Valakunja Nagaraja

Histone H3 lysine 79 (H3K79) methylation catalysed by methyltransferase (MTase) DOT1L is involved in maintaining chromatin organization and regulation of gene expression to mount proinflammatory response upon pathogen infection. Rv2067c, a Mycobacterium tuberculosis MTase also trimethylates H3K79 at loci distinct from those of DOT1L to regulate the expression of several different genes. Apart from upregulating SESTRIN3, TMTC1, and NLRC3, which regulate reactive oxygen species, calcium homeostasis and NF-κB signaling respectively it also represses DOT1L expression. The DOT1L repression and the consequent decrease in H3K79me3 is likely due to deposition of repressive mark H3K9me3 at the DOT1L promoter during M. tuberculosis infection. However, the mechanism by which the repression of the host MTase is brought about by Rv2067c is unknown. Here, we have elucidated the mechanisms of DOT1L repression by Rv2067c. We demonstrate that Rv2067c interacts with the host epigenetic regulator HP1γ, facilitating the recruitment of H3K9 MTase SUV39H1 to the DOT1L promoter to add the repressive H3K9me3 mark. Parallelly, SUV39H1 methylates DOT1L decreasing its catalytic proficiency towards H3 methylation. This combinatorial action effected by Rv2067c essentially ensures the attenuation of proinflammatory response directed by DOT1L upon M. tuberculosis infection.

SUMMARY: DNA topoisomerases are essential for maintaining the integrity and functionality of the genome. By homeostatically controlling the genome topology in bacteria, these enzymes influence essential cellular processes such as gene expression, replication, recombination, and chromosome segregation. Although DNA topoisomerases were initially considered to act as stand-alone enzymes, they are now being recognized as integral components of dynamic protein complexes that perform diverse functions in vivo . The discovery of several proteinaceous modulators of DNA topoisomerases has expanded the understanding of their functional versatility. These are encoded either by the bacterial chromosome or extrachromosomal elements like plasmids and bacteriophages. Chromosomally encoded modulators are engaged in fine-tuning topoisomerase activity, enabling them to carry out specialized cellular functions beyond their canonical enzymatic roles. Crucially, they prevent genomic instability that may arise from dysregulated topoisomerase activity. By altering DNA topology, modulatory proteins can influence transcriptional bursting, leading to epigenetic heterogeneity within the bacterial populations, crucial for bacterial stress adaptation. In contrast, topoisomerase interacting proteins encoded by plasmids and phages have dedicated roles in facilitating plasmid maintenance and phage propagation by suppressing host defenses and promoting horizontal gene transfer. We present a comprehensive analysis of the topoisomerase modulatory proteins discovered so far, offering insights into the mechanisms by which DNA topology and homeostasis are maintained within the intracellular environment. We project the potential of topoisomerase modulator-based drug design as a promising strategy for combating multidrug-resistant bacterial pathogens.

The nucleoid-associated proteins (NAPs) are the primary organizers of chromosome architecture, function, and regulators of gene expression in bacteria. Mycobacterium tuberculosis (Mtb) encodes a limited set of NAPs, among which HU (MtHU) is essential for the organism. While MtHU’s DNA-binding properties are well documented, the genome-wide regulatory circuit by which it coordinates transcription remains to be elucidated. Here, we combine CRISPR interference (CRISPRi), chemical inhibition, ChIP-seq, RNA-seq, and RNA polymerase (RNAP) occupancy experiments to dissect MtHU function. Conditional depletion of MtHU resulted in transcriptional deregulation of over 800 genes involved in various cellular processes, indicating its dominant role in controlling gene expression. ChIP-seq analysis revealed widespread MtHU binding across the genome, including intergenic regions and within gene bodies. The large-scale occupation of MtHU within open reading frames (ORFs) hampers RNA polymerase elongation during transcription. MtHU depletion thus resulted in upregulation of genes, indicating its predominant negative regulatory role. Limiting MtHU during Mtb infection resulted in decreased intracellular survival and decreased antibiotic tolerance, linking gene regulation to host-pathogen dynamics. Our findings expand the functional landscape of MtHU, uncovering its multifaceted roles in transcriptional control, immune modulation, and antibiotic tolerance, opening up potential opportunities for the development of new treatment regimens.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) belonging to global strains such as ST-131 pose a significant health challenge. To understand the evolutionary landscape and molecular mechanisms defining ST-131 UPEC, the complete genome of E. coli NS30 was generated and analyzed. The complete genome assembly of E. coli NS30, belonging to high-risk ST-131, C2 subclade, revealed a chromosome and two plasmids. A large conjugative plasmid, pNS30-1, harboured a multi-drug resistance (MDR) cassette within a Tn402-like class 1 integron, which was functionally demonstrated to be transferable. Comparative genomic analysis identified four distinct genomic islands (GIs) that are absent in its closest ST-131 neighbour. Two of these, including a novel pathogenicity island (PAI), were acquired from other E. coli lineages, harbouring Virulence factors (VFs) and efflux pump genes. The remaining two GIs are phage-like elements contributing to genome plasticity. E. coli NS30 is distinct from the other ST-131 UPEC genomes by the acquisition of novel GIs. The presence of GIs, virulence factors and AMR genes in a conjugative MDR plasmid has driven its evolution into a formidable uropathogen with a high potential to spread resistance and virulence traits.

Being a persistent and deadly infection, tuberculosis (TB) caused by Mycobacterium tuberculosis remains a global health challenge. Despite having a well-established 4-drug combination therapy for drug-sensitive TB, the emergence of drug-resistant TB has rendered the treatment less effective. Although fluoroquinolones (FQs) are among the prominent drugs in the second-line treatment for multidrug-resistant tuberculosis (MDR-TB), FQ resistance has readily emerged in cases of extensively drug-resistant tuberculosis (XDR-TB). Other than the mutations in DNA gyrase, a universally conserved bacterial enzyme targeted by FQs, several mechanisms contribute to the emergence of FQ resistance. Recently, post-translational modification of DNA gyrase is implicated as one of the mechanisms for FQ resistance. Here, we describe succinylation of GyrB by a promiscuous acetyltransferase, Eis of M. tuberculosis, as a new mechanism contributing to FQ resistance in mycobacteria. Lysine succinylation of GyrB results in a reduced interaction of FQs with the enzyme, thereby decreasing the DNA cleavage by DNA gyrase in the presence of FQs. Accordingly, Eis overexpressing mycobacterial strains exhibit increased minimum inhibitory concentration (MIC) to FQs. Thus, succinylation of DNA gyrase is yet another resistance mechanism against the FQs.

Topoisomerase I (TopoI) is the sole DNA relaxase in Mycobacterium tuberculosis. Despite being a validated drug target and indispensable to the pathogen, only a limited repertoire of inhibitors targeting the enzyme have been identified. We employed monoclonal antibodies (mAbs) to address this shortfall. From the pool of a large number of mAbs, we describe an inhibitory mAb specific to mycobacterial TopoI with a distinct mechanism of action. Among the various steps of the TopoI reaction cycle, the mAb does not interfere with DNA binding but impedes DNA cleavage. It does not alter the religation activity of TopoI; however, it inhibits its strand passage activity. Probing with the mAb, we show the precise step at which the topology of the DNA is changed during DNA relaxation reaction. Surprisingly, instead of the initial strand scission action of the enzyme, the subsequent strand passage followed by the second transesterification entails the alteration in DNA topology. With their selective and specific inhibitory properties, the mAb and its derived single-chain variable fragment (ScFv) would serve to probe the structure of mycobacterial TopoI and as a starting point in designing peptide inhibitors with therapeutic potential to combat the rampant drug-resistant M. tuberculosis.

Tuberculosis (TB) continues to be a major cause of death worldwide despite having an effective combinatorial therapeutic regimen and vaccine. Being one of the most successful human pathogens, Mycobacterium tuberculosis retains the ability to adapt to diverse intracellular and extracellular environments encountered by it during infection, persistence, and transmission. Designing and developing new therapeutic strategies to counter the emergence of multidrug-resistant and extensively drug-resistant TB remains a major task. DNA topoisomerases make up a unique class of ubiquitous enzymes that ensure steady-state level supercoiling and solve topological problems occurring during DNA transactions in cells. They continue to be attractive targets for the discovery of novel classes of antibacterials and to develop better molecules from existing drugs by virtue of their reaction mechanism. The limited repertoire of topoisomerases in M. tuberculosis, key differences in their properties compared to topoisomerases from other bacteria, their essentiality for the pathogen's survival, and validation as candidates for drug discovery provide an opportunity to exploit them in drug discovery efforts. The present review provides insights into their organization, structure, function, and regulation to further efforts in targeting them for new inhibitor discovery. First, the structure and biochemical properties of DNA gyrase and Topoisomerase I (TopoI) of mycobacteria are described compared to the well-studied counterparts from other bacteria. Next, we provide an overview of known inhibitors of DNA gyrase and emerging novel bacterial topoisomerase inhibitors (NBTIs). We also provide an update on TopoI-specific compounds, highlighting mycobacteria-specific inhibitors.

In every bacterium, nucleoid‐associated proteins (NAPs) play crucial roles in chromosome organization, replication, repair, gene expression, and other DNA transactions. Their central role in controlling the chromatin dynamics and transcription has been well‐appreciated in several well‐studied organisms. Here, we review the diversity, distribution, structure, and function of NAPs from the genus Mycobacterium. We highlight the progress made in our understanding of the effects of these proteins on various processes and in responding to environmental stimuli and stress of mycobacteria in their free‐living as well as during distinctive intracellular lifestyles. We project them as potential drug targets and discuss future studies to bridge the information gap with NAPs from well‐studied systems.