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Understanding cell state transitions and purposefully controlling them to improve therapies is a longstanding challenge in biological research and medicine. Here, we identify a transcriptional signature that distinguishes activated macrophages from TB-susceptible and TB-resistant mice. We then apply the cSTAR (cell State Transition Assessment and Regulation) approach to data from screening-by-RNA sequencing to identify chemical perturbations that shift the. transcriptional state of the TB-susceptible macrophages towards that of TB-resistant cells. Finally, we demonstrate that the compounds identified with this approach enhance resistance of the TB-susceptible mouse macrophages to virulentM. tuberculosis.

Mycobacterium tuberculosis (Mtb) employs diverse strategies to survive inside the host macrophages. In this study, we have identified a conserved hypothetical protein of Mtb; Rv0674, which is present in the mitochondria of the host cell. The genetic knock-out of rv0674 (Mtb-KO) showed increased growth of Mtb. The intracellular infection with recombinant Mycobacterium smegmatis (MSMEG) expressing Rv0674 (MS_Rv0674), established that the protein is involved in promoting the apoptotic cell death of the macrophage. To investigate the mechanism incurred in mitochondria, we observed that the protein physically interacts with the control region (D-loop) of the mitochondrial DNA (LSP and HSP promoters of the loop) of the macrophages and facilitates the increased expression of mRNA in all the complexes of mitochondrial encoded OXPHOS subunits. The changes in OXPHOS levels corroborated with the ATP synthesis, mitochondrial membrane potential and superoxide production. The infection with MS_Rv0674 confirmed the role of this protein in effecting the intracellular infection. The fluorescent and confocal microscopy confirmed that the protein is localized in the mitochondria of infected macrophages and in the cells of BAL of TB patients. Together these findings indicate towards the novel function of the protein which is unlike to the earlier established mechanisms of mycobacterial physiology.

The causative agent of novel coronavirus disease (COVID-19) is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 possesses RNA as a genetic material with 79% of the match with the bat SARS-CoV genome, which became epidemic in 2002. The SARS-CoV-2 peripheral Spike-Fc protein binds specifically to the ACE2 receptors present on bronchial epithelial cells and alveolar pneumocytes to downmodulates its expression which leads to severe acute respiratory failure. The disease is super infectious from human to human and the symptoms are similar to flu. The old aged and immunocompromised population are severely affected, and healthcare providers globally applied various strategies for treatment including the repurposing of drugs including antimalarial drug, hydroxychloroquine and anti-viral drugs.Herein, we described the SARS-CoV-2 pandemic, immune responses, possible drug targets, vaccines under the trials and correlated the possibility of trained immunity induced by BCG vaccination over control of SARS-CoV-2 infection. The countries with constraint BCG vaccination policy are struggling badly compared to countries with BCG vaccination policy. The BCG vaccination policy supports either lowering the total number of COVID-19 cases or the increasing recovery rate.

Summary Macrophages contribute to host immunity and tissue homeostasis via alternative activation programs. M1-like macrophages control intracellular bacterial pathogens and tumor progression. In contrast, M2-like macrophages shape reparative microenvironments that can be conducive for pathogen survival or tumor growth. An imbalance of these macrophages phenotypes may perpetuate sites of chronic unresolved inflammation, such as infectious granulomas and solid tumors. We have found that plant-derived and synthetic rocaglates sensitize macrophages to low concentrations of the M1-inducing cytokine IFN-gamma and inhibit their responsiveness to IL-4, a prototypical activator of the M2-like phenotype. Treatment of primary macrophages with rocaglates enhanced phagosome-lysosome fusion and control of intracellular mycobacteria. Thus, rocaglates represent a novel class of immunomodulators that can direct macrophage polarization toward the M1-like phenotype in complex microenvironments associated with hypofunction of type 1 and/or hyperactivation of type 2 immunity, e.g., chronic bacterial infections, allergies, and, possibly, certain tumors., Graphical abstract, Highlights • Rocaglates sensitize MΦ to IFNγ proportional to their translation inhibition activity • Active rocaglates inhibit MΦ response to IL-4 and CSF1 • Rocaglates upregulate stress response and host defense pathways • Rocaglates induced autophagy in MΦ and improved control of virulent mycobacteria, Immunology; Microbiology

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase (PtpA) has so far been known to control intracellular survival of mycobacteria; whereas the ATP synthase which is essential for mycobacterial growth has recently been contemplated in developing a breakthrough anti-TB drug, diarylquinoline. Since both of these enzymes have been established as validated drug targets; we report a robust and functional relationship between these two enzymes through a series of experiments using Mtb H37Ra. In the present study we report that the mycobacterial ATP synthase alpha subunit is regulated by PtpA. We generated gene knock-out for the enzyme PtpA and subjected to determine the mycobacterial replication and the proteome profile of wild type, mutant (ΔptpA) and complemented (ΔptpA:ptpA) strains of Mtb H37Ra. A substantial amount of decrease in the protein level of ATP synthase alpha subunit (AtpA) in case of mutant H37Ra was observed, while the levels of the enzyme were either increased or remained unchanged, in wild type and in the complemented strains.

The correlation of mycobactericidal property of macrophages with its potential to deliver bacteria to hydrolytic lysosomes, augmented with ubiquitin-derived peptides (Ub2), activates the process of autophagy. This leads to the formation of phagolysosomes supported by factor involving increased cationic charges which regulate the acidic pH causing elimination of Mycobacterium. To better understand this interaction of cationic-rich ubiquitin-derived peptides with mycobacteria and to identify putative mycobacterial intrinsic resistance mechanisms for phagolysosome formation, we have synthesized a new series of Ub2 peptides, wherein the Gly residues are replaced with azaGly with the aim to improve metabolic stability. In addition to that a new methodology is reported for the synthesis of heteroaryl tethered peptides using azaGly as a linker. We have demonstrated that positive puncta (directly proportional to the acidification of lysosome) in cytosol was significantly increased after 6 hours on the treatment of macrophage with Ub2 peptide derivatives (1, 6, 10, and 11) causing the higher intensity of lysosome observed through LysoTracker Red Dye. The circular dichroism spectral studies are carried out in water and water:TFE mixture and demonstrated that the Ub2 peptides have helix-forming tendency in the presence of TFE. The recognizable intracellular killing of Mycobacterium tuberculosis by Ub2 peptides provides a new approach for host-directed therapy.

Pulmonary infections caused by the group of nontuberculosis mycobacteria (NTM), Mycobacterium avium complex (MAC), are a growing public health concern with incidence and mortality steadily increasing globally. Granulomatous inflammation is the hallmark of MAC lung infection, yet reliable correlates of disease progression, susceptibility, and resolution are poorly defined. Unlike widely used inbred mouse strains, mice that carry the mutant allele at the genetic locus sst1 develop human-like pulmonary tuberculosis featuring well-organized caseating granulomas. We characterized pulmonary temporospatial outcomes of intranasal and left intrabronchial M. avium spp. hominissuis (M.av) induced pneumonia in B6.Sst1S mice, which carries the sst1 mutant allele. We utilized traditional semi-quantitative histomorphological evaluation, in combination with fluorescent multiplex immunohistochemistry (fmIHC), whole slide imaging, and quantitative digital image analysis. Followingintrabronchiolar infection with the laboratory M.av strain 101, the B6.Sst1S pulmonary lesions progressed 12–16 weeks post infection (wpi), with plateauing and/or resolving disease by 21 wpi. Caseating granulomas were not observed during the study. Disease progression from 12–16 wpi was associated with increased acid-fast bacilli, area of secondary granulomatous pneumonia lesions, and Arg1+ and double positive iNOS+/Arg1+ macrophages. Compared to B6 WT, at 16 wpi, B6.Sst1S lungs exhibited an increased area of acid-fast bacilli, larger secondary lesions with greater Arg1+ and double positive iNOS+/Arg1+ macrophages, and reduced T cell density. This morphomolecular analysis of histologic correlates of disease progression in B6.Sst1S could serve as a platform for assessment of medical countermeasures against NTM infection.

Tuberculosis remains a critical infectious disease world-wide. The development of novel therapeutic strategies requires greater understanding of host factors that contribute to disease susceptibility. A major unknown in TB pathogenesis is the mechanism of necrosis in TB granulomas that leads to the massive lung tissue damage and cavity formation necessary for the pathogen transmission. In humans, TB progression has been linked to hyperactivity of type I IFN (IFN-I) pathway, the primary cause of which remains elusive.We studied the mechanistic drivers of pulmonary TB progression using a unique model B6J.C3-Sst1C3HeB/Fej Krmn mice that develop human-like necrotic TB granulomas and IFN-I hyperactivity. We established that IFNβ super-induction occurred in the susceptible macrophages in response to continuous TNF stimulation in the context of a dysregulated antioxidant defense. We observed that unresolving oxidative stress amplified the induction of IFNβ through JNK activation and induced the Integrated Stress Response via PKR activation as a compensatory pathway. Subsequently, PKR amplifies IFNβ upregulation, forming a positive feedback loop, maintaining the hyperinflammatory state in susceptible macrophages and leading to mitochondrial dysfunction. Thus, within the inflammatory milieu, a cell-intrinsic mechanism of chronic regulatory dysfunction and unresolved stress gradually weakens the macrophage and ultimately promotes the necrotization of TB granulomas. The aberrant macrophage response to TNF can be prevented by an iron chelator and inhibitor of lipid peroxidation, ferrostatin-1. Moreover, ferrostatin treatment increased macrophage survival and boosted bacterial control in the TNF-stimulated macrophages infected with virulent Mtb. These findings identify targets for host-directed therapeutics to interrupt necrotization in TB granulomas.

Macrophages contribute to host immunity and tissue homeostasis via alternative activation programs. M1-like macrophages control intracellular bacterial pathogens and tumor progression. In contrast, M2-like macrophages shape reparative microenvironments that can be conducive for pathogen survival or tumor growth. An imbalance of these macrophages phenotypes may perpetuate sites of chronic unresolved inflammation, such as infectious granulomas and solid tumors.We have found that plant-derived and synthetic rocaglates sensitize macrophages to low concentrations of the M1-inducing cytokine IFN-gamma and inhibit their responsiveness to IL-4, a prototypical activator of the M2-like phenotype. Treatement of primary macrophages with rocaglates increased their resilience to oxidative stress, stimulated autophagy and killing of intracellular mycobacteria. Thus, rocaglates represent a novel class of immunomodulators that can direct macrophage polarization towards the M1-like phenotype in complex microenvironments associated with hypofunction of type 1 and/or hyperactivation of type 2 immunity, e.g. chronic bacterial infections, allergies and, possibly, certain tumors.

Isoprinosine (Inos) or immunovir is a synthetic purine derivative with immune-modulatory and antiviral properties. The drug shows apparent in vivo enhancement of host immune responses by inducing pro-inflammatory cytokines and rapid proliferation of T-cell subsets. Strikingly, the cytokines induced by Inos also play crucial roles in providing immune resistance against Mycobacterium tuberculosis (Mtb). Inos has been licensed for several antiviral diseases; however, its efficacy against Mtb has not been tested yet. Since Mtb subverts the host immune system to survive within the host. Therefore, we hypothesized that the immune-stimulatory properties of Inos can be explored as an adjunct therapy for the management of tuberculosis. We have also outlined a systematic direction of study to evaluate if Inos could be repurposed for tuberculosis. The in vivo studies for therapeutic evaluation of Inos alone or in combination with the first line anti-TB drugs in a suitable TB disease model would provide a clearer picture of its utility as a host-directed anti-TB drug and may endow us with a new application of an existing drug to combat tuberculosis.

The ESAT-6 family proteins of Mycobacterium tuberculosis are regarded as the key mediators in mycobacterial virulence and are largely considered as antigens that can improve TB vaccines and diagnostics. We have characterized Rv3444c and Rv3445c proteins of the ESX-4 system of ESAT-6 family of M. tuberculosis H37Rv, and have experimentally established that these two proteins interact to form a heterodimeric complex. Complex formation resulted in induction of α-helical conformation and stability against chemical denaturation. To evaluate the immunogenic potential, we have immunized mice with Rv3444c or Rv3445c along with Freund's incomplete adjuvant (FIA). Immunization with Rv3444c-FIA or Rv3445c-FIA resulted in long term humoral responses. Re-stimulation of splenocytes from immunized mice resulted in significant lymphocyte proliferation with induction of TNF-α and IL-6. Further, the humoral responses to Rv3444c and Rv3445c antigens in Indian patients with active pulmonary TB (n = 44), and healthy individuals (n = 20), were investigated. Compared to healthy individuals, high levels of IgG against Rv3444c and Rv3445c were observed in TB patient's sera, indicating that these proteins are actively produced during the active phase of TB. Cellular immune responses to these proteins in active pulmonary TB patients (n = 5) were also investigated using peripheral blood mononuclear cells (PBMCs). Both the proteins induce significant lymphocyte proliferation and up-regulate the induction of TNF-α and IL-6 in TB patients.

Early secretory antigenic target protein (ESAT-6) is an important virulent factor which plays a crucial role in Mycobacterium tuberculosis (MTB) pathogenesis. Here, we demonstrate the role of ESAT-6 in phagocytosis and intracellular survival of mycobacteria through a mechanism mediated by regulation of a host protein; Peroxiredoxin-1 (Prdx-1). Prdx-1 is an anti-apoptotic and stress response protein which protects cells from damage by ROS and H 2 O 2 . The J774 A.1 cells infected with MTB or over-expressing ESAT-6 through eukaryotic promoter vector showed elevated expression of Prdx-1. Further investigation revealed that the up-regulation of Prdx-1 is mediated through the activation of one of the MAP kinases, p38. The NRF-2, a transcriptional activator of Prdx-1 is translocated to the nucleus upon phosphorylation by p38 and subsequently, regulates expression of Prdx-1. Inhibition of the p38 MAPK by a specific inhibitor, SB203580, abrogates the ESAT-6 mediated induction of Prdx-1 expression as well as the phosphorylation of NRF-2 in a time-dependent manner. The inhibition of Prdx-1 expression by specific siRNA in J774 A.1 cells resulted in the reduced bacterial uptake and intracellular survival of the mycobacteria. This is the first report proclaiming that the ESAT-6 regulates Prdx-1 which is involved in the increase of mycobacterial uptake and survival. The intermediate mechanisms involve the increased Prdx-1 production in macrophages through the activation of p38 and NRF-2 dependent signaling.

Background: In the recent years mitochondria has emerged as one of the main target for Mycobacterium tuberculosis (Mtb) because of its multiple biological function. Herein we have investigated the Mtb proteins translocating to the mitochondria of the host macrophages. Objective: The aim of the study was to identify the mycobacterial protein translocating to the mitochondria of the host macrophages after infection and its possible role in tuberculosis pathogenesis. Methods: We confirmed the translocation of several mycobacterial proteins from cytosol to mitochondria using TB patient sera. 1D Nano LC MS/MS was employed further to identify the proteins in mitochondrial fractions. Identified protein, Rv0674 was cloned, purified and over expressed in Mycobacterium smegmatis (MSMEG). The role of this protein was evaluated for the growth, intracellular survival, mitochondrial membrane potential, apoptosis and ATP production. Results and Conclusion: We identified a conserved hypothetical protein (Rv0674) from Mtb infected mitochondrial fractions of macrophages. The gene of this protein was decoded and characterized through a series of molecular approaches by recombinantly expressing it in MSMEG. The protein was found to be localized in the mitochondria of infected macrophages, in the macrophages of BAL fluid and drug resistant cases. Rv0674 plays significant roles in the apoptosis of infected macrophages, regulating mitochondrial proteins of the OXPHOS complexes which lead to ATP production and mitochondrial membrane potential. It is responsible for prolonging the growth of MSMEG and promoting its survival in infected macrophages

The remarkable ability of Mycobacterium tuberculosis (Mtb) to survive inside human macrophages is attributed to the presence of a complex sensory and regulatory network. PrrA is a DNA-binding regulatory protein, belonging to an essential two-component system (TCS), PrrA/B, which is required for early phase intracellular replication of Mtb. Despite its importance, the mechanism of PrrA/B-mediated signaling is not well understood. In the present study, we demonstrate that the binding of PrrA on the promoter DNA and its consequent activation is cumulatively controlled via dual phosphorylation of the protein. We have further characterized the role of terminal phospho-acceptor domain in the physical interaction of PrrA with its cognate kinase PrrB. The genetic deletion of prrA/B in Mycobacterium smegmatis was possible only in the presence of ectopic copies of the genes, suggesting the essentiality of this TCS in fast-growing mycobacterial strains as well. The overexpression of phospho-mimetic mutant (T6D) altered the growth of M. smegmatis in an in vitro culture and affected the replication of Mycobacterium bovis BCG in mouse peritoneal macrophages. Interestingly, the Thr6 site was found to be conserved in Mtb complex, whereas it was altered in some fast-growing mycobacterial strains, indicating that this unique phosphorylation might be predominant in employing the regulatory circuit in M. bovis BCG and presumably also in Mtb complex.

Rhamnolipids (RLs) are the bacterial derived biosurfactants and known for a wide range of industrial and therapeutic applications. They exhibit potent anti-bacterial activity against various gram positive, gram negative and acid fast bacteria including Mycobacterium tuberculosis . Since, Pseudomonas is one of the largest known genuses containing a variety of rhamnolipid producing strains. Therefore, in this study, we selectively isolated the Pseudomonas aeruginosa strains from the rhizospheric soil of the Indian plants of medicinal value, e.g. Azadirachta Indica and Ficus spp ., and evaluated them for their natural ability to produce antibacterial rhamnolipids. The bacteria were identified on the basis of 16s rRNA sequencing and biochemical characterization. Among 33 of P. aeruginosa isolates from different soil samples, four isolates showed potent inhibitory activity against methicillin resistant Staphylococcus aureus (MRSA) and fast grower mycobacterial spp. The inhibitory potential of the isolates was found to be correlated with their ability to produce RLs in the medium. The industrial viability of the strains was assessed on the basis of cytotoxicity determining alternative allele, exoS/exoU and cell mediated cytotoxicity against murine macrophages J774.1. The newly isolated strains harbor exoS allele and exhibits lower cell mediated cytotoxicity on macrophage cell line as compared to the clinical strains PA-BAA-427 and PA-27853 used as a control in this study. Evaluation of antimycobacterial rhamnolipid production from non-cytotoxic strains of Pseudomonas aeruginosa isolated from rhizospheric soil of medicinal plants