Research Projects

Mycobacterium tuberculosis (Mtb) is a pulmonary pathogen that spreads through aerosol droplets and can remain inside the host in an active or dormant form. During infection, mycobacteria thrive inside tissue macrophages and withstand significant amounts of acidic, starvation, hypoxic, nitrosative, and oxidative stress (1). To successfully infect a host, the pathogen must overcome all the stresses by activating its defense mechanisms, achieved by changes in its transcriptome (2). Mycobacterium has a genome size of 4.4 million bases that encodes ~4000 genes (3). Under different stresses, more than a thousand genes are differentially expressed (4), which could explain the pathogen's ability to adapt upon infection. In prokaryotes, the transcription process has three steps: initiation, elongation, and termination. The most crucial regulatory step is the initiation of transcription, which involves the core RNAP and the initiation factor Sigma (σ). Sigma factors determine promoter specificity and initiate transcription from associated promoters. Unlike E. coli, Mtb encodes for 13 sigma factors, of which 12 are non-essential for bacterial growth and survival in vitro. These sigma factors are divided into 4 groups; Group 1 comprises σA, Group 2 has σB, Group 3 consists of σF, rest of the sima factors are in group 4, which are also known as extracellular function (ECFs) sigma factors (4,5). Although few studies have identified some promoters that are recognized by most of the ECFs sigma factors, a comprehensive study detailing the transcriptional regulation under different stress conditions has not been performed. The project aims to delineate how the ECF sigma factors help Mtb modulate its transcription network under stress conditions. We aim to generate or obtain gene replacement mutants for all the ECFs and evaluate growth profiles under different in vitro stress conditions. We would perform RNAseq, whole-cell proteomics, and Chromatin Immuno-precipitation followed by sequencing (ChIP-seq) experiments under appropriate stress conditions. The RNAseq data would provide the transcriptome profile change. In contrast, the ChIP-seq data would provide information on the promoters occupied by the sigma factors under normal growth and stress growth conditions. We would also perform interactome experiments to identify other accessory factors that associate with the sigma factor under different growth conditions. Finally, we will evaluate the ex vivo and in vivo infections to determine their role in intracellular survival in the host.