Tuberculosis (TB) remains a leading cause of mortality globally, driven by the infectious pathogen, Mycobacterium tuberculosis (M.tb). A novel DNA methyltransferase (DNA MTase), encoded by the Rv1509 gene and involved in TB pathogenesis, has been identified as a promising therapeutic target of anti-TB drugs. The present research employs an in silico approach to identify potential inhibitors of the Rv1509-encoded DNA MTase using a computational drug design pipeline. A multi-stage virtual screening of ZINC natural compounds was conducted against Rv1509. These phytomolecules were retrieved from the ZINC database, following computationally intensive docking and analysis of Absorption, Distribution, Metabolism, and Excretion (ADME) properties, top hits with a docking score ≤ -8.0 kcal/mol and favourable predicted pharmacokinetic profiles were prioritized. Subsequently, molecular dynamics simulations (MD) and principal component analysis (PCA) were employed to corroborate these hits. Of these, ZINC00338392, ZINC01662782, ZINC04104877, and ZINC96316367 ligands exhibited hydrogen bond formation with functional residue of DNA MTase, indicating biological relevance of binding. MD analysis revealed stable protein–ligand complexes during a 200 ns simulation. These computational analyses suggest that natural compounds bind with high predicted affinity to the active site of the Rv1509-encoded DNA MTase, warranting future experimental validation of their potential as novel leads for anti-TB drug development.

BACKGROUND:Mycobacterium tuberculosis ( M.tb ) remains a leading global cause of mortality. The current Bacillus Calmette-Guérin vaccine lacks efficacy in adults and fails to generate a long-term memory response. With the rise of multidrug-resistant strains, there is an urgent need for novel vaccines that can provide broader protection. This study aimed to design a multiepitope vaccine (MEV) targeting hallmark proteins involved in different aspects of M.tb virulence. METHODS:Four unique M.tb proteins, Rv1507A (role in memory response), Rv1509 (role in phagolysosomal escape), Rv1954A (role in macrophage activation/antigen presentation), and Rv2231A (role in persistence) were selected. In silico analyses were performed to identify epitopes with high-binding affinity for Toll-like receptors (TLRs). Two MEVs were optimized for codon and were linked with adjuvants that could bind with TLR4 or TLR2 (TLR4-laterosporulin and TLR2-PorB). Physicochemical properties, allergenicity, toxicity, and structural stability were evaluated, followed by molecular docking with TLR receptors, molecular dynamic (MD) simulation, in silico cloning, and immune simulations. RESULTS:Both MEVs exhibited favorable biophysical properties and high structural stability. Molecular docking confirmed strong binding affinities with TLR2 and TLR4 receptors, suggesting a robust activation of innate and adaptive immunity. Immunological simulations predicted a potent immune response characterized by high cytokine production and memory cell differentiation. The designed MEV demonstrated approximately 90% global population coverage. CONCLUSIONS:The designed MEVs effectively bridge gaps in existing TB immunization by targeting multiple aspects of M.tb pathogenesis. These in silico leads provide a promising framework for preclinical studies, potentially moving toward a more effective clinical solution against TB.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major global health challenge, exacerbated by prolonged treatment regimens, variable vaccine efficacy, and the emergence of drug-resistant strains. Beyond conventional pathogen-targeted antibiotics, increasing attention has focused on host-directed therapies (HDTs) that aim to enhance intrinsic immune mechanisms to improve disease control and treatment outcomes. Among these, autophagy, a conserved cellular process responsible for the degradation and recycling of damaged organelles, proteins, and intracellular pathogens, has emerged as a promising, yet complex, target in TB. Autophagy contributes to host defense by restricting intracellular M. tuberculosis survival, shaping innate and adaptive immune responses, and intersecting with antimicrobial effector pathways. However, the literature presents seemingly contradictory findings, with autophagy reported as both protective and insufficient, or even subverted, during infection. In the present Perspective, we critically examine these discrepancies and reconcile them by highlighting the influence of infection burden, disease stage, host cell type, and experimental context on autophagic outcomes. We further discuss how M. tuberculosis actively modulates autophagy to promote persistence and how the host counterbalances these strategies through interconnected immune pathways. Importantly, we position autophagy modulation within the broader framework of HDT for TB, critically evaluating pharmacological agents known to influence autophagic pathways, their potential therapeutic benefits, and their current limitations. We also address key translational challenges, including strain heterogeneity, cell-specific targeting, and drug delivery to infected macrophages. Finally, we outline future directions required to safely and effectively harness autophagy as an adjunctive strategy to shorten treatment duration and improve clinical outcomes in TB.

TB is a serious health challenge globally, with more than 10 million new cases every year and rising MDR and XDR variants diminishing the efficacy of existing chemotherapeutics. Although BCG vaccination confers protection in children, its limited ability to prevent adult pulmonary TB reveals the urgent need for next-generation vaccines proficient in inducing robust and durable immunity. PE and PPE protein families of M. tb, constituting nearly 10

We observed a high proportion of proteins in pathogenic Mycobacterium species that can potentially undergo liquid-liquid phase separation (LLPS) mediated biomolecular condensate formation, compared to nonpathogenic species. These proteins mainly include the PE-PPE and PE-PGRS families of proteins that have nucleic acid and protein-protein binding functions, typical of LLPS proteins. We also mapped identified LLPS proteins in M. tuberculosis (M.tb) drug-resistant databases PubMLST and TBProfiler, based upon the WHO 2023 catalogue of resistance-associated mutations. High sequence conservation of LLPS-associated proteins in various multiple drug-resistant M.tb isolates points to their potentially important role in virulence and host-pathogen interactions during pathogenic evolution. This analysis provides a perspective on the role of protein phase separation in the evaluation of M.tb pathogenesis and offers avenues for future research aimed at developing innovative strategies to combat M.tb infection.

Tuberculosis (TB) remains a global health burden, particularly because of the limited efficacy of the Bacillus Calmette-Guérin (BCG) vaccine against adult pulmonary TB. To improve immunogenicity, we developed a recombinant BCG strain expressing the M. tuberculosis -specific antigen Rv1507A (rBCG_Rv1507A) and evaluated its immune-enhancing potential. rBCG_Rv1507A-infected human PBMCs and murine macrophages exhibited enhanced co-stimulatory marker expression and Th1-skewed cytokine profiles in vitro. The vaccine stimulated the expansion of T follicular helper (TFH) cells and both central and effector memory T cells. Intratracheal immunisation induced systemic and mucosal antibody responses, localized memory B cell formation, and enrichment of lung-resident memory T cells in vivo. Importantly, rBCG_Rv1507A promoted macrophage apoptosis and suppressed autophagy, which may support cross-antigen presentation. Furthermore, it induces features of trained immunity, including hematopoietic progenitor expansion and metabolic reprogramming of macrophages. These immunological enhancements were compartmentalized to the lungs, the primary site of TB infection, due to mucosal delivery. Collectively, rBCG_Rv1507A demonstrated potential as a next-generation TB vaccine by integrating durable adaptive memory with innate immune training. However, further studies are required to confirm its protective efficacy.

The BCG vaccine represents a significant milestone in the prevention of tuberculosis (TB), particularly in children. Researchers have been developing recombinant BCG (rBCG) variants that can trigger lasting memory responses, thereby enhancing protection against TB in adults. The breakdown of immune surveillance is a key link between TB and other communicable and non-communicable diseases. Notably, TB is more prevalent among people with comorbidities such as HIV, diabetes, cancer, influenza, COVID-19, and autoimmune disorders. rBCG formulations have the potential to address both TB and HIV co-pandemics. TB increases the risk of lung cancer and immunosuppression caused by cancer can reactivate latent TB infections. Moreover, BCG's efficacy extends to bladder cancer treatment and blood glucose regulation in patients with diabetes and TB. Additionally, BCG provides cross-protection against unrelated pathogens, emphasizing the importance of BCG-induced trained immunity in COVID-19 and other respiratory diseases. Furthermore, BCG reduced the severity of pulmonary TB-induced influenza virus infections. Recent studies have proposed innovations in BCG delivery, revaccination, and attenuation techniques. Disease-centered research has highlighted the immunomodulatory effects of BCG on TB, HIV, cancer, diabetes, COVID-19, and autoimmune diseases. The complex relationship between TB and comorbidities requires a nuanced re-evaluation to understand the shared attributes regulated by BCG. This review assessed the interconnected relationships influenced by BCG administration in TB and related disorders, recommending the expanded use of rBCG in healthcare. Collaboration among vaccine research stakeholders is vital to enhance BCG's efficacy against global health challenges.

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB) is responsible for millions of deaths annually. BCG, the only vaccine against TB, fails to evoke memory response and is ineffective to provide protection among adults. The emergence of multi drug resistant strains of M.tb has necessitated development of new vaccines that can confer wider degree and long-term protection against TB. In this study, we have designed a novel multi-epitope vaccine (MEV) using the M.tb proteins, Rv1507A, Rv1509, Rv1954A and Rv2231A that are not only unique to this pathogen but are hallmark as these modulate different aspects of M.tb physiology. Rv1507A and Rv1954A elicit memory response in host while Rv1509 and Rv2231A have roles in virulence. These four proteins were chosen so as to develop a MEV that fills the gaps in existing vaccine against TB. We carried out in-silico analyses of various combinations of epitopes from these proteins, after codon optimization, to ensure that there is no toxicity and allergenicity while maintaining the highest level of immunogenicity of the MEV construct. Two MEV constructs were designed which can pair with different TLR-binding adjuvants: Laterosporulin (TLR-4) and PorB (TLR-2). In silico analysis of the MEV constructs revealed favorable biophysical and physicochemical properties, stability in structure and strong interactions with TLR receptors, suggesting their potential to trigger a robust immune response. Immunological simulations showed that MEV elicits robust immune response and exhibits nearly 90% coverage for global population due to its conserved nature. This study provides an important lead that can be validated through in vitro and in vivo assays, which may translate into tangible vaccine against TB.

IntroductionTuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of mortality worldwide. A crucial factor in Mtb's virulence is the ESX-5 secretion system, which transports PE/PPE proteins such as PE18 and PPE26. These proteins modulate host-pathogen interactions, immune responses, and intracellular survival mechanisms. Despite their importance, the roles and molecular interactions of PE18 and PPE26 in Mtb pathogenesis require further investigation.MethodsWe explored the roles of PE18 and PPE26 using recombinant Mycobacterium smegmatis (Msmeg) as a model organism. Protein-protein interactions were analyzed biochemically to identify partners within the ESX-5 secretion system, including EspG5 and other PE/PPE proteins. Subcellular localization of these proteins was assessed via cell fractionation studies. Functional assays, including in vitro cytokine production and antigen presentation studies, were performed using TLR2/Myd88 knockout and wild-type macrophages. In vivo experiments were conducted to assess effector T-cell activation and intracellular survival. Mechanistic insights into endosome-phagosome maturation and actin cytoskeleton dynamics were obtained through fluorescence microscopy.ResultsOur biochemical analyses confirmed interactions between PE18/PPE26, PE18/PPE27, PE19/PPE25, and EspG5/PPE, highlighting their involvement in ESX-5-mediated secretion. Cell fractionation studies revealed that PE/PPE proteins predominantly localize to the cell wall, with PE18 also secreted extracellularly. In vitro and in vivo experiments demonstrated that PE18 and PPE26 activate cytokine production and antigen presentation via TLR2/Myd88-dependent signaling pathways, inducing robust effector memory T-cell responses. Recombinant Msmeg expressing PE18, PPE26, or their combination exhibited enhanced intracellular survival by disrupting endosome-phagosome maturation, likely through interference with actin cytoskeletal organization.DiscussionOur findings elucidate the pivotal roles of PE18 and PPE26 in Mtb pathogenesis, emphasizing their contributions to immune modulation and intracellular persistence. The observed disruption of actin dynamics and endosome-phagosome maturation underscores a novel mechanism by which Mtb evades host defenses. The ability of PE18 and PPE26 to induce effector T-cell responses highlights their potential as targets for host-directed therapies or vaccine development against TB. Further studies focusing on their structure-function relationships and interactions with host proteins could accelerate the development of innovative therapeutic strategies.

Mtb subverts host immune surveillance by damaging phagolysosomal membranes, exploiting them as replication niches. In response, host cells initiate a coordinated LDR, integrating membrane repair, selective autophagy, and de novo biogenesis. This review delineates a systems-level model of lysosomal quality control governed by three critical regulatory axes: LGALS3/8/9, TRIM E3 ubiquitin ligases, and the AMPK-TFEB signaling pathway. LGALSs detect exposed glycans on ruptured membranes, triggering ESCRT-mediated repair and recruiting ARs. TRIM proteins mediate context-specific ubiquitination, enhancing cargo selection and facilitating transcriptional reprogramming via TFEB. Simultaneously, AMPK-TFEB signaling links metabolic stress to lysosomal regeneration, reinforcing immune defense and cellular adaptation. We highlight emerging mechanisms, including ATG8ylation, CASM, Ca2 + leakage, and SG formation, that refine this multilayered response. Mtb virulence factors selectively disrupt these pathways, revealing their relevance to pathogen persistence. Beyond infection, this triadic network maintains lysosomal integrity in neurodegeneration, inflammation, and lysosomal storage disorders. Understanding its modular design reveals novel therapeutic targets and HDTs for combatting drug-resistant TB. This review integrates recent advances into a coherent framework that redefines lysosomal function as a dynamic, immune-regulatory hub essential for cellular resilience under infectious and metabolic stress.

Mycobacterium tuberculosis (M. tb) genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis and treatment of tuberculosis, it is imperative to comprehend the pathomechanism of the disease and host-pathogen interactions to identify new drug targets for intervention strategies. Using in-silico comparative genome analysis, we identified one of the M. tb genes, Rv1509, as a signature protein exclusively present in M. tb. To explore the role of Rv1509, a likely methyl transferase, we constructed a knock-in Mycobacterium smegmatis (M. smegmatis) constitutively expressing Rv1509 (Ms_Rv1509). The Ms_Rv1509 led to differential expression of many transcriptional regulator genes as assessed by RNA-seq analysis. Further, in-vitro and in-vivo studies demonstrated an enhanced survival of Ms_Rv1509 inside the host macrophages. Ms_Rv1509 also promoted phagolysosomal escape inside macrophages to boost bacterial replication and dissemination. In-vivo infection studies revealed that Ms_Rv1509 survives better than BCG and causes pathological manifestations in the pancreas after intraperitoneal infection. Long-time survival of Ms_Rv1509 resulted in lymphocyte migration, increased T regulatory cells, giant cell formation, and likely granuloma formation in the pancreas, pointing toward the role of Rv1509 in M. tb pathogenesis.

Autophagy is a crucial immune defense mechanism that controls the survival and pathogenesis of M. tb by maintaining cell physiology during stress and pathogen attack. The E3-Ub ligases (PRKN, SMURF1, and NEDD4) and autophagy receptors (SQSTM1, TAX1BP1, CALCOCO2, OPTN, and NBR1) play key roles in this process. Galectins (LGALSs), which bind to sugars and are involved in identifying damaged cell membranes caused by intracellular pathogens such as M. tb, are essential. These include LGALS3, LGALS8, and LGALS9, which respond to endomembrane damage and regulate endomembrane damage caused by toxic chemicals, protein aggregates, and intracellular pathogens, including M. tb. They also activate selective autophagy and de novo endolysosome biogenesis. LGALS3, LGALS9, and LGALS8 interact with various components to activate autophagy and repair damage, while CGAS-STING1 plays a critical role in providing immunity against M. tb by activating selective autophagy and producing type I IFNs with antimycobacterial functions. STING1 activates cGAMP-dependent autophagy which provides immunity against various pathogens. Additionally, cytoplasmic surveillance pathways activated by ds-DNA, such as inflammasomes mediated by NLRP3 and AIM2 complexes, control M. tb. Modulation of E3-Ub ligases with small regulatory molecules of LGALSs and TRIM proteins could be a novel host-based therapeutic approach for controlling TB.

Mycobacterium tuberculosis (M. tb) is one of the most successful human pathogens, causing a severe and widespread infectious disease. The frequent emergence of multidrug-resistant (MDR) strains has exacerbated this public health crisis, particularly in underdeveloped regions. M. tb employs a sophisticated array of virulence factors to subvert host immune responses, both innate and adaptive. It utilizes the early secretory antigenic target (ESAT6) secretion system 1 (ESX-1) type VII secretion system (T7SS) and cell wall lipids to disrupt phagosomal integrity, inhibiting phagosome maturation, and fusion with lysosomes. Although host cells activate mechanisms such as ubiquitin (Ub), Ub-ligase, and cyclic GMP-AMP synthase-stimulator of interferon genes 1 (CGAS-STING1)-mediated autophagy to inhibit M. tb survival within macrophages, the pathogen counteracts these defenses with its own virulence factors, thereby inhibiting autophagy and dampening host-directed responses. T7SSs are critical for transporting proteins across the complex mycobacterial cell envelope, performing essential functions, including metabolite uptake, immune evasion, and conjugation. T7SS substrates fall into two main families: ESAT-6 system proteins, which are found in both Firmicutes and Actinobacteria, and proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) proteins, which are unique to mycobacteria. Recent studies have highlighted the significance of T7SSs in mycobacterial growth, virulence, and pathogenesis. Understanding the mechanisms governing T7SSs could pave the way for novel therapeutic strategies to combat mycobacterial diseases, including tuberculosis (TB).

Mycobacterium tuberculosis (M. tb) employs an extensive network of more than 90 toxin–antitoxin systems, and among them, VapC toxins are the most abundant. While most VapCs function as classical RNases with toxic effects, a significant number of them do not exhibit toxicity. However, these non-toxic VapCs may retain specific RNA binding abilities as seen in case of VapC16, leading to ribosome stalling at specific codons and reprofiling M. tb's proteome to aid in the bacterium's survival under different stressful conditions within the host. Here, we challenge the conventional classification of all VapC toxins as RNases and highlight the complexity of M. tb's strategies for survival and adaptation during infection.

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and β-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

Mycobacterium tuberculosis (M.tb) remains a formidable global health threat. The increasing drug resistance among M.tb clinical isolates is exacerbating the current tuberculosis (TB) burden. In this study we focused on identifying novel repurposed drugs that could be further investigated as potential anti-TB drugs. We utilized M.tb RNA methyltransferase Rv3366 (spoU) as a potential drug target due to its imperative activity in RNA modification and no structural homology with human proteins. Using computational modeling approaches the structure of Rv3366 was determined followed by high throughput virtual screening of Food and Drug Administration (FDA) approved drugs to screen potential binders of Rv3366. Molecular dynamics (MD) simulations were performed to assess the drug-protein binding interactions, complex stability and rigidity. Through this multi-step structure-based drug repurposing workflow two promising inhibitors of Rv3366 were identified, namely, Levodopa and Droxidopa. This study highlights the significance of targeting M.tb RNA methyltransferases to combat drug-resistant M.tb. and proposes Levodopa and Droxidopa as promising inhibitors of Rv3366 for future pre-clinical investigations.