M. tuberculosis (MTB) is a facultative intracellular pathogen that can reside within professional phagocytes such as macrophages and dendritic cells in the infected host. A number of virulence genes are known to be induced in intracellular bacteria at different stages following uptake by the host. Gene expression is altered in the host cells as well during infection. Our major interest is to understand how intracellular MTB regulates host gene expression. Chromatin modifications play a major role in the regulation of eukaryotic gene expression. Acetylation of histones is a major epigenetic modification and is maintained by two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs are generally associated with upregulation of gene expression, and HDACs, with repression of gene expression. Intracellular bacteria can manipulate host defense gene expression by epigenetic modifications to facilitate infection and survival inside the host cell.
Recently we have shown that Rv3423.1 of MTB is a histone acetyltransferase, secreted by intracellular bacteria, that directly enters the nucleus of macrophages and acetylates histone H3 at K9/K14 positions (Jose et al., 2015). Employing ChIP seq, we have identified some genes in the infected macrophages to whose promoters this HAT is recruited to upregulate their expression.
In collaboration with Dr Ramandeep Singh (THSTI, Faridabad) we have generated a knockout strain of MTB that lacks Rv3423.1 gene (DST-funded project). Infection of macrophages and guinea pig with wild-type and knockout strains is expected to give us an idea about the role played by this gene product during infection.
As mentioned before, while histone acetyltransferases add acetyl moieties to histones, HDACs remove them! We have shown that MTB infection causes significant upregulation of histone deacetylase 1 (HDAC1) in macrophages. Many host genes are known to be repressed during MTB infection. We have shown that some genes whose products play a key role in the initiation of Th1 responses following MTB infection are repressed by the recruitment of HDAC1 to their promoters. We demonstrated that HDAC1 hypoacetylates histone H3 at the promoter of IL-12B gene leading to the downregulation of its expression in macrophages infected with live, virulent MTB H37Rv, and not in those infected with MTB H37Ra or heat-killed MTB H37Rv (Chandran et al., 2015). Significantly our study showed that c-Jun is essential for HDAC1 expression, and is recruited to the promoter of HDAC1 gene only in macrophages infected with live MTB H37Rv. Our goal is to understand how HDAC1 is recruited to the target promoters. We are currently studying the functional significance of a few proteins (of MTB and the host) that we found to associate with HDAC1 during infection. Hopefully our studies will lead to the identification of novel targets in the host (host-directed therapy) and the pathogen for therapeutic intervention to fight TB.

MTB is capable of remaining in a state of dormancy in the infected host for a long time, leading to latent TB infection (LTBI). One of the reasons why TB still remains a major challenge is the ability of MTB to remain dormant in the body. The dormant bacilli are resistant to most of the anti-TB drugs, and become activated when the immune system of the body becomes weak. This is a major hindrance to TB control programmes. We carried out a comparative proteomic analysis to identify proteins that are differentially expressed during dormancy and reactivation of MTB. We made actively growing MTB dormant using Wayne's dormancy model, and developed a simple system to reactivate dormant bacteria. We extracted proteins at different stages of dormancy and reactivation, and carried out a label-free, one-dimensional LC-MS/MS analysis. Proteins were isolated from normoxic control, two phases of dormancy, and two phases of reactivation. One thousand, eight hundred and seventy one proteins (47% of the MTB proteome) were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. We analyzed various biological functions during these conditions. Fluctuations in the relative quantities of proteins involved in various metabolic processes such as energy metabolism, amino acid metabolism, lipid biosynthesis, lipid biodegradation, DNA replication, repair, transcription, protein synthesis etc. were observed during dormancy and reactivation (Gopinath et al., 2015). We are interested in proteins that are uniquely expressed or upregulated during reactivation, and have selected a few hypothetical proteins that are predicted to be transcriptional regulators for further studies. Functional and structural characterization of these proteins are essential to enhance our understanding of the biology of MTB which will enable us to devise more effective strategies to combat tuberculosis, especially to prevent reactivation of latent tuberculosis.
One such transcriptional regulator that we characterized recently is Rv0474 which was shown to be a copper-responsive transcriptional regulator that negatively regulates expression of RNA polymerase subunit in Mycobacterium tuberculosis (FEBS J. 2018 Oct; 285(20):3849-3869).
Another mycobacterial transcriptional regulator that we characterized is Rv3334. It is a multiple stress responsive transcriptional regulator and we showed that it is an auto-repressor and a positive regulator of kstR. (FEBS J. 283(16):3056-71).

Despite the availability of effective anti-TB drugs and BCG vaccine, the incidence of TB has increased at an alarming rate in the past few decades. Currently BCG is the only approved vaccine against TB. However, it is found to be less effective in preventing the disease in adults, and the efficacy is relatively low in tropical countries. Long duration of treatment, and emergence of multidrug resistant (MDR), extensively drug-resistant (XDR) and recently of totally drug-resistant (TDR) strains of MTB render the current mode of treatment of TB very difficult, prohibitively expensive and often completely ineffective. The situation is made worse by the co-infection with HIV. This scenario calls for the development of novel and more effective anti-TB drugs at least until better vaccines or other forms of treatment are available. With financial support from OSDD/CSIR, we have screened hundreds of actinomycetes isolated from different parts of Kerala and Tamil Nadu for their inhibitory activity against MTB. Employing different chromatographic techniques we isolated a potent antimycobacterial molecule (Chrysomycin A) from a Streptomyces sp. which was found to have an MIC of 3.125 microgram/ml on virulent laboratory strain of MTB in vitro (Muralikrishnan et al., 2017). It is equally effective against intracellular MTB too. Identification of its target and the mode of action are under way.

Isoniazid (INH) is a first-line anti-TB drug. Actually it is a prodrug that is converted into its active form (INH-NADH adduct) with the help of bacterial KatG protein. While actively dividing Mtb is sensitive to INH, dormant Mtb is resistant to this drug. It has been believed that this is due to the inability of the dormant bacterium to convert the prodrug into its active form. However we have shown that this is not the case. Dormant bacterium can indeed convert INH into its active adduct (The Journal of Antibiotics. 05 September 2018). The real reason for dormant bacterium to be INH resistant is something else!
INH interferes with the mycolic acid biosynthesis which in turn inhibits the formation of a healthy mycobacterial cell wall. Usually INH resistant Mtb strains will have mutations in katG, inhA, ahpC, kasA, and ndh genes. However there are INH resistant strains which do not have mutations in any of these genes. We have found that Mtb can acetylate INH and can render it inactive. We propose that acetylation is an alternative mechanism of INH resistance at least in some cases.