Research Projects

Tuberculosis (TB) is one of the oldest diseases known to mankind, however, it continues to pose new challenges to researchers worldwide. The emergence of drug-resistant TB and HIV/TB co-infection has surpassed the current treatment modalities and calls for development of new drugs. To strengthen the current drug regimen, research on Mycobacterium tuberculosis (Mtb), needs to be done in a new perspective/paradigm. The role of epigenetics in Mtb-host interaction might prove to be beneficial in this regard. Hemavathy et al in 1995, reported presence of 6-methyl adenine and 5-methyl cytosine in Mtb genome by HPLC. While 6-methyl adenine modification is linked to restriction modification systems that form innate immune mechanisms of bacterium, the actual biological function of 5-methyl cytosine in Mtb remains elusive. In our previous work, we have found CpG methylation in Mtb genomic DNA using Oxford Nanopore sequencing (unpublished data). All these changes make Mtb epigenome highly dynamic. It was found that the DNA methylation landscape changes as Mtb adapts to intracellular lifestyle in the host. The CpG DNA methylation signature from Mtb isolated post infection in THP-1 macrophages was significantly different from the signature in in vitro grown Mtb. The genes belonging to intermediary metabolism and respiration category showed the highest number of CpG methylation sites in the intraphagosomal bacteria. Careful dissection of the data revealed that most of these genes belonged to alternative Carbon metabolism category. From the previously published studies, we know that Mtb undergoes massive transcriptome changes when it is inside the host. We propose that Mtb epigenome might be key driver for these transcriptome changes. In this project, we aim to find the 5’methyl cytosine methyltransferase/es, which are responsible for change in CpG landscape of Mtb when it is inside the host. Targeting methyltransferase/es, will prove beneficial as it will make Mtb crippled, and it would not be able to adapt inside the host and will be cleared from the host immune system. The main strategy is to first select, annotated DNA/RNA/possible and hypothetical methyl transferases of Mtb. CRISPR knockdowns will be generated for these putative methyltransferases. The THP1 macrophages will be infected with BSL2 safe strains of Mtb and specific knockdown will be selected for whole genome bisulfite sequencing in intracellular bacteria and comparison of CpG methylation in the wildtype and the knockdown strain. Specific enzyme assays will be performed to validate the enzymatic activity. Infection studies will be performed with the wildtype and the knockdown strain to see whether lack of CpG methylation leads to changes in the virulence of Mtb. This study will help us to find out the hitherto unknown 5’ methyl cytosine methyltransferase of Mtb, which is crucial in priming the bacterium for an intracellular life in the host.