Mtb Growth Research Articles

Tobacco smoke exposure recruits inflammatory airspace monocytes that establish permissive lung niches for Mycobacterium tuberculosis.

Tobacco smoking doubles the risk of active tuberculosis (TB) and accounts for up to 20% of all active TB cases globally. How smoking promotes lung microenvironments permissive to Mycobacterium tuberculosis (Mtb) growth remains incompletely understood. We investigated primary bronchoalveolar lavage cells from current and never smokers by performing single-cell RNA sequencing (scRNA-seq), flow cytometry, and functional assays. We observed the enrichment of immature inflammatory monocytes in the lungs of smokers compared with nonsmokers. These monocytes exhibited phenotypes consistent with recent recruitment from blood, ongoing differentiation, increased activation, and states similar to those with chronic obstructive pulmonary disease. Using integrative scRNA-seq and flow cytometry, we identified CD93 as a marker for a subset of these newly recruited smoking-associated lung monocytes and further provided evidence that the recruitment of monocytes into the lung was mediated by CCR2-binding chemokines, including CCL11. We also show that these cells exhibit elevated inflammatory responses upon exposure to Mtb and accelerated intracellular growth of Mtb compared with mature macrophages. This elevated Mtb growth could be inhibited by anti-inflammatory small molecules, providing a connection between smoking-induced pro-inflammatory states and permissiveness to Mtb growth. Our findings suggest a model in which smoking leads to the recruitment of immature inflammatory monocytes from the periphery to the lung, which results in the accumulation of these Mtb-permissive cells in the airway. This work defines how smoking may lead to increased susceptibility to Mtb and identifies host-directed therapies to reduce the burden of TB among those who smoke.

Computer-aided identification of Mycobacterium tuberculosis resuscitation-promoting factor B (RpfB) inhibitors from Gymnema sylvestre natural products

Tuberculosis (TB), an infectious disease caused by multi-drug resistant Mycobacterium tuberculosis (Mtb), has been a global health concern. Mtb affects over a third of the world’s population, causing two million deaths annually due to its dormancy and propensity to spread infection during this period. Resuscitation-promoting factor B (RpfB) plays a pivotal role in the growth of Mtb during dormant periods, making it a critical target for eliminating Mtb and curing TB. Gymnema sylvestre is a famous medicinal plant with several medicinal properties, including antimicrobial activity; however, the therapeutic potential of the various reported metabolites of this plant against Mtb has not yet been explored. The aim of this study was to explore the reported natural products of G. sylvestre against the RpfB of the Mtb. A total of 131 reported secondary metabolites of this plant were collected and virtually screened against the RpfB. We particularly targeted the Glu292 residue of RpfB as it is crucial for the catalysis of this protein. From our in-house library, 114 compounds showed a binding affinity higher than the standard drug. The binding stability of the top three lead compounds was further confirmed through MD simulation analysis. Drug likeness analyses indicated that the ten hits had zero violations of the Lipinski rule of five. In addition, analyses of pharmacokinetics, toxicity, and target prediction revealed that the top compounds are devoid of toxicity and do not affect human proteins. Additionally, they reflect multifaceted approach as anti-TB agents. Our selected hits not only exhibit molecular properties favoring physiological compatibility but also exhibit properties enhancing their potential efficacy as therapeutic candidates. The compounds investigated here are worthy of experimental validation for the discovery of novel treatments against TB. Further, this study also provides a promising avenue for research on the pharmacological potential of G. sylvestre.

Mycobacterial FtsZ and inhibitors: a promising target for the anti-tubercular drug development.

The emergence of multidrug-resistant tuberculosis (MDR-TB) strains has rendered many anti-TB drugs ineffective. Consequently, there is an urgent need to identify new drug targets against Mycobacterium tuberculosis (Mtb). Filament Forming Temperature Sensitive Gene Z (FtsZ), a member of the cytoskeletal protein family, plays a vital role in cell division by forming a cytokinetic ring at the cell's center and coordinating the division machinery. When FtsZ is depleted, cells are unable to divide and instead elongate into filamentous structures that eventually undergo lysis. Since the inactivation of FtsZ or alterations in its assembly impede the formation of the Z-ring and septum, FtsZ shows promise as a target for the development of anti-mycobacterial drugs. This review not only discusses the potential role of FtsZ as a promising pharmacological target for anti-tuberculosis therapies but also explores the structural and functional aspects of the mycobacterial protein FtsZ in cell division. Additionally, it reviews various inhibitors of Mtb FtsZ. By understanding the importance of FtsZ in cell division, researchers can explore strategies to disrupt its function, impeding the growth and proliferation of Mtb. Furthermore, the investigation of different inhibitors that target Mtb FtsZ expands the potential for developing effective treatments against tuberculosis.

A novel multistage antigens ERA005f confer protection against Mycobacterium tuberculosis by driving Th-1 and Th-17 type T cell immune responses.

Tuberculosis (TB) is a major threat to human health. In 2021, TB was the second leading cause of death after COVID-19 among infectious diseases. The Bacillus Calmette-Guérin vaccine (BCG), the only licensed TB vaccine, is ineffective against adult TB. Therefore, there is an urgent need to develop new effective vaccines. In this study, we developed a novel multistage subunit vaccine (ERA005f) comprising various proteins expressed in metabolic states, based on three immunodominant antigens (ESAT-6, Rv2628, and Ag85B). We utilized the E. coli prokaryotic expression system to express ERA005f and subsequently purified the protein using nickel affinity chromatography and anion exchange. Immunogenicity and protective efficacy of ERA005f and ERA005m were evaluated in BALB/c mice. ERA005f was consistently expressed as an inclusion body in a prokaryotic expression system, and a highly pure form of the protein was successfully obtained. Both ERA005f and ERA005m significantly improved IgG titers in the serum. In addition, mice immunized with ERA005f and ERA005m generated higher titers of antigen-specific IgG2a than the other groups. Elispot results showed that, compared with other groups, ERA005f increased the numbers of IFN-γ-secreting and IL-4-secreting T cells, especially the number of IFN-γ-secreting T cells. Meanwhile, ERA005f induced a higher number of IFN-γ+ T lymphocytes than ERA005m did. In addition, ERA005f improved the expression of cytokines, including IFN-γ, IL-12p70, TNF-α, IL-17, and GM-CSF and so on. Importantly, both ERA005f and ERA005m significantly inhibited the growth of Mtb. The novel multistage antigen ERA005f elicited a strong antigen-specific humoral response and Th-1 and Th-17 cell-mediated immunity in mice. Meanwhile, it can effectively inhibit H37Rv growth in vitro, and represents a correlate of protection in vivo, indicating that ERA005f may exhibit excellent protective efficacy against Mycobacterium tuberculosis H37Rv infection. Our study suggests that ERA005f has the potential to be a promising multistage tuberculosis vaccine candidate.

Early innate role for CD8αα+ cells in tuberculosis.

Cell types that mediate early control of Mycobacterium tuberculosis (Mtb) infection are not well understood. Winchell and Nyquist et al. (https://doi.org/10.1084/jem.20230707) show that CD8αα+ lymphocytes have a major role in the innate suppression of Mtb growth in the lungs of macaques.

Single-cell analysis reveals a weak macrophage subpopulation response to Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb) infection remains one of society's greatest human health challenges. Macrophages integrate multiple signals derived from ontogeny, infection, and the environment. This integration proceeds heterogeneously during infection. Some macrophages are infected, while others are not; therefore, bulk approaches mask the subpopulation dynamics. We establish a modular, targeted, single-cell protein analysis framework to study the immune response to Mtb. We demonstrate that during Mtb infection, only a small fraction of resting macrophages produce tumor necrosis factor (TNF) protein. We demonstrate that Mtb infection results in muted phosphorylation of p38 and JNK, regulators of inflammation, and leverage our single-cell methods to distinguish between pathogen-mediated interference in host signaling and weak activation of host pathways. We demonstrate that the inflammatory signal magnitude is decoupled from the ability to control Mtb growth. These data underscore the importance of developing pathogen-specific models of signaling and highlight barriers to activation of pathways that control inflammation.

Mycobacterium tuberculosis suppresses host DNA repair to boost its intracellular survival

Mycobacterium tuberculosis (Mtb) triggers distinct changes in macrophages, resulting in the formation of lipid droplets that serve as a nutrient source. We discover that Mtb promotes lipid droplets by inhibiting DNA repair responses, resulting in the activation of the type-I IFN pathway and scavenger receptor-A1 (SR-A1)-mediated lipid droplet formation. Bacterial urease C (UreC, Rv1850) inhibits host DNA repair by interacting with RuvB-like protein 2 (RUVBL2) and impeding the formation of the RUVBL1-RUVBL2-RAD51 DNA repair complex. The suppression of this repair pathway increases the abundance of micronuclei that trigger the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway and subsequent interferon-β (IFN-β) production. UreC-mediated activation of the IFN-β pathway upregulates the expression of SR-A1 to form lipid droplets that facilitate Mtb replication. UreC inhibition via a urease inhibitor impaired Mtb growth within macrophages and invivo. Thus, our findings identify mechanisms by which Mtb triggers a cascade of cellular events that establish a nutrient-rich replicative niche.

Unnatural Endotype B PPAPs as Novel Compounds with Activity against Mycobacterium tuberculosis.

Pre-SARS-CoV-2, tuberculosis was the leading cause of death by a single pathogen. Repetitive exposure of Mycobacterium tuberculosis(Mtb) supported the development of multidrug- and extensively drug-resistant strains, demanding novel drugs. Hyperforin, a natural type A polyprenylated polycyclic acylphloroglucinol from St. John's wort, exhibits antidepressant and antibacterial effects also against Mtb. Yet, Hyperforin's instability limits the utility in clinical practice. Here, we present photo- and bench-stable type B PPAPs with enhanced antimycobacterial efficacy. PPAP22 emerged as a lead compound, further improved as the sodium salt PPAP53, drastically enhancing solubility. PPAP53 inhibits the growth of virulent extracellular and intracellular Mtb without harming primary human macrophages. Importantly, PPAP53 is active against drug-resistant strains of Mtb. Furthermore, we analyzed the in vitro properties of PPAP53 in terms of CYP induction and the PXR interaction. Taken together, we introduce type PPAPs as a new class of antimycobacterial compounds, with remarkable antibacterial activity and favorable biophysical properties.

Macrophage targeted iron oxide nanodecoys augment innate immunological and drug killings for more effective Mycobacterium Tuberculosis clearance

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, is still one of the top killers worldwide among infectious diseases. The escape of Mtb from immunological clearance and the low targeting effects of anti-TB drugs remain the substantial challenges for TB control. Iron is particularly required for Mtb growth but also toxic for Mtb in high dosages, which makes iron an ideal toxic decoy for the ‘iron-tropic’ Mtb. Here, a macrophage-targeted iron oxide nanoparticles (IONPs)-derived IONPs-PAA-PEG-MAN nanodecoy is designed to augment innate immunological and drug killings against intracellular Mtb. IONPs-PAA-PEG-MAN nanodecoy exhibits preferential uptake in macrophages to significantly increase drug uptake with sustained high drug contents in host cells. Moreover, it can serve as a specific nanodecoy for the ‘iron-tropic’ Mtb to realize the localization of Mtb contained phagosomes surrounding the drug encapsulated nanodecoys and co-localization of Mtb with the drug encapsulated nanodecoys in lysosomes, where the incorporated rifampicin (Rif) can be readily released under acidic lysosomal condition for enhanced Mtb killing. This drug encapsulated nanodecoy can also polarize Mtb infected macrophages into anti-mycobacterial M1 phenotype and enhance M1 macrophage associated pro-inflammatory cytokine (TNF-α) production to trigger innate immunological responses against Mtb. Collectively, Rif@IONPs-PAA-PEG-MAN nanodecoy can synergistically enhance the killing efficiency of intracellular Mtb in in vitro macrophages and ex vivo monocyte-derived macrophages, and also significantly reduce the mycobacterial burdens in the lung of infected mice with alleviated pathology. These results indicate that Rif@IONPs-PAA-PEG-MAN nanodecoy may have a potential for the development of more effective therapeutic strategy against TB by manipulating augmented innate immunity and drug killings.Graphic

Drug-Tolerant Mycobacterium tuberculosis Adopt Different Survival Strategies in Alveolar Macrophages of Patients with Pulmonary Tuberculosis.

The rapid spread of drug-resistant M. tuberculosis (Mtb) strains and the phenomenon of phenotypic tolerance to drugs present challenges toward achieving the goal of tuberculosis (TB) elimination worldwide. By using the ex vivo cultures of alveolar macrophages obtained from lung tissues of TB patients after intensive antimicrobial chemotherapy before surgery, different subpopulations of multidrug-tolerant Mtb with a spectrum of phenotypic and growth features were identified in the same TB lesions. Our results are indicative of not only passive mechanisms generating nonheritable resistance of Mtb to antibiotics, which are associated mainly with a lack of Mtb growth, but also some active mechanisms of Mtb persistence, such as cell wall and metabolic pathway remodeling. In one of the subpopulations, non-acid-fast Mtb have undergone significant reprogramming with the restoration of acid-fastness, lipoarabinomannan expression and replication in host cells of some patients after withdrawal of anti-TB drugs. Our data indicate the universal stress protein Rv2623 as a clinically relevant biomarker of Mtb that has lost acid-fastness in human lungs. The studies of Mtb survival, persistence, dormancy, and resumption and the identification of biomarkers characterizing these phenomena are very important concerning the development of vaccines and drug regimens with individualized management of patients for overcoming the resistance/tolerance crisis in anti-TB therapy.

Discovery, Synthesis, and Optimization of 1,2,4-Triazolyl Pyridines Targeting Mycobacterium tuberculosis.

The rise in multidrug resistant tuberculosis cases underscores the urgent need to develop new treatment strategies for tuberculosis. Herein, we report the discovery and synthesis of a new series of compounds containing a 3-thio-1,2,4-triazole moiety that show inhibition of Mycobacterium tuberculosis (Mtb) growth and survival. Structure-activity relationship studies led us to identify several potent analogs displaying low micromolar to nanomolar inhibitory activity, specifically against Mtb. The potent analogs demonstrated no cytotoxicity in mammalian cells at over 100 times the effective concentration required in Mtb and were bactericidal against Mtb during infection of macrophages. In the exploratory ADME investigations, we observed suboptimal ADME characteristics, which prompted us to identify potential metabolic liabilities for further optimization. Our preliminary investigations into the mechanism of action suggest that this series is not engaging the promiscuous targets that arise from many phenotypic screens. We selected for resistant mutants with the nanomolar potent nitro-containing compound 20 and identified resistant isolates with mutations in genes required for coenzyme F420 biosynthesis and the nitroreductase Ddn. This suggests that the aromatic nitro-1,2,4-triazolyl pyridines are activated by F420-dependent Ddn activity, similar to the nitro-containing TB drug pretomanid. We were able to circumvent the requirement for F420-dependent Ddn activity using compounds that contained non-nitro groups, identifying a key feature to be modified to avoid this predominant resistance mechanism. These studies provide the foundation for the development of a new class of 1,2,4-triazole compounds for the treatment of tuberculosis.

A tRNA modification in Mycobacterium tuberculosis facilitates optimal intracellular growth.

Diverse chemical modifications fine-tune the function and metabolism of tRNA. Although tRNA modification is universal in all kingdoms of life, profiles of modifications, their functions, and physiological roles have not been elucidated in most organisms including the human pathogen, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To identify physiologically important modifications, we surveyed the tRNA of Mtb, using tRNA sequencing (tRNA-seq) and genome-mining. Homology searches identified 23 candidate tRNA modifying enzymes that are predicted to create 16 tRNA modifications across all tRNA species. Reverse transcription-derived error signatures in tRNA-seq predicted the sites and presence of nine modifications. Several chemical treatments prior to tRNA-seq expanded the number of predictable modifications. Deletion of Mtb genes encoding two modifying enzymes, TruB and MnmA, eliminated their respective tRNA modifications, validating the presence of modified sites in tRNA species. Furthermore, the absence of mnmA attenuated Mtb growth in macrophages, suggesting that MnmA-dependent tRNA uridine sulfation contributes to Mtb intracellular growth. Our results lay the foundation for unveiling the roles of tRNA modifications in Mtb pathogenesis and developing new therapeutics against tuberculosis.

A tRNA modification in Mycobacterium tuberculosis facilitates optimal intracellular growth

Diverse chemical modifications fine-tune the function and metabolism of tRNA. Although tRNA modification is universal in all kingdoms of life, profiles of modifications, their functions, and physiological roles have not been elucidated in most organisms including the human pathogen, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To identify physiologically important modifications, we surveyed the tRNA of Mtb, using tRNA sequencing (tRNA-seq) and genome-mining. Homology searches identified 23 candidate tRNA modifying enzymes that are predicted to create 16 tRNA modifications across all tRNA species. Reverse transcription-derived error signatures in tRNA-seq predicted the sites and presence of nine modifications. Several chemical treatments prior to tRNA-seq expanded the number of predictable modifications. Deletion of Mtb genes encoding two modifying enzymes, TruB and MnmA, eliminated their respective tRNA modifications, validating the presence of modified sites in tRNA species. Furthermore, the absence of mnmA attenuated Mtb growth in macrophages, suggesting that MnmA-dependent tRNA uridine sulfation contributes to Mtb intracellular growth. Our results lay the foundation for unveiling the roles of tRNA modifications in Mtb pathogenesis and developing new therapeutics against tuberculosis.

Differentiating the roles of Mycobacterium tuberculosis substrate binding proteins, FecB and FecB2, in iron uptake.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, mechanisms through which Mtb acquires host iron represent attractive therapeutic targets. Mtb scavenges host iron via Mtb siderophore-dependent and heme iron uptake pathways. While multiple studies describe the import of heme and ferric-siderophores and the export of apo-siderophores across the inner membrane, little is known about their transport across the periplasm and cell-wall environments. Mtb FecB and FecB2 are predicted periplasmic binding proteins implicated in host iron acquisition; however, their precise roles are not well understood. This study sought to differentiate the roles FecB and FecB2 play in Mtb iron acquisition. The crystallographic structures of Mtb FecB and FecB2 were determined to 2.0 Å and 2.2 Å resolution, respectively, and show distinct ligand binding pockets. In vitro ligand binding experiments for FecB and FecB2 were performed with heme and bacterial siderophores from Mtb and other species, revealing that both FecB and FecB2 bind heme, while only FecB binds the Mtb sideophore ferric-carboxymycobactin (Fe-cMB). Subsequent structure-guided mutagenesis of FecB identified a single glutamate residue-Glu339-that significantly contributes to Fe-cMB binding. A role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis and Mtb pull-down assays, which revealed interactions between FecB and members of the mycobacterial siderophore export and import machinery. Similarly, pull-down assays with FecB2 confirms its role in heme uptake revealing interactions with a potential inner membrane heme importer. Due to ligand preference and protein partners, our data suggest that Mtb FecB plays a role in siderophore-dependent iron and heme acquisition pathways; in addition, we confirm that Mtb FecB2 is involved in heme uptake.

Homodimeric Granzyme A Opsonizes Mycobacterium tuberculosis and Inhibits Its Intracellular Growth in Human Monocytes via Toll-Like Receptor 4 and CD14.

Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete granzyme A (GzmA) protective against intracellular Mtb growth. However, GzmA-enzymatic activity is unnecessary for pathogen inhibition, and the mechanisms of GzmA-mediated protection remain unknown. We show that GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes, and subsequent pathogen clearance within the phagolysosome. Although monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4). Without access to surface-expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1 was associated with inhibitory activity. Furthermore, GzmA colocalized with and was regulated by protein disulfide isomerase AI (PDIA1), which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify a previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis, and elimination of Mtb in human monocytes, and they highlight PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.

Impaired mitophagy induces antimicrobial responses in macrophages infected with Mycobacterium tuberculosis

BackgroundMitophagy, mitochondrial selective autophagy, plays a pivotal role in the maintenance of cellular homeostasis in response to cellular stress. However, the role of mitophagy in macrophages during infection has not been elucidated. To determine whether mitophagy regulates intracellular pathogen survival, macrophages were infected with Mycobacterium tuberculosis (Mtb), an intracellular bacterium.ResultsWe showed that Mtb-infected macrophages induced mitophagy through BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) activation. In contrast, BNIP3-deficient macrophages failed to induce mitophagy, resulting in reduced mitochondrial membrane potential in response to Mtb infection. Moreover, the accumulation of damaged mitochondria due to BNIP3 deficiency generated higher levels of mitochondrial reactive oxygen species (mROS) compared to the control, suppressing the intracellular survival of Mtb. We observed that siBNIP3 suppressed intracellular Mtb in mice lungs.ConclusionWe found that BNIP3 plays a critical role in the regulation of mitophagy during Mtb infection. The inhibition of mitophagy suppresses Mtb growth in macrophages through increased mROS production. Therefore, BNIP3 might be a novel therapeutic target for tuberculosis treatment.

Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of redox homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is evolutionarily equipped to resist exogenous reactive oxygen species (ROS) but shows vulnerability to an increase in endogenous ROS (eROS). Since eROS is an unavoidable consequence of aerobic metabolism, understanding how Mtb manages eROS levels is essential yet needs to be characterized. By combining the Mrx1-roGFP2 redox biosensor with transposon mutagenesis, we identified 368 genes (redoxosome) responsible for maintaining homeostatic levels of eROS in Mtb. Integrating redoxosome with a global network of transcriptional regulators revealed a hypothetical protein (Rv0158) as a critical node managing eROS in Mtb. Disruption of rv0158 (rv0158 KO) impaired growth, redox balance, respiration, and metabolism of Mtb on glucose but not on fatty acids. Importantly, rv0158 KO exhibited enhanced growth on propionate, and the Rv0158 protein directly binds to methylmalonyl-CoA, a key intermediate in propionate catabolism. Metabolite profiling, ChIP-Seq, and gene-expression analyses indicate that Rv0158 manages metabolic neutralization of propionate toxicity by regulating the methylcitrate cycle. Disruption of rv0158 enhanced the sensitivity of Mtb to oxidative stress, nitric oxide, and anti-TB drugs. Lastly, rv0158 KO showed poor survival in macrophages and persistence defect in mice. Our results suggest that Rv0158 is a metabolic integrator for carbon metabolism and redox balance in Mtb.

Mycobacterium tuberculosis Rv2324 is a multifunctional feast/famine regulatory protein involved in growth, DNA replication and damage control

Feast/famine regulatory proteins (FFRPs) are multifunctional regulators. We show that Mtb Rv2324 is important for growth, survival, and countering DNA damage in Mycobacterium tuberculosis (Mtb). DNA-relaxation activity against linear and supercoiled substrates suggest its involvement in transcription activation, while its high affinity for recombination, replication and repair substrates suggest a role there too. Small-Angle-X-ray scattering supports the adoption of an ‘open’ quaternary association in response to amino-acid binding. Size-exclusion-chromatography and glutaraldehyde cross-linking identify the adoption of diverse oligomers modulated by amino-acid binding, and DNA interactions. We tested G52A, G101T and D104A mutants which correspond to highly conserved residues, distal to the DNA-binding site, and are important for amino acids binding. G101T exhibits increased DNA affinity, while G52A and D104A exhibit weak DNA-binding thereby suggesting that they mediate effector-binding, and DNA binding activities. Gain and loss-of-function studies show that Rv2324 overexpression promotes growth-rate, while its knock-down leads to retarded growth. Rv2324 down-regulation lowers Mtb survival inside resting and IFN-ϒ-activated macrophages. Rv2324 protects the pathogen from DNA damage, as evidenced by the reduction in the knockdown strain's survival following treatment with H2O2 and UV light. Overall, we show that Rv2324 plays a crucial role in regulating survival and growth of Mtb.

Discovery and characterization of antimycobacterial nitro-containing compounds with distinct mechanisms of action and in vivo efficacy.

Nitro-containing compounds have emerged as important agents in the control of tuberculosis (TB). From a whole-cell high-throughput screen for Mycobacterium tuberculosis (Mtb) growth inhibitors, 10 nitro-containing compounds were prioritized for characterization and mechanism of action studies. HC2209, HC2210, and HC2211 are nitrofuran-based prodrugs that need the cofactor F420 machinery for activation. Unlike pretomanid which depends only on deazaflavin-dependent nitroreductase (Ddn), these nitrofurans depend on Ddn and possibly another F420-dependent reductase for activation. These nitrofurans also differ from pretomanid in their potent activity against Mycobacterium abscessus. Four dinitrobenzamides (HC2217, HC2226, HC2238, and HC2239) and a nitrofuran (HC2250) are proposed to be inhibitors of decaprenyl-phosphoryl-ribose 2'-epimerase 1 (DprE1), based on isolation of resistant mutations in dprE1. Unlike other DprE1 inhibitors, HC2250 was found to be potent against non-replicating persistent bacteria, suggesting additional targets. Two of the compounds, HC2233 and HC2234, were found to have potent, sterilizing activity against replicating and non-replicating Mtb in vitro, but a proposed mechanism of action could not be defined. In a pilot in vivo efficacy study, HC2210 was orally bioavailable and efficacious in reducing bacterial load by ~1 log in a chronic murine TB infection model.

NCoR1 controls Mycobacterium tuberculosis growth in myeloid cells by regulating the AMPK-mTOR-TFEB axis.

Mycobacterium tuberculosis (Mtb) defends host-mediated killing by repressing the autophagolysosome machinery. For the first time, we report NCoR1 co-repressor as a crucial host factor, controlling Mtb growth in myeloid cells by regulating both autophagosome maturation and lysosome biogenesis. We found that the dynamic expression of NCoR1 is compromised in human peripheral blood mononuclear cells (PBMCs) during active Mtb infection, which is rescued upon prolonged anti-mycobacterial therapy. In addition, a loss of function in myeloid-specific NCoR1 considerably exacerbates the growth of M. tuberculosis in vitro in THP1 differentiated macrophages, ex vivo in bone marrow-derived macrophages (BMDMs), and in vivo in NCoR1MyeKO mice. We showed that NCoR1 depletion controls the AMPK-mTOR-TFEB signalling axis by fine-tuning cellular adenosine triphosphate (ATP) homeostasis, which in turn changes the expression of proteins involved in autophagy and lysosomal biogenesis. Moreover, we also showed that the treatment of NCoR1 depleted cells by Rapamycin, Antimycin-A, or Metformin rescued the TFEB activity and LC3 levels, resulting in enhanced Mtb clearance. Similarly, expressing NCoR1 exogenously rescued the AMPK-mTOR-TFEB signalling axis and Mtb killing. Overall, our data revealed a central role of NCoR1 in Mtb pathogenesis in myeloid cells.