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Abstract 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. The graphical abstract illustrates the multilayered host defense network countering Mtb-induced lysosomal and phagosomal injury. Upon membrane disruption, LGALS3 binds exposed glycans and initiates membrane repair by recruiting ESCRT components such as PDCD6IP and CHMP4B to the damaged phagosomal membrane. Beyond canonical repair, LGALS3 also activates ESCRT-independent pathways, coordinating the recruitment of SGs via LC3 lipidation (ATG8ylation), which stabilize membranes and halt local translation, reinforcing repair dynamics. In parallel, LGALS9 senses lysosomal damage and activates the MAP3K7-AMPK axis, leading to MTOR inhibition and transcriptional activation of lysosomal and ATG genes through TFEB. TRIM16, an E3 Ub ligase, is recruited by LGALS3 to damaged compartments, where it amplifies selective autophagy for removal of compromised lysosomes and promotes lysosomal gene expression by facilitating TFEB activation. A critical layer of control involves the conjugation of LC3-II to phagosomal membranes. This lipidation event enables recruitment and activation of the MCOLN1 Ca²⁺ channel, resulting in localized Ca²⁺ release. Elevated Ca²⁺ activates calcineurin, which dephosphorylates and translocates TFEB to the nucleus, triggering expression of ATG and lysosomal biogenesis genes, culminating in de novo lysosome formation. Simultaneously, LC3-II engagement with IRGM suppresses the RRAG GTPase-Ragulator complex, leading to MTOR inhibition. This relieves repression on TFEB, further driving a transcriptional program for lysosomal expansion and autophagic flux. Together, this dynamic and interlinked response, centered on LGALSs, TRIMs, and TFEB activation, constitutes a finely tuned system for damage recognition, repair, removal, and renewal of host endomembranes under microbial assault.

Tuberculosis (TB), caused by (Mtb), remains a leading cause of mortality worldwide. A crucial factor in 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 pathogenesis require further investigation. We explored the roles of PE18 and PPE26 using recombinant ( ) 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 cytokine production and antigen presentation studies, were performed using TLR2/Myd88 knockout and wild-type macrophages. 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. Our 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 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 expressing PE18, PPE26, or their combination exhibited enhanced intracellular survival by disrupting endosome-phagosome maturation, likely through interference with actin cytoskeletal organization. Our findings elucidate the pivotal roles of PE18 and PPE26 in 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 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.

( ) persists as latent infection in nearly a quarter of the global population and remains the leading cause of death among infectious diseases. While BCG is the only vaccine for TB, its inability to provide complete protection makes it imperative to engineer BCG such that it expresses immunodominant antigens that can enhance its protective potential. comparative genomic analysis of Mycobacterium species identified Rv1507A as a \"signature protein\" found exclusively in . (cell lines) and experiments carried out in mice, using purified recombinant Rv1507A revealed it to be a pro-inflammatory molecule, eliciting significantly high levels of IL-6, TNF-α, and IL-12. There was increased expression of activation markers CD69, CD80, CD86, antigen presentation molecules (MHC I/MHCII), and associated Th1 type of immune response. Rv1507A knocked-in also induced significantly higher pro-inflammatory Th1 response and higher survivability under stress conditions, both (macrophage RAW264.7 cells) and (mice). Sera derived from human TB patients showed significantly enhanced B-cell response against Rv1507A. The ability of Rv1507A to induce immuno-modulatory effect, B cell response, and significant memory response, renders it a putative vaccine candidate that demands further exploration.

Dissecting the function(s) of proteins present exclusively in Mycobacterium tuberculosis ( M.tb ) will provide important clues regarding the role of these proteins in mycobacterial pathogenesis. Using extensive computational approaches, we shortlisted ORFs/proteins unique to M.tb among 13 different species of mycobacteria and identified a hypothetical protein Rv1509 as a ‘signature protein’ of M.tb . This unique protein was found to be present only in M.tb and absent in all other mycobacterial species, including BCG. In silico analysis identified numerous putative T cell and B cell epitopes in Rv1509. Initial in vitro experiments using innate immune cells demonstrated Rv1509 to be immunogenic with potential to modulate innate immune responses. Macrophages treated with Rv1509 exhibited higher activation status along with substantial release of pro-inflammatory cytokines. Besides, Rv1509 protein boosts dendritic cell maturation by increasing the expression of activation markers such as CD80, HLA-DR and decreasing DC-SIGN expression and this interaction was mediated by innate immune receptor TLR2. Further, in vivo experiments in mice demonstrated that Rv1509 protein promotes the expansion of multifunctional CD4+ and CD8+T cells and induces effector memory response along with evoking a canonical Th1 type of immune response. Rv1509 also induces substantial B cell response as revealed by increased IgG reactivity in sera of immunized animals. This allowed us to demonstrate the diagnostic efficacy of this protein in sera of human TB patients compared to the healthy controls. Taken together, our results reveal that Rv1509 signature protein has immunomodulatory functions evoking immunological memory response with possible implications in serodiagnosis and TB vaccine development.

( ), the intracellular pathogen causing tuberculosis, has developed mechanisms that endow infectivity and allow it to modulate host immune response for its survival. Genomic and proteomic analyses of non-pathogenic and pathogenic mycobacteria showed presence of genes and proteins that are specific to . studies predicted that Rv1954A is a hypothetical secretory protein that exhibits intrinsically disordered regions and possess B cell/T cell epitopes. Treatment of macrophages with Rv1954A led to TLR4-mediated activation with concomitant increase in secretion of pro-inflammatory cytokines, IL-12 and TNF-α. studies showed that rRv1954A protein or Rv1954A knock-in (Ms_Rv1954A) activates macrophages by enhancing the expression of CD80 and CD86. An upregulation in the expression of CD40 and MHC I/II was noted in the presence of Rv1954A, pointing to its role in enhancing the association of APCs with T cells and in the modulation of antigen presentation, respectively. Ms_Rv1954A showed increased infectivity, induction of ROS and RNS, and apoptosis in RAW264.7 macrophage cells. Rv1954A imparted protection against oxidative and nitrosative stress, thereby enhancing the survival of Ms_Rv1954A inside macrophages. Mice immunized with Ms_Rv1954A showed that splenomegaly and primed splenocytes restimulated with Rv1954A elicited a Th1 response. Infection of Ms_Rv1954A in mice through intratracheal instillation leads to enhanced infiltration of lymphocytes in the lungs without formation of granuloma. While Rv1954A is immunogenic, it did not cause adverse pathology. Purified Rv1954A or Rv1954A knock-in (Ms_Rv1954A) elicited a nearly two-fold higher titer of IgG response in mice, and PTB patients possess a higher IgG titer against Rv1954A, also pointing to its utility as a diagnostic marker for TB. The observed modulation of innate and adaptive immunity renders Rv1954A a vital protein in the pathophysiology of this pathogen.

A growing body of evidence supports the hypothesis that intrinsically disordered proteins often mediate host–pathogen interactions and modulate host functions for pathogen survival and virulence. Mycobacterium tuberculosis (M.tb) has evolved largely through reductive evolution, with a few exceptions such as the glycine–alanine‐rich PE–PPE/PGRS protein family, which has been expanding in pathogenic mycobacteria. Here, our analyses of the M.tb proteome and secretome revealed that the PE–PGRS subfamily is enriched for disordered regions and disordered binding sites, pointing to their importance in host–pathogen interactions. As a case study, the secondary structure of PE35–PPE68 and PE32–PPE65 of the pathogenesis‐related RD1 and RD8 regions was analyzed through Fourier‐transform infrared spectroscopy. These disordered proteins displayed a considerable structural shift from disordered to ordered while engaged in the formation of complexes. While these proteins are immunogenic individually and enhance the pro‐pathogen response, their corresponding complexes enhanced the responses manifold as displayed here by PE35 and PPE68. It is likely that M.tb exploits such disorder–order structural dynamics as a strategy to mount a pro‐pathogen response and subvert host defense for productive infection. This functional gain also serves as a means to compensate genomic content loss due to reductive evolution. Mycobacterium tuberculosis exploits disordered proteins to subvert the host immune response. Bioinformatic analysis of various protein families of M. tuberculosis revealed that PE/PPE–PGRS family proteins are highly disordered and interact with cognate partners to achieve ordered structure as validated by FTIR. Immunization of mice indicated that the ordered protein complex exhibits gain of function by augmenting the immune response compared to that produced against disordered proteins.

Considering the BCG's efficacy against childhood tuberculosis (TB), meningeal TB and leprosy, efforts to remove BCG from the repertoire of childhood vaccinations in the developing world in order to replace it with a new TB vaccine are not likely to succeed. Several attempts have been made to modify the vaccine potential of BCG by generating recombinant strains expressing cytokines, pore-forming listeriolysin/perfringolysin, immunodominant antigens or additional antigens missing from the genome of the avirulent Mycobacterium bovis BCG genome. Subtraction hybridization, DNA microarrays and bacterial artificial chromosome arrays have led to identification of about 168 open reading frames that are absent from various strains of BCG (RD1-RD16). In principle, potency of BCG vaccines could be improved by reintroducing the genes for the immunodominant RD antigens to BCG or to use the immunodominant RD proteins as adjunctive subunit vaccine. The later approach is comparatively safe as this strategy is endowed with dual characteristics of both a live vaccine and a subunit vaccine. For subunit vaccines, use of native/recombinant immunodominant proteins is associated with number of limitations as compared to synthetic peptides representing the immunodominant epitopes. However the most common problem associated with T cell based epitope vaccines is MHC restriction that can be overcome by use of 'promiscuous epitopes' that are recognized in the context of more than one MHC and recognized by more than one T cell clones. In this context, bioinformatics tools that search protein sequences for promiscuous epitopes are able to accelerate the development of epitope based vaccines that effectively protect genetically diverse human populations.The aim of the present study was to determine the possibility of using pools of various promiscuous peptides from RD proteome of M. tuberculosis as multi epitope subunit vaccines to supplement the current BCG for better prophylaxis and also devise a peptide based therapeutic vaccine for tuberculosis. A predictive approach of all RD proteins harboring a secretion signal of the classical SecA pathway, but lacking membrane spanning segments, was carried out, resulting in the identification of 22 secretory RD proteins. The promiscuous peptides of these predicted secretory antigens as well as five non-classically secreted/immunodominant RD proteins known in literature were identified by HLA prediction analysis using the ProPred and ProPred 1 server. The peptides that were predicted to bind >50% HLA-DR alleles included in ProPred were considered as 'promiscuous'. Numerous peptides from each protein were found to be promiscuous but only a single peptide, binding to maximum no. of alleles was selected out of each protein. Similar approach was used to mine out the promiscuous CTL epitopes from these RD proteins by analyzing them with ProPred 1. The peptide binding to maximum number of alleles was taken as promiscuous. Moreover peptide regions were selected from RD proteins by assembling their minimal CD8+ and CD4+ T cell epitope constructs, in a single stretch as a 'promiscuous region' that were expected to be more effective for activating both Th as well as Tc subsets. Total 18 such peptide regions (R1-R18) were selected from secretory RD proteome of M. tuberculosis H37RV and included in the study to be evaluated as potential prophylactic and therapeutic vaccine candidates after screening for their immunoreactivity in TB patients, household contacts and healthy controls.In order to evaluate the immunoreactivity in terms of Thl-Th2 cytokines and CTL response to the selected promiscuous peptides (R1-R18), peripheral blood mononuclear cells from donors consisting of 25 healthy PPD+ve household contacts (HHC), 25 PPD_ve healthy controls and 25 sputum smear positive pulmonary TB patients at the baseline before the start of chemotherapy (F0), as well as after 2 (FI) and 4 (F2) months of therapy were in vitro stimulated. Biased strong Thl response was observed to all the 18 regions in healthy household contacts upon stimulation with peptides (R1-R18), while as it was either weak Thl, a Thl-Th2 balance or biased Th2 response in case of tuberculosis patients. Moreover upon follow up of patients it was observed that median IFNγ concentrations increased significantly after therapy for many of the peptides like R2, R3, R9, RIO, R12, R13, R15 and R18. In contrast, IL-10 concentrations decreased significantly after 2 and 4 months of chemotherapy in case of R2, R4, R6, RIO, R15, R16 and R18. Majority of peptides were also found to induce prominent autologous macrophage cytotoxicity and cytotoxicity was significantly increased in patients after follow up against many of the peptides like R3, R4, R5, RIO, R12, R13, R15, R18 indicating increase in CTL response following treatment.Considering the convention where proteins that are immunodominant and recognized both by patients as well as contacts are expected to be good candidates for vaccine development, three peptides were selected to formulate multi epitope vaccine (MEV I), which consisted of R3, Rll and R14. Another approach has been to identify antigens which stimulate strong biased Thl response in healthy PPD+ve patient contacts and there is minimal Thl response in patients. On this basis another set of three peptides (R2, R12 and R15) was selected that formulated MEV II. Furthermore antituberculous experimental vaccines inducing both Thl and Th2 response following immunization have been hypothesized as most effective. In case of HHCs, all the peptides resulted in biased Thl response, therefore those regions were selected which led to balanced Thl-Th2 response in patients with moderate/weak Thl response in contacts. This led to identification of a set of three more peptides (Rl, R15 and R16) which were used to formulate MEV III.After selection of three sets of vaccine candidates on the basis of immune recognition in human subjects, these vaccine candidates were screened for their immunogenic potential in BALB/c mice after 4 and 8 weeks of immunization using Dimethyldioctadecylammonium bromide (DDA) and Monophosphoryl lipid A (MPL) as adjuvants. These vaccines were used along with BCG in a mix-boost strategy in which first immunization was done with both BCG as well as MEV followed by boosting with MEV only. Evaluation of cytokines from splenocyte, peritoneal exudate cells (PECs) and bone marrow derived dendritic cells (BMDCs) cultures revealed these vaccines to be broadly immunogenic at both time points with significant induction of Thl and minimal Th2 response, together with significantly elevated macrophage cytotoxicity. Immunoprophylactic potential of these selected multi epitope vaccines adjuvanted with DDA-MPL, when supplemented to BCG in mix-boost approach, was evaluated in BALB/c mice. All the multi epitope vaccine combinations imparted significant protection at 4 and 8 weeks as compared to unvaccinated controls against aerosol challenge with M. tuberculosis H37RV (-100 bacilli). Vaccination with BCG+MEV III imparted maximum protection at both 4 and 8 weeks p.im in terms of reduction of log10 CPU (colony forming units) as well as less number of pathological lesions in lungs as compared to other vaccine formulations tested in this study, followed by BCG+MEV I. BCG+MEV II was least protective of the three combinations but was still able to impart significant protection (p<0.01; p<0.001) when compared to BCG alone.On evaluation of comparative immune responses generated by the stimulation of these peptides in tuberculosis patients, a therapeutic combination (METV) that consisted of RIO, R15 and R18 was selected based on increase in IFNy and CTL response and decrease in IL-10 response following treatment. After 4 and 8 weeks of commencement of treatment of infected mice, it was observed that all the constituent peptides of METV were able to induce high Thl response in infected splenocytes and treatment with METV induced significant reduction in log10 bacilli when compared to untreated controls. Moreover administration of METV with drugs displayed adjunct effect and elicited robust therapeutic efficacy over drugs alone or METV alone which was corroborated by histological studies.Thus current study is an effort to validate reverse vaccinology in the field of tuberculosis, whereby an optimal promiscuous peptide based multi epitope vaccine used along with BCG in mix-boost strategy resulted in significant vaccine efficacy in experimental tuberculosis. Moreover a therapeutic vaccine candidate based on promiscuous peptides has also been identified from RD proteins that can be used as an adjunct to antituberculous drugs.