| dc.description.abstract | DNA encounters topological constraints during essential cellular processes such as replication, transcription, and chromosome segregation. To ensure facile execution of these processes, a ubiquitous class of enzymes known as topoisomerases play a crucial role. Topoisomerases resolve topological constraints by introducing transient breaks in the DNA strands, changing the topology, and subsequently resealing the breaks. They are classified broadly as type I and type II based on their structure and mechanism. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a formidable global health challenge claiming millions of lives annually. This pathogen possesses a single representative of both type I and type II topoisomerases. Earlier studies from the laboratory have underscored the indispensability of both the topoisomerases in mycobacteria. While DNA gyrase, a type II topoisomerase, has been extensively exploited as a target for antibacterial agents, the landscape of compounds specifically targeting bacterial topoisomerase I (TopoI) remains limited. The emergence of drug resistance undermines the potential of known anti-tuberculosis drugs, necessitating additional studies and identification of more inhibitors with therapeutic potential. The limited repertoire of topoisomerases in M. tuberculosis, their essentiality for the pathogen’s survival, and validation as candidates for drug discovery provides an opportunity to exploit them in new drug discovery efforts. The previous studies in the Nagaraja laboratory revealed distinct properties of DNA gyrase from mycobacteria compared to gyrases from other bacteria. Given the difference in their properties, it is likely that the assembly of the holoenzyme from the subunits is also different in mycobacteria that has not been studied in detail previously. Hence, I have investigated the assembly of DNA gyrase from M. tuberculosis a pathogenic species and Mycobacterium smegmatis, a fast-growing non-pathogenic species belonging to the genus Mycobacterium which is often used as a surrogate for M. tuberculosis. The results show that in addition to the distinct structural, biochemical, and regulatory features of DNA gyrase from mycobacteria, the enzyme assembly also follows a different pattern than what is seen in other bacteria such as Escherichia coli. Moreover, efforts were made to identify and characterize new inhibitors against mycobacterial topoisomerases, aiming to bridge this therapeutic gap and potentially contribute to the development of novel antimicrobial strategies.
Part I : Distinct subunit architecture and assembly pattern of DNA gyrase from mycobacteria
In this study, the assembly of mycobacterial DNA gyrase from its individual subunits was investigated. This study reveals that, GyrA from M. tuberculosis and M. smegmatis forms tetramers (A4) in solution unlike in E. coli and other bacteria where GyrA exists as a dimer. GyrB, however, persists as a monomer, resembling the pattern found in E. coli. GyrB in both mycobacterial species interacts with GyrA and triggers the dissociation of the GyrA tetramer to facilitate the formation of catalytically active A2B2. Despite oligomerization, the GyrA tetramer retained its DNA binding ability, and DNA binding had no effect on GyrA's oligomeric state in both the species. Moreover, the presence of DNA facilitated the assembly of holoenzyme in the case of M. smegmatis by stabilizing the GyrA2B2 tetramer but with little effect in M. tuberculosis. Thus, in addition to the distinct organization and regulation of the gyr locus in mycobacteria, the enzyme assembly also follows a different pattern.
Part II: Selective inhibition of mycobacterial topoisomerase I cleavage and strand passage activity
In this part, a monoclonal antibody (mAb) was characterised (from a previously generated pool) that specifically inhibits mycobacterial TopoI by a novel mechanism. This mAb did not cross-react with or inhibit E. coli TopoI (Ec TopoI). Among different steps of TopoI reaction cycle, mAb did not interfere with the DNA binding step. Instead, it selectively impeded the cleavage step of TopoI. Additionally, the mAb did not alter the religation property of TopoI, however, it inhibited the strand passage. Further, the variable regions of the mAb were sequenced using 5’ RACE (rapid amplification of cDNA ends) method and reconstructed into a single chain variable fragment (ScFv) expression construct. The ScFv was expressed and purified from E. coli C43 cells using two different strategies, which exhibited efficient inhibitory activity similar to the full length mAb. With its selective and specific inhibitory properties, this mAb can serve as a useful molecular tool to understand the biochemical as well as structural aspects of mycobacterial TopoI and help in designing the peptide inhibitors with therapeutic potential.
Part III: Preliminary structural studies of M. tuberculosis TopoI-Fab complex
Here, I have purified the antigen binding fragment (Fab) of the mAb (described in chapter 3) and evaluated for its ability to retain the inhibitory properties of the full-length antibody. Subsequently, this Fab was employed in structural studies of M. tuberculosis TopoI (Mt TopoI). The study details the optimization of a protocol for purifying a stable ternary complex comprising Mt TopoI, DNA, and Fab for cryo-electron microscopy (cryo-EM) investigations. These efforts are a substantial progress towards unravelling the structural details of Mt TopoI and elucidating epitope-paratope interactions, as well as the understanding of the inhibitory mechanism of cleavage and strand passage activities of Mt TopoI by the mAb. We surmise that the epitope-paratope interaction details will serve as a template to design peptide inhibitors against this essential enzyme. | en_US |
