, 7, 14, and 21 d) postinfection for organ collection and evaluation of mycobacterial survival. Following anesthesia induction in mice via graded CO2 asphyxiation (30% chamber volume displacement/min), without chemical anesthetics, and the surgical plane of anesthesia was confirmed by abolition of the pedal withdrawal reflex. Immediate cervical dislocation was performed postphlebotomy to ensure irreversible euthanasia prior to potential anesthetic recovery. Organ harvesting (liver, spleen, kidney, lung) were executed post-mortem. 0.1 g of the lungs, livers, spleens, and kidneys were lysed using a high-throughput frozen tissue mill, diluted, and plated on Middlebrook 7H10 plates to count CFUs. And the remaining organs of mice were obtained and fixed with 4% formaldehyde, cut into thin sections, and stained with hematoxylin and eosin for histopathological analysis.

Statistical Analysis

All data are presented as the mean ± the standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, USA). Experimental data were analyzed by a t-test and one-way analysis of variance (ANOVA) in GraphPad Prism 8 software. A P-value of <0.05 was considered statistically significant and denoted as follows: P < 0.05 (*), P < 0.01 (**), P < 0.001­(***).

Results

Transcriptomics and MRA_0776 Target Genes Analysis in MTB H37Ra

To identify gene expression differences between the pMV262-H37Ra and pMV262-MRA_0776-H37Ra strains, we performed Illumina sequencing on cDNA libraries derived from three biological replicates. Analysis showed that the overexpression of MRA_0776 induced significant changes in the expression of 234 genes; of these, 111 genes were up-regulated and 123 genes were downregulated (Figure A) (Table S2). KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment revealed that the genes exhibiting significant changes participate in fatty acid metabolism, glycerolipid metabolism, the two-component system, gluconeogenesis, the citrate cycle (TCA cycle), arginine biosynthesis, and so on (Figure B). The GO result shows that the genes showing significant changes are involved with the cell membrane, membrane part, intrinsic component of membrane, integral component of membrane, and oxidoreductase activity and so on (Figure C). These results suggested that MRA_0776 is likely to be a pleiotropic regulatory protein.

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Transcriptomic analysis of MRA_0776 and target genes analysis. (A) Volcano plot showing the relative abundance of transcripts in the pMV262-MRA_0776-H37Ra strain in comparison with the pMV262-H37Ra strain, p < 0.05. (B) KEGG enrichment, the spectrum from blue to red represents corrected p-values. (C) GO enrichment analysis. Plots of GO term enrichment, the spectrum from blue to red represents corrected p-values. The numbers of genes in the top 30 genes identified by GO and pathway enrichment analysis are listed on the left side of Figure (B) and (C), respectively. (D) Relative gene expression analysis for MRA_0776 target genes with qRT-PCR. The relative expression level is given as fold change of the expression level in pMV262-MRA_0776-H37Ra overexpression strain versus the expression level of pMV262-H37Ra strain. The vertical coordinate represents the fold change. Standard deviations (calculated for three biological replicates) are indicated (n = 3 biological replicates).

Based on the results of the MRA_0776 overexpression transcriptome (Table S2), we selected 20 genes with significant fold changes and important physiological functions as candidates for the qRT-PCR assay. Relative gene expression analysis for MRA_0776 target genes was performed with qRT-PCR. Comparing with the expression level of target genes in pMV262-H37Ra. The eight genes MRA_2046 (homologous with rv2031c, hspX), MRA_2023 (homologous with rv2007c, fdxA), MRA_3165 (homologous with rv3133c, dosR), MRA_0225 (homologous with rv0217c, lipW), MRA_0174 (homologous with rv0166, fadD5), MRA_1826 (homologous with rv1814, erg3), MRA_3465 (homologous with rv3425, ppe57), MRA_0909 (homologous with rv0902c, prrB) were up-regulated, and 12 genes were down-regulated MRA_2553 (homologous with rv2526c, vapB17), MRA_2405 (homologous with rv2382c, mbtC), MRA_0136 (homologous with rv0129c, fbpC), MRA_1606 (homologous with rv1596, nadC), MRA_1021 (homologous with rv1013, pks16), MRA_2178 (homologous with rv2163c, pbpB), MRA_3301 (homologous with rv3260c, whiB2), MRA_2763 (homologous with rv2737c, recA), MRA_1664 (homologous with rv1653, argJ), MRA_2834 (homologous with rv2810c), MRA_0836 (homologous with rv0826), and MRA_1964 (homologous with rv1954c) in pMV262-MRA_0776-H37Ra overexpression strains (Figure D, Table S3).

Validation of Transcriptomic Data by In Vitro DNA-Binding Assays and Conserved Motifs Prediction

Based on the results of the MRA_0776 overexpression transcriptome, we selected 26 promoters with significant fold changes and important physiological functions as candidates for the in vitro binding assay with purified MRA_0776 recombinant protein, which show high amino acids homologous with Rv0767 and MSMEG_5860 (Figure S1). The tested genes contained 19 up-regulated genes including MRA_0174, MRA_0225, MRA_1096 ((2Z,6E)-farnesyl diphosphate synthase), MRA_1826, MRA_3165, MRA_2046, MRA_2023 (fdxA), MRA_0582 (oxidoreductase), MRA_1095 (hemolysin-like protein), MRA_1412 (methyltransferase), MRA_1414 (methyltransferase), MRA_1693, MRA_2440, MRA_2542, MRA_3784 (pe34), MRA_0909, MRA_2228 (adenylyl cyclase), MRA_2667 (rsbV) and MRA_2927 (rnhB) and 7 down-regulated genes including MRA_2554 (antitoxin VapB17), MRA_1973 (mce), MRA_3232 (ATP-Binding protein), MRA_3465, MRA_3559 (coenzyme F420-dependent oxidoreductase), MRA_0773 (cyp51), and MRA_1715 (ppe23). We predicted 0–300 bp upstream of the transcription start site of the gene as the promoter sequence region and used SnapGene for prediction. The primers used for amplifying the promoters of selected target genes are shown in Table S1. EMSA revealed that as protein was added, a DNA–protein complex was formed, thus indicating that MRA_0776 binds with the promoters of targeted genes (Figure , Table S4).

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As shown in Figure , as increasing levels of MRA_0776 protein with the final concentration of 0, 8, 16 μM were added into the reaction mixtures, a stepwise decrease was clearly observed in the levels of shifted DNA, which indicates that MRA_0776 could directly bind with operon promoter regions of target genes. Positions of free DNA (F) and protein-DNA complexes (B) are highlighted. MRA_0050 and MRA_2812 are negative controls.

MRA_0776 Specifically Bound to the dosR Promoter In Vitro and In Vivo

Considering the important role of dosR in the latent infection and physiological activities of MTB, we selected this gene for further analysis. To determine the binding specificity of MRA_0776 to the dosR promoter, we validated the specific competition inhibition by EMSA. A series of unlabeled cold probes, as shown in Figure A, was added to the reaction as competitors. The excess of cold probes bound to the MRA_0776 protein, thus resulting in the reduction of a discernible complex. These results suggested that MRA_0776 could specifically bind to the dosR promoter. To further confirm the binding of MRA_0776 to the dosR promoter in vitro, we performed BLI assays; analysis showed that MRA_0776 bound to the dosR promoter with a KD (dissociation constant) value of 2.882 × 10–6 M (Figure B). These results indicated that MRA_0776 specifically bound to the dosR promoter in vitro. This KD value indicates that the binding ability between MRA_0776 and the dosR promoter is moderate, which falls within the typical range for transcription factor–promoter interactions. And this interaction suggests that MRA_0776 may dynamically regulate the expression of dosR under physiological conditions in response to environmental signals such as hypoxia or metal ions and so on, thereby modulating the dormancy and adaptive responses of MTB. ChIP-PCR results showed that MRA_0776 bound to the dosR promoter in M.tuberculosis H37Ra cells and demonstrated that dosR is the target of MRA_0776 (Figure C).

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MRA_0776 bound with the dosR promoter both in vitro and in vivo. (A) EMSA identification of the specific binding of MRA_0776 with final concentration of 10 μM to the dosR promoter. The addition of a cold probe allowed Cy-5 labeled dosR to be out-competed due to its inability to bind the protein. (B) BLI assay. Biotin-labeled dosR promoter DNA was used to interact with a gradient dilution of MRA_0776. BLI results showed good binding activity with a dissociation constant of 2.882 × 10–6. (C) ChIP-PCR verifying that MRA_0776 bound to the dosR promoter in vivo. M: DNA Marker, Input: supernatant of lysed cells; P: preimmunized antigen serum, I: serum after immunization.

Ligand Specificity of MRA_0776

Next, we screened the small molecules that might affect the binding of MRA_0776 at 10 μM to the dosR promoter using EMSA assays. Analysis showed that Lys, Arg, Tyr, CuCl2, PbCl2, and FeCl3 all inhibited the binding of MRA_0776 to the dosR promoter (Figure ) at various concentrations: Lys and Tyr at 40 mM, Arg at 10 mM, CuCl2 at 2.5 mM, PbCl2 at 5 mM, and FeCl3 at 0.05 mM. Collectively, these results demonstrated that MRA_0776 senses a wide range of ligands to modulate binding affinity.

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Effect of different ligands on MRA_0776 DNA binding to the dosR promoter, as detected by EMSAs. The protein concentration was 10 μM, and the cofactors added are displayed on the top of the figure: (A) Lys; (B) Arg; (C) Tyr; (D) Cu2+; (E) Fe3+; (F) Pb2+.

Structure Prediction and Molecular Docking of MRA_0776

The 3D structure of MRA_0776 was analyzed by Alphafold2 (Figure A); the structure consisted of 10 helices; residues 18–70 formed helices 1–4 and residues 74–204 formed helices 5–10. A Ramachandran plot of the 3D structure of MRA_0776 was computed by using Chimera 1.16. A Ramachandran plot was used to predict the amount of protein (Figure B); the dots in the plot indicate amino acids in the protein; usually the amino group is distributed within the largest contour. We found that all amino acids fell within the high contour region, thus indicating that the predicted structure of MRA_0776 possessed rationality. Detailed intervals of the amino acid residues are shown in Table S5.

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Structural analysis of MRA_0776 modeling results and molecular docking of MRA_0776 with ligands. (A) MRA_0776. (B) Ramachandran plot for MRA_0776. Dark blue dots represent residues in favored regions. 3D binding models between Rv0767 and (C) Arg; (D) Lys; (E) Tyr; (F) Cu2+; (G) Fe3+; and (H) Pb2+. Details of the 3D interactions between ligands and the MRA_0776 protein. The cyan stick indicates small molecule compounds, the orb indicates metal ions, the yellow stick indicates amino acids around small molecules.

Next, the interaction models of ligands (Arg, Lys, Tyr, Fe3+, Cu2+, and Pb2+) and MRA_0776 were analyzed by molecular docking. Arg interacted with Arg-90 of MRA_0776 to form hydrogen bonding, while the carboxyl group on the Arg and Thr-125 of MRA_0776 formed hydrogen bonding (Figure C). The carboxyl group on the ligand Lys bound with Thr-125 and Leu-176 of MRA_0776 to form hydrogen bonding (Figure D). The carboxyl group on the Tyr ligand bound with Thr-125 of MRA_0776 to form hydrogen bonding (Figure E). Cu2+ interacted with the carbonyl group on Thr-125 of MRA_0776 as well as with the phenolic hydroxyl group on Tyr-186 and Gly-121 of MRA_0776 to stabilize the binding of the metal ion and protein (Figure F). The binding site of Fe3+-MRA_0776 was similar to that of the Cu2+–MRA_0776 complex (Figure G). The formation of Fe3+ was observed with the carbon groups on Thr-125 of MRA_0776, as well as the phenolic hydroxyl groups on Tyr-186 and Gly-121, thus providing stabilization. The interaction between Pb2+ and the amide bonds on Ala-127 and Asn-75 of MRA_0776 stabilized the binding of MRA_0776 with Pb2+ (Figure H). In summary, Thr-125 and Tyr-186 were predicted as potential amino acid sites with the highest frequency of interaction with the tested ligands.

In addition, the negative binding energies of the ligands and proteins indicated the possibility of binding. A lower binding score indicates a higher binding affinity (Table S6). As shown in Table S6, all binding energy values were negative, thus indicating that the MRA_0776 protein interacted with the ligands.

MRA_0776 Promoted the Colonization of MTB and Induced Organ Injury in Mice

To investigate whether MRA_0776 plays a role in the colonization of MTB and pathological damage in mice, we used a mouse model of MTB infection. We injected C57BL/6 mice with 5.0 × 106 CFUs of Ms per mouse via tail vein injection.

Colony counts were carried out after the aseptic isolation of liver, spleen, lungs, and kidneys at 4, 7, 14, and 21 d postinfection. Analysis revealed that at 4 and 7 d postinfection, the bacterial burdens in the livers, spleens, lungs, and kidneys of mice in the pMV262-MRA_0776-Ms group were significantly higher (p < 0.05) than that in the pMV262-Ms group (Figure A). At 14 days postinfection, there was no significant difference in the bacterial load in the organs (Figure ). These results suggested that the MRA_0776 protein promoted the colonization of Ms in mouse organs at early postinfection.

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Colony counts in the organs of mice infected with Ms, as determined by colonization assays. (A) Liver; (B) Spleen; (C) Lungs; (D) Kidneys. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), not significant (ns).

To further investigate the organ injury induced by MRA_0776 protein, mice from the pMV262-Ms group and pMV262-MRA_0776-Ms group were dissected 7 days later, and the livers, spleens, lungs, and kidneys of the mice were isolated for pathological analysis (Figure A). Analysis showed that liver hemorrhage was more severe in the pMV262-MRA_0776-Ms group than in the pMV262-Ms group. The spleen was enlarged in the pMV262-MRA_0776-Ms group when compared with the pMV262-Ms group. The pMV262-MRA_0776-Ms infected group had more swollen kidneys than the pMV262-Ms control group (Figure A).

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(A) Macroscopic lesions of mice infected with pMV262-Ms and pMV262-MRA_0776-Ms strains. (B) Histopathology in the organs of mice infected with MTB. The scale bars are 200 μm. Images are pseudocolored representations at a ×200 magnification.

Histopathology (Figure B) revealed a large number of hepatocytes with multifocal necrosis, cytosolic nuclei consolidation, and lysis; a small number of infiltrating lymphocytes were evident in liver tissues from the pMV262-MRA_0776-Ms infection group (red arrows); a large number of hepatocytes with hydropic degeneration were seen (blue arrows), and an increased number of hydropic degeneration was seen in the hepatocytes when compared with the liver tissues of pMV262-Ms. No significant difference was seen in the spleen tissue of the pMV262-MRA_0776-Ms infected group when compared to the pMV262-Ms group. Lung tissues from the pMV262-MRA_0776-Ms infection group showed infiltration of alveolar wall granulocytes; eosinophils were seen in the alveoli (purple arrows); a few leukocytes were seen in the blood vessels (green arrows); and the number of alveolar wall granulocytes was reduced when compared with that of the pMV262-Ms group. Kidney tissues in the pMV262-MRA_0776-Ms infection group mostly showed hydropic degeneration of renal tubular epithelial cells, cellular swelling, and infiltration of lymphocytes and granulocytes; vascular stasis was seen in the interstitium. Compared with kidneys from the pMV262-Ms-infected group, vascular stasis was seen in the interstitium. Collectively, these results suggested that the MRA_0776 protein promoted pathological organ damage in Ms-infected mice. It is important to note that the data relating in vivo colonization and pathology were obtained using a mouse model infected with Ms overexpressing the MRA_0776 protein. Even though this model provides valuable preliminary insights for this transcription regulator, the exact function of MRA_0776 will be deciphered in the MTB infection model in the future.

Discussion

The family of TetR regulators plays important regulatory roles in various physiological activities in bacteria, including antibiotic synthesis, the regulation of surface osmolarity, metabolism, drug resistance, and bacterial pathogenicity. Some bacterial TetR regulators control only a single or a few target genes, whereas others regulate a vast number of genes that are involved in various pathways. Primary transcriptomic analysis of M. tuberculosis H37Ra-pMV262-MRA_0776 revealed that 111 genes were up-regulated and 123 genes were down-regulated. Moreover, the genes exhibiting significant changes in expression were associated with fatty acid metabolism, glycerolipid metabolism, and the two-component system. Our analysis identified 25 new binding sites for MRA_0776 and demonstrated that MRA_0776 is a much more versatile regulator than originally thought. Generally, the family of TetR transcriptional regulators usually acts as repressors to inhibit the transcription of target genes, with some exceptions, acting as activators or playing roles that are unrelated to transcription.3 In our study, qRT-PCR revealed that MRA_0776 can act as a repressor as well as an activator.

In this study, we demonstrated the specific binding of MRA_0776 to the dosR promoter, both in vitro and in vivo. Our analysis demonstrated that MRA_0776 directly regulated the transcription of dosR. dosR is critical for MTB infection and persistence in the host under hypoxic conditions. Our analysis indicates that the regulatory role of DosR in MTB is more complex and extensive than previously thought. DosR regulates amino acid synthesis and metabolism, maintains ribosome stability, induces TLR2 pathway-dependent macrophage activation during hypoxia, , participates in nitrate metabolism and deoxyribonucleotide synthesis processes, and is involved in the adaptation to oxidative stress and the maintenance of redox homeostasis. ,

MTB adapts to environmental changes by sensing small molecules and using transcriptional regulatory proteins to initiate its own regulatory system. The known ligands for the family of TetR transcriptional regulatory proteins are diverse, including bile acids, antibiotics, proteins, fatty acids and their derivatives, cell signaling molecules, carbon sources, and metal ions. − The ability to bind to multiple types of ligands increases the functional diversity of the TetR family of transcriptionally regulated proteins. In a previous in vitro study, Li et al. verified that c-di-GMP could inhibit the DNA-binding activity of HpoR (Ms5860), a homologous protein of MRA_0776. In addition, c-di-GMP could inhibit the binding ability of HpoR with LtmA. Our study identified additional ligands, including Arg, Lys, Tyr, Cu2+, Fe3+, and Pb2+, that can all influence the binding activity of MRA_0776 with the dosR promoter. However, the regulatory mechanisms mediated by ligands with MRA_0776 must be investigated further. Arginine is widely present in both hosts and bacteria and is involved in a variety of physiological processes, and arginine decarboxylation provides a carbon and nitrogen source for the survival of many bacteria. Under vitamin C or isoniazid-induced reactive oxygen species (ROS) stress, MTB initiates the process of arginine de novo synthesis as an early adaptive strategy to resist host antimicrobial functions. In addition, DosR is a novel arginine regulator that can bind to the promoter region of argC (rv1652) to regulate arginine synthesis.

Metal ions are elements that play important roles in all living organisms, exerting catalytic, structural and allosteric roles for many enzymes and transcription factors. Proteins and enzymes require metal ions as cofactors for their life activities or structural modifications and play important roles not only in growth, replication, and cellular metabolism but also in the proliferation of pathogens as well as in host infestation. , Iron and copper are essential for the survival of MTB, are closely related to the pathophysiological metabolism of TB, and are limiting factors for infection. ,

The interactions of the TetR family of transcriptional regulatory proteins with ligands greatly increase the complexity of the process by which they interact with gene promoters. In this study, we predicted the three-dimensional patterns of six ligands to model docking on predicted crystal structures. This analysis revealed that 75-Asn, 90-Arg, 121-Gly, 125-Thr, 127-Ala, 176-Leu, and 186-Tyr were all involved in ligand interactions with MRA_0776, and all seven interacting residues were located in the C-terminal domain of MRA_0776. Because Thr125 and Tyr186 are identified as predicted potential key residue candidates, these predictions remain hypothetical until validated by site-directed mutagenesis and functional assays in future studies.

It is noteworthy that the in vivo infection model employed in this study utilized the pMV262-Rv0767c-Ms overexpress strain. Because Ms shares the high genetic homology with MTB, it can be widely used in animal model to primarily study the protein function of MTB. In this study, MRA_0776 overexpression enhanced colonization and tissue damage in the animal model; it is supposed that it may play as the “virulence factor,” but its precise role will be deciphered and analyzed by using a virulent strain like H37Rv in the future.

In summary, this study establishes MRA_0776 as a pleiotropic regulator that directly controls the dormancy-associated gene dosR and responds to a spectrum of ligands. This study primarily suggested that MRA_0776 overexpression leads to colonization and pathogenicity of MTB. Future work involving knockdown mutants in virulent MTB will be performed to decipher the physical functionality and regulatory role of MRA_0776 and enhance our understanding of the role of transcription regulators. And the deeper studies employing knockout/knockdown mutants in MTB, combined with in vivo investigation of the identified ligands, will be crucial to fully clarify the physiological function of MRA_0776 and to assess its potential as a novel therapeutic target for TB.

Conclusions

In this study, we demonstrated that the TetR family regulator MRA_0776 functions as a versatile regulator in MTB. We identified 25 new binding sites for MRA_0776, revealing its broad regulatory influence on genes associated with fatty acid metabolism, glycerol lipid metabolism, and two-component systems. Crucially, MRA_0776 directly binds to the dosR promoter, an important regulator of MTB hypoxia and dormancy adaptation, latent infection, and oxidative stress responses.

DNA-binding activity of MRA_0776 is modulated by interactions with ligands, including amino acids (Arg, Lys, Tyr) and metal ions (Cu2+, Fe3+, Pb2+). Structural modeling predicted Thr125 as a potential key residue candidate for ligand interaction, and the importance of this finding with regard to binding ability needs to be verified in future studies by point mutation. Additionally, the overexpression of MRA_0776 in Ms enhanced bacterial colonization and virulence, highlighting its potential contribution to virulence.

In conclusion, our analyses demonstrate that MRA_0776 is a pleiotropic regulator, and its overexpression is associated with enhanced colonization and pathological damage, suggesting a potential role in mycobacterial pathogenicity. Future studies should validate its mechanisms in animal models and explore small-molecule inhibitors targeting its ligand-binding domain. This work deepens our understanding of transcription regulator-mediated pathogenesis and provides a foundation for novel antituberculosis strategies. Finally, the observed enhancement of colonization and pathology in an Ms mouse model provides a foundational framework for investigating MRA_0776 physiological function. Future studies employing virulent M. tuberculosis strains are warranted to translate these findings into the context of tuberculosis infection.

Supplementary Material

ao5c10658_si_001.pdf (282.6KB, pdf)

Acknowledgments

The authors appreciate the Key Laboratory of Respiratory Tract Pathogens and Drug Therapy, Shandong Second Medical University, which provided the facilities for this research.

All of the sequence reads have been deposited at NCBl, and can be found below: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1099405/, and PRJNA1099405.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c10658.

  • Amino acid sequence alignment of MRA_0776, Rv0767, and MSMEG_5860; primers used in this study; all significantly changed genes in the MRA_0776 overexpressed strain; expression level of potential regulons of MRA_0776 by qRT-PCR; potential regulons of MRA_0776 by EMSA; detailed intervals of amino acid residues of MRA_0776; and binding energy between ligands and MRA_0776 (PDF)

N.S.: Conceptualization, funding acquisition, supervision, writingoriginal draft preparation, writingreviewing and editing. Z.L.: Validation, writingreviewing and editing. S.W.: Methodology, data curation, writingoriginal draft preparation, writingreviewing and editing. L.Z.: Writingreviewing and editing. R.W.: Writingreviewing and editing. H.W.: Software, writingreviewing and editing. M.S.: Writingreviewing and editing. Y.Z.: Writingreviewing and editing. J.X.: Methodology, writingreviewing and editing. R.F.: Formal analysis, writingreviewing and editing. X.L.: Writingreviewing and editing. Y.W.: Writingreviewing and editing. All authors reviewed the final version, read and approved the finally submitted manuscript.

This work was supported by the Shandong Provincial Natural Science Foundation (No. ZR2024MC10), Youth Innovation Team Project for Talent Introduction and Cultivation in Universities of Shandong Province, China (No. 05430201); Weifang Special expert talent program (No. 00000710), Weifang science and technology development program (No. 2024JZ0014), and by the National Natural Science Foundation of China (No. 31873014).

Ethics Approval: Animal experiments were performed in strict accordance with the animal protocols approved by the Ethical Committee of Animal Experiments of Shandong Second Medical University (Ethical Committee Approval number 2022SDL109).

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao5c10658_si_001.pdf (282.6KB, pdf)

Data Availability Statement

All of the sequence reads have been deposited at NCBl, and can be found below: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1099405/, and PRJNA1099405.

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