ABSTRACT
Novel anti-tuberculosis drugs are essential to manage drug-resistant tuberculosis, caused by Mycobacterium tuberculosis. We recently reported the antimycobacterial activity of chrysomycin A in vitro and in infected macrophages. In this study, we report that it inhibits the growth of drug-resistant clinical strains of M. tuberculosis and acts in synergy with anti-TB drugs such as ethambutol, ciprofloxacin, and novobiocin. In pursuit of its mechanism of action, it was found that chrysomycin A is bactericidal and exerts this activity by interacting with DNA at specific sequences and by inhibiting the topoisomerase I activity of M. tuberculosis. It also exhibits weak inhibition of the DNA gyrase enzyme of the pathogen.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Aminoglycosides , Antitubercular Agents/pharmacology , DNA Topoisomerases, Type I , Humans , Microbial Sensitivity TestsABSTRACT
Rv1019, a member of an uncharacterized tetracycline resistance regulator family of transcriptional regulators of Mycobacterium tuberculosis H37Rv, was found to be differentially expressed during dormancy and reactivation in vitro. In this study, we show that this protein binds to its own promoter and acts as a negative regulator of its own expression. It forms dimers in vitro which is essential for the DNA binding activity. We also show that Rv1019 and downstream genes Rv1020 (mfd) and Rv1021 (mazG) are cotranscribed. Constitutive expression of Rv1019 in M. smegmatis downregulated MSMEG_5423 (mfd) and MSMEG_5422 (mazG), suggesting that Rv1019 negatively regulates these downstream genes which encode key proteins involved in DNA repair. M. smegmatis expressing Rv1019 was found to be sensitive to DNA-damaging environments, suggesting its role in regulating the DNA damage response in mycobacterium.
Subject(s)
Bacterial Proteins/genetics , DNA Repair , DNA, Bacterial/genetics , Gene Expression Regulation, Bacterial , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/genetics , Operon , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Cloning, Molecular , DNA Damage , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Hydrogen Peroxide/pharmacology , Models, Molecular , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tetracycline/chemistry , Tetracycline/pharmacology , Tetracycline Resistance/genetics , Transcription, GeneticABSTRACT
We characterize Rv0474, a putative transcriptional regulatory protein of Mycobacterium tuberculosis, which is found to function as a copper-responsive transcriptional regulator at toxic levels of copper. It is an autorepressor, but at elevated levels (10-250 µm) of copper ions the repression is relieved resulting in an increase in Rv0474 expression. Copper-bound Rv0474 is recruited to the rpoB promoter leading to its repression resulting in the growth arrest of the bacterium. Mutational analysis showed that the helix-turn-helix and leucine zipper domains of Rv0474 are essential for its binding to Rv0474 and rpoB promoters, respectively. The mechanism of Rv0474-mediated rpoB regulation seems to be operational only in pathogenic mycobacteria that can persist inside the host.
Subject(s)
Bacterial Proteins/genetics , Copper/pharmacology , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Gene Expression Regulation, Bacterial/drug effects , Mycobacterium tuberculosis/metabolism , Transcription Factors/genetics , Amino Acid Sequence , Bacterial Proteins/metabolism , Humans , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Promoter Regions, Genetic , Sequence Homology , THP-1 Cells , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Rv3334 protein of Mycobacterium tuberculosis belongs to the MerR family of transcriptional regulators and is upregulated during hypoxia and other stress conditions. Employing GFP reporter constructs, mobility shift assays and ChIP assays, we demonstrate that Rv3334 binds to its own promoter and acts as an autorepressor. We were able to locate a 22 bp palindrome in its promoter that we show to be the cognate binding sequence of Rv3334. Using chase experiments, we could conclusively prove the requirement of this palindrome for Rv3334 binding. Recombinant Rv3334 readily formed homodimers in vitro, which could be necessary for its transcriptional regulatory role in vivo. Although the DNA-binding activity of the protein was abrogated by the presence of certain divalent metal cations, the homodimer formation remained unaffected. In silico predictions and subsequent assays using GFP reporter constructs and mobility shift assays revealed that the expression of ketosteroid regulator gene (kstR), involved in lipid catabolism, is positively regulated by Rv3334. ChIP assays with aerobically grown M. tuberculosis as well as dormant bacteria unambiguously prove that Rv3334 specifically upregulates expression of kstR during dormancy. Our study throws light on the possible role of Rv3334 as a master regulator of lipid catabolism during hypoxia-induced dormancy.
Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Mycobacterium tuberculosis/genetics , Repressor Proteins/metabolism , Bacterial Proteins/genetics , DNA/metabolism , Inverted Repeat Sequences , Metals, Heavy/metabolism , Mycobacterium tuberculosis/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Multimerization , Up-RegulationABSTRACT
We isolated an 8 kDa mycobacterial hypothetical protein, Rv3423.1, from the chromatin of human macrophages infected with Mycobacterium tuberculosis H37Rv. Bioinformatics predictions followed by in vitro biochemical assays with purified recombinant protein showed that Rv3423.1 is a novel histone acetyltransferase that acetylates histone H3 at the K9/K14 positions. Transient transfection of macrophages containing GFP-tagged histone H1 with RFP-tagged Rv3423.1 revealed that the protein co-localizes with the chromatin in the nucleus. Co-immunoprecipitation assays confirmed that the Rv3423.1-histone interaction is specific. Rv3423.1 protein was detected in the culture filtrate of virulent but not avirulent M. tuberculosis. Infection of macrophages with recombinant Mycobacterium smegmatis constitutively expressing Rv3423.1 resulted in a significant increase in the number of intracellular bacteria. However, the protein did not seem to offer any growth advantage to free-living recombinant M. smegmatis. It is highly likely that, by binding to the host chromatin, this histone acetyltransferase from M. tuberculosis may manipulate the expression of host genes involved in anti-inflammatory responses to evade clearance and to survive in the intracellular environment.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Histone Acetyltransferases/metabolism , Mycobacterium tuberculosis/enzymology , Acetyl Coenzyme A/chemistry , Bacterial Proteins/chemistry , Chromatin/metabolism , Computer Simulation , Gene Expression Regulation, Bacterial , Histone Acetyltransferases/chemistry , Histone Acetyltransferases/genetics , Histones/metabolism , Humans , Macrophages/microbiology , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/pathogenicity , NAD/metabolism , Protein ConformationABSTRACT
Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic intervention to prevent reactivation of latent tuberculosis.
