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Background: Mycobacterium tuberculosis resides inside host macrophages during infection and adapts to resilient stresses generated by the host immune system. As a response, M. tuberculosis codes for short-chain dehydrogenases/ reductases (SDRs). These SDRs are nicotinamide adenine dinucleotide-reliant oxidoreductases involved in cell homeostasis. The precise function of oxidoreductases in bacteria especially M. tuberculosis were not fully explored. This study aimed to know the detail functional role of one of the oxidoreductase Rv0148 in M. tuberculosis. Results: In silico analysis revealed that Rv0148 interacts with Htdy (Rv3389) and the protein interactions were confirmed using far western blot. Gene knockout mutant of Rv0148 in M. tuberculosis was constructed by specialized transduction. Macrophage cell line infection with this knockout mutant showed increased expression of pro-inflammatory cytokines. This knockout mutant is sensitive to oxidative, nitrogen, redox and electron transport inhibitor stress agents. Drug susceptibility testing of the deletion mutant showed resistance to first-line drugs such as streptomycin and ethambutol and second-line aminoglycosides such as amikacin and kanamycin. Based on interactorme analysis for Rv0148 using STRING database, we identified 220 most probable interacting partners for Htdy protein. In the Rv0148 knockout mutants, high expression of htdy was observed and we hypothesize that this would have perturbed the interactome thus resulting in drug resistance. Finally, we propose that Rv0148 and Htdy are functionally interconnected and involved in drug resistance and cell homeostasis of M. tuberculosis. Conclusions:Our study suggests that Rv0148 plays a significant role in various functional aspects such as intermediatory metabolism, stress, homeostasis and also in drug resistance.

The emergence of drug-resistant mycobacteria, including Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria (NTM), poses an increasing global threat that urgently demands the development of new potent anti-mycobacterial drugs. One of the approaches toward the identification of new drugs is fragment-based drug discovery (FBDD), which is the most ingenious among other drug discovery models, such as structure-based drug design (SBDD) and high-throughput screening. Specialized techniques, such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and many others, are part of the drug discovery approach to combat the Mtb and NTM global menaces. Moreover, the primary drawbacks of traditional methods, such as the limited measurement of biomolecular toxicity and uncertain bioavailability evaluation, are successfully overcome by the FBDD approach. The current review focuses on the recognition of fragment-based drug discovery as a popular approach using virtual, computational, and biophysical methods to identify potent fragment molecules. FBDD focuses on designing optimal inhibitors against potential therapeutic targets of NTM and Mtb (PurC, ArgB, MmpL3, and TrmD). Additionally, we have elaborated on the challenges associated with the FBDD approach in the identification and development of novel compounds. Insights into the applications and overcoming the challenges of FBDD approaches will aid in the identification of potential therapeutic compounds to treat drug-sensitive and drug-resistant NTMs and Mtb infections.

Mycobacterium tuberculosis, which causes tuberculosis, is one of the leading infectious agents worldwide with a high rate of mortality. Following aerosol inhalation, M. tuberculosis primarily infects the alveolar macrophages, which results in a host immune response that gradually activates various antimicrobial mechanisms, including the production of reactive oxygen species (ROS), within the phagocytes to neutralize the bacteria. OxyR is the master regulator of oxidative stress response in several bacterial species. However, due to the absence of a functional oxyR locus in M. tuberculosis, the peroxidase stress is controlled by alkylhydroperoxidases. M. tuberculosis expresses alkylhydroperoxide reductase to counteract the toxic effects of ROS. In the current study, we report the functional characterization of an orthologue of alkylhydroperoxidase family member, Rv2159c, a conserved protein with putative peroxidase activity, during stress response and virulence of M. tuberculosis. We generated a gene knockout mutant of M. tuberculosis Rv2159c (MtbΔ2159) by specialized transduction. The MtbΔ2159 was sensitive to oxidative stress and exposure to toxic transition metals. In a human monocyte (THP-1) cell infection model, MtbΔ2159 showed reduced uptake and intracellular survival and increased expression of pro-inflammatory molecules, including IL-1β, IP-10, and MIP-1α, compared to the wild type M. tuberculosis and Rv2159c-complemented MtbΔ2159 strains. Similarly, in a guinea pig model of pulmonary infection, MtbΔ2159 displayed growth attenuation in the lungs, compared to the wild type M. tuberculosis and Rv2159c-complemented MtbΔ2159 strains. Our study suggests that Rv2159c has a significant role in maintaining the cellular homeostasis during stress and virulence of M. tuberculosis.

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