Exploring the roles of nucleoid-associated protein HU and two of its interacting proteins in Mycobacterium tuberculosis pathogenesis

Abstract

Embargo up to Jan 29, 2026

The genome in prokaryotes is restricted to a membrane-less compartment termed nucleoid. The genome is maintained in the compacted state within the nucleoid with the help of three key cellular factors viz, macromolecular crowding, DNA supercoiling, and DNA binding by nucleoid-associated proteins (NAPs). Due to their analogous role in genome compaction, NAPs are referred to as the prokaryotic counterparts of histones. NAPs are small basic proteins that help genome organization by virtue of their various DNA binding modes like bridging, wrapping, coating, and bending. A change in cellular concentrations of NAPs or their post-translation modification (PTM) during different growth phases of bacteria can lead to a change in genome compaction. The dynamic changes in genome compaction are required during cellular processes like replication, transcription, repair, etc. NAPs also contribute to maintaining control over gene expression, which is crucial for any pathogenic bacteria to adapt to the varying environments encountered when infecting its host. Therefore, understanding the regulons of NAPs is necessary to better comprehend their role in fine-tuning the transcriptomic network and pathophysiology of the bacteria. In this work, the cellular processes controlled by a mycobacterial NAP, HU (MtHU), have been identified, and its role in Mycobacterium tuberculosis (Mtb) pathogenesis. Additionally, the characterization of MtHU interacting proteins and their functional significance is investigated in the present thesis. The first chapter of the thesis provides a general introduction to tuberculosis disease, its epidemiology, and the current drug regime. It also includes an overview of Mtb, the causative agent of tuberculosis, its cellular features, pathogenesis, and mode of survival within the host. Further, the role of mycobacterial nucleoid-associated proteins as genome organizers and global gene expression regulators has been presented. Compared to other well-studied bacteria, there is an underrepresentation of NAPs in Mtb, which may impose additional responsibility on Mtb NAPs, rendering them more valuable in Mtb compared to other bacteria. Unlike 16-18 NAPs in other well-studied bacteria, only 7 NAPs have been identified in Mtb, and 4 among these NAPs are essential, indicating their greater importance in Mtb pathophysiology. One of these essential NAPs is MtHU. Its biological importance has been investigated with the help of a conditional knockdown (cKD) strain and MtHU-specific inhibitor and is presented in the second chapter. MtHU-depleted cells displayed growth retardation in vitro and compromised survival inside macrophages. Analysis of transcriptomic data suggests that MtHU is a global regulator. It controls the expression of various cellular pathways that could account for the phenotypic changes in the cKD strain. Also, the altered expression of virulence factors in the cKD strain contributes to its clearance from infected macrophages and mice, indicating that MtHU serves as a virulence factor. Hence, MtHU targeting inhibitor SD1 can be refined further to be used in combination with anti-TB drugs against Mtb. In the third chapter, the role of one of the MtHU interacting proteins, Rv3816c, has been evaluated. Rv3816c has been found to possess all four characteristic motifs of acylglycerol-3-phosphate acyltransferase (AGPAT), exist as a membrane-bound enzyme, and function as 1-acylglycerol-3-phosphate acyltransferase. The enzyme can transfer the acyl group to acylglycerol-3-phosphate (LPA) to produce phosphatidic acid by utilizing monounsaturated fatty acyl-coenzyme A of chain lengths 16 or 18. It can complement the Escherichia coli PlsC mutant, indicating its functions as AGPAT in vivo. Its active site mutants were incapable of performing AGPAT reaction and rescuing the growth defect of E. coli PlsC mutant. Taken together, it has been demonstrated that Rv3816c, although do not modify MtHU, performs an important catalytic step in the triacylglycerol (TAG) synthesis pathway in Mtb. The product synthesized by Rv3816c is an important branchpoint intermediate for the synthesis of TAG and phospholipids. TAG is vital to mycobacteria, serving as a cell envelope component and energy reservoir during latency. This makes Rv3816c a valuable enzyme for Mtb. In the fourth chapter, the functional characterization of another MtHU interacting protein, Rv0731c, is presented. Through a combination of biochemical and structural approaches, it has been demonstrated that Rv0731c is a fatty acid modifying enzyme that selectively methylates the carboxylate moiety of long-chain fatty acids (LCFA), revealing its catalytic promiscuity. High-resolution crystal structure analysis revealed that Rv0731c acts as a directional molecular ruler in guiding only cognate LCFAs into the active site while restricting M/SCFA from reaching the catalytic center of the enzyme. Additionally, the interaction of Rv0731c with MtHU could be a moonlighting function where Rv0731c interferes with the methylation of MtHU from host methyltransferase. It is demonstrated that the knockout of Rv0731c does not render Mtb incompetent in its intracellular survival or drug susceptibility due to the presence of redundant carboxy-methyltransferases (CMTs) homologous to Rv0731c. However, the products synthesized by Rv0731c (methylated fatty acids) are capable of shifting Mtb-infected macrophages from M1 to M2 phenotype, suggesting that CMTs are helpful to Mtb to better survive inside the macrophage. In conclusion, the work embodied in my thesis has enabled an understanding of how the highly abundant and essential NAP MtHU regulates gene expression in Mtb. Also, by characterizing two of MtHU’s interacting proteins, this work has uncovered the importance of the large repertoire of modification enzymes possessed by Mtb.