Chrysomycin A inhibits the topoisomerase I of Mycobacterium tuberculosis.

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.

Transcription Repressor Protein ZBTB25 Associates with HDAC1-Sin3a Complex in Mycobacterium tuberculosis-Infected Macrophages, and Its Inhibition Clears Pathogen by Autophagy.

Downregulation of host gene expression is a key strategy employed by intracellular pathogens for their survival in macrophages and subsequent pathogenesis. In a previous study, we have shown that histone deacetylase 1 (HDAC1) levels go up in macrophages infected with Mycobacterium tuberculosis, and it hypoacetylates histone H3 at the promoter of IL-12B gene, leading to its downregulation. We now show that after infection with M. tuberculosis, HDAC1 is phosphorylated, and the levels of phosphorylated HDAC1 (pHDAC1) increase significantly in macrophages. We found that transcriptional repressor protein zinc finger and BTB domain 25 (ZBTB25) and transcriptional corepressor Sin3a associate with the HDAC1 silencing complex, which is recruited to the promoter of IL-12B to downregulate its expression in infected macrophages. Knocking down of ZBTB25 enhanced release of IL-12p40 from infected macrophages. Inhibition of HDAC1 and ZBTB25 promoted colocalization of M. tuberculosis and LC3 (microtubule-associated protein 1A/1B-light chain 3) in autophagosomes. Induction of autophagy resulted in the killing of intracellular M. tuberculosis Enhanced phosphorylation of JAK2 and STAT4 was observed in macrophages upon treatment with HDAC1 and ZBTB inhibitors, and inhibition of JAK2/STAT4 negated the killing of the intracellular pathogen, suggesting their role in the autophagy-mediated killing of intracellular M. tuberculosis In view of the emergence of drug resistance in M. tuberculosis, host-directed therapy is an attractive alternative strategy to combat tuberculosis (TB). HDACs have been proposed to be host targets for TB treatment. Our study indicates that ZBTB25, a functional subunit of the HDAC1/Sin3a repressor complex involved in IL-12B suppression, could be an alternative target for host-directed anti-TB therapy.IMPORTANCE Following infection with M. tuberculosis, levels of HDAC1 go up in macrophages, and it is recruited to the promoter of IL-12B where it hypoacetylates histone H3, leading to the downregulation of the gene. Here, we show that host transcriptional repressor protein ZBTB25 and transcriptional corepressor Sin3a associate with HDAC1 in the silencing complex. Knocking down of ZBTB25 prevented the recruitment of the complex to the promoter and consequently enhanced the gene expression and the release of IL-12p40 from infected macrophages. Pharmacological inhibition of ZBTB25 in infected macrophages resulted in the induction of autophagy and killing of intracellular M. tuberculosis Drug-resistant TB is a serious challenge to TB control programs all over the world which calls for finding alternative therapeutic methods. Host-directed therapy is gaining significant momentum in treating infectious diseases. We propose that ZBTB25 is a potential target for host-directed treatment of TB.

Comparative label-free lipidomic analysis of Mycobacterium tuberculosis during dormancy and reactivation.

Mycobacterium tuberculosis employs several strategies to combat and adapt to adverse conditions encountered inside the host. The non-replicative dormant state of the bacterium is linked to drug resistance and slower response to anti-tubercular therapy. It is known that alterations in lipid content allow dormant bacteria to acclimatize to cellular stress. Employing comparative lipidomic analysis we profiled the changes in lipid metabolism in M. tuberculosis using a modified Wayne's model of hypoxia-induced dormancy. Further we subjected the dormant bacteria to resuscitation, and analyzed their lipidomes until the lipid profile was similar to that of normoxially grown bacteria. An enhanced degradation of cell wall-associated and cytoplasmic lipids during dormancy, and their gradual restoration during reactivation, were clearly evident. This study throws light on distinct lipid metabolic patterns that M. tuberculosis undergoes to maintain its cellular energetics during dormancy and reactivation.

Dormant Mycobacterium tuberculosis converts isoniazid to the active drug in a Wayne's model of dormancy.

Isoniazid (INH) is one among the four first-line drugs used in the treatment of tuberculosis. The bactericidal activity of INH is due to its ability to inhibit mycolic acid synthesis, which is an integral component of the mycobacterial cell wall. Non-replicating Mycobacterium tuberculosis (MTB) is phenotypically resistant to INH. The exact mechanism of this resistance is not clear, although the inability of dormant MTB to convert the pro-drug into an active form is thought to be one of the possible reasons. Employing targeted metabolomics approach, we show that dormant MTB can metabolize INH into its active INH-NAD+ adduct form. Further we show that the dormant bacteria have unaltered gene expression levels of katG and inhA (INH metabolizing enzymes). Transcript levels of drug efflux pump proteins which were low during dormancy did not increase in response to INH treatment. These findings point to an alternative mechanism for INH resistance in dormant MTB, which needs to be further elucidated.

Rv0474 is a copper-responsive transcriptional regulator that negatively regulates expression of RNA polymerase ß subunit in Mycobacterium tuberculosis.

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.

The multiple stress responsive transcriptional regulator Rv3334 of Mycobacterium tuberculosis is an autorepressor and a positive regulator of kstR.

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.

Hypothetical protein Rv3423.1 of Mycobacterium tuberculosis is a histone acetyltransferase.

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.

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation.

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.