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A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop clinical, mostly pulmonary, TB. To dissect mechanisms of susceptibility in immunocompetent individuals, we developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: development of necrotic lung lesions after infection with virulent Mtb. In this study, we explored the connectivity of the sst1-regulated pathways during prolonged macrophage activation with TNF. We determined that the aberrant response of the sst1-susceptible macrophages to TNF was primarily driven by conflicting Myc and antioxidant response pathways that resulted in a coordinated failure to properly sequester intracellular iron and activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled IFNß superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. Our findings suggest a novel link between metabolic dysregulation in macrophages and susceptibility to TB, offering insights into potential therapeutic targets aimed at modulating macrophage function and improving TB control.
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In the ongoing battle against antimicrobial resistance, phenotypic drug tolerance poses a formidable challenge. This adaptive ability of microorganisms to withstand drug pressure without genetic alterations further complicating global healthcare challenges. Microbial populations employ an array of persistence mechanisms, including dormancy, biofilm formation, adaptation to intracellular environments, and the adoption of L-forms, to develop drug tolerance. Moreover, molecular mechanisms like toxin-antitoxin modules, oxidative stress responses, energy metabolism, and (p)ppGpp signaling contribute to this phenomenon. Understanding these persistence mechanisms is crucial for predicting drug efficacy, developing strategies for chronic bacterial infections, and exploring innovative therapies for refractory infections. In this comprehensive review, we dissect the intricacies of drug tolerance and persister formation, explore their role in acquired drug resistance, and highlight emerging therapeutic approaches to combat phenotypic drug tolerance. Furthermore, we outline the future landscape of interventions for persistent bacterial infections.
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Antibacterianos , Bactérias , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Tolerância a Medicamentos , Farmacorresistência Bacteriana , Estresse Oxidativo/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , FenótipoRESUMO
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 the tuberculosis (TB) susceptible and 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 tumor necrosis factor (TNF)-activated TB-susceptible macrophages toward that of TB-resistant cells, i.e., prevents their aberrant activation without suppressing beneficial TNF responses. Last, we demonstrate that the compounds identified with this approach enhance the resistance of the TB-susceptible mouse macrophages to virulent Mycobacterium tuberculosis.
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Mycobacterium tuberculosis , Tuberculose , Camundongos , Animais , Tuberculose/microbiologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/genética , Suscetibilidade a Doenças , Fator de Necrose Tumoral alfa/genéticaRESUMO
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 virulent M. tuberculosis .
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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.
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Infecção por Mycobacterium avium-intracellulare , Pneumonia , Animais , Modelos Animais de Doenças , Progressão da Doença , Suscetibilidade a Doenças , Granuloma , Camundongos , Camundongos Endogâmicos , Mycobacterium avium , Complexo Mycobacterium avium , Infecção por Mycobacterium avium-intracellulare/epidemiologiaRESUMO
Here, we present a streamlined protocol for assessing intracellular Mycobacterium tuberculosis (Mtb) loads in macrophages. This protocol describes the simultaneous assessment of macrophage viability using automated microscopy. Further, we detail the quantification of mycobacterial loads using a rapid, inexpensive, and accurate approach for mycobacterial DNA isolation from paraformaldehyde-fixed macrophages. Simultaneous assessment of the bacterial loads using internal standard and macrophage viability allows for precise quantification of the effects of perturbations on Mtb and host cells while accounting for technical artifacts. For complete details on the use and execution of this protocol, please refer to Chatterjee et al. (2021).
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Mycobacterium tuberculosis , Macrófagos/microbiologia , Mycobacterium tuberculosis/genéticaRESUMO
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.
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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.
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Vacina BCG/uso terapêutico , Vacinas contra COVID-19/uso terapêutico , COVID-19/terapia , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Antivirais/uso terapêutico , Vacina BCG/imunologia , COVID-19/epidemiologia , COVID-19/mortalidade , Vacinas contra COVID-19/imunologia , Humanos , Vacinação em Massa , Mycobacterium bovis/imunologia , Pandemias , SARS-CoV-2/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
Mycobacterium is known for subverting the host defense machinery, and one such mechanism is the inhibition of autophagy. Here, we have demonstrated that Mycobacterium tuberculosis (MTB) secretes a virulence factor; an early secretory antigenic target protein (ESAT-6) into the phagosome, which induces the expression and activity of mitochondrial superoxide dismutase (SOD-2) of macrophages. Using a series of experiments, and Mycobacterium bovis BCG as a model strain (where ESAT-6 protein is not expressed), we have delineated that the protein regulates SOD-2 of macrophages. The expression and augmentation of SOD-2 activity were confirmed by either incubating the macrophages with ESAT-6 protein, transfection of macrophage by esat6 gene using a eukaryotic promoter vector, or by infection with different mycobacterial strains. The induction of acidification of phagosomal compartment containing bacteria was observed in cells that express low levels of SOD-2. This was further confirmed by observing a significant decrease in the M. bovis BCG intracellular load in the sod-2 knocked-down macrophages.
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Antígenos de Bactérias/metabolismo , Autofagia , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Infecções por Mycobacterium/metabolismo , Infecções por Mycobacterium/microbiologia , Mycobacterium bovis/fisiologia , Superóxido Dismutase/metabolismo , Animais , Autofagossomos , Linhagem Celular , Ativação Enzimática , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Viabilidade Microbiana , Recombinação Genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
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Proteínas de Bactérias/fisiologia , ATPases Bacterianas Próton-Translocadoras/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Proteínas Tirosina Fosfatases/fisiologia , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Diarilquinolinas/farmacologia , Técnicas de Inativação de Genes , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Proteínas Tirosina Fosfatases/genéticaRESUMO
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.
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Antituberculosos/síntese química , Peptídeos/química , Ubiquitina/química , Sequência de Aminoácidos , Animais , Antituberculosos/química , Antituberculosos/farmacologia , Compostos Aza/química , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Lisossomos/química , Lisossomos/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/farmacologia , Estrutura Secundária de ProteínaRESUMO
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.
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Antituberculosos/uso terapêutico , Inosina Pranobex/uso terapêutico , Tuberculose/tratamento farmacológico , Adjuvantes Imunológicos/uso terapêutico , Quimioterapia Adjuvante , Citocinas/metabolismo , Reposicionamento de Medicamentos , Humanos , Modelos Imunológicos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologiaRESUMO
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 (nâ¯=â¯44), and healthy individuals (nâ¯=â¯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 (nâ¯=â¯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.
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Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adulto , Animais , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/metabolismo , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Feminino , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunização , Imunogenicidade da Vacina , Índia , Interleucina-6/sangue , Interleucina-6/imunologia , Ativação Linfocitária , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Mycobacterium tuberculosis/metabolismo , Conformação Proteica em alfa-Hélice , Desnaturação Proteica , Estabilidade Proteica , Baço/imunologia , Baço/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/metabolismo , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/imunologia , Adulto JovemRESUMO
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.
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Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Líquido Intracelular/metabolismo , Mycobacterium tuberculosis/metabolismo , Ativação Transcricional/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Líquido Intracelular/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/genética , Fosforilação/fisiologia , CoelhosRESUMO
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 H2O2. 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.