Derailing the aspartate pathway of Mycobacterium tuberculosis to eradicate persistent infection

Abstract A major constraint for developing new anti-tuberculosis drugs is the limited number of validated targets that allow eradication of persistent infections. Here, we uncover a vulnerable component of Mycobacterium tuberculosis (Mtb) persistence metabolism, the aspartate pathway. Rapid death of threonine and homoserine auxotrophs points to a distinct susceptibility of Mtb to inhibition of this pathway. Combinatorial metabolomic and transcriptomic analysis reveals that inability to produce threonine leads to deregulation of aspartate kinase, causing flux imbalance and lysine and DAP accumulation. Mtb’s adaptive response to this metabolic stress involves a relief valve-like mechanism combining lysine export and catabolism via aminoadipate. We present evidence that inhibition of the aspartate pathway at different branch-point enzymes leads to clearance of chronic infections. Together these findings demonstrate that the aspartate pathway in Mtb relies on a combination of metabolic control mechanisms, is required for persistence, and represents a target space for anti-tuberculosis drug development.

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Obesity-Induced Dysbiosis Exacerbates IFN-γ Production and Pulmonary Inflammation in the Mycobacterium tuberculosis Infection

Cells ◽

10.3390/cells10071732 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1732

Author(s):

Sandra Patricia Palma Albornoz ◽

Thais Fernanda de Campos Fraga-Silva ◽

Ana Flávia Gembre ◽

Rômulo Silva de Oliveira ◽

Fernanda Mesquita de Souza ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Gut Microbiota ◽

Inflammatory Diseases ◽

Pulmonary Inflammation ◽

Host Susceptibility ◽

Chronic Infections ◽

Mycobacterium Tuberculosis Infection ◽

Pulmonary Damage ◽

Ifn Γ

The microbiota of the gut–lung axis affects local and far-reaching immune responses and might also trigger chronic and inflammatory diseases. We hypothesized that gut dysbiosis induced by obesity, which coexists in countries with a high tuberculosis burden, aggravates the host susceptibility and the pulmonary damage tolerance. To assess our hypothesis, we used a model of high-fat diet (HFD)-induced obesity, followed by infection of C57BL/6 mice with Mycobacterium tuberculosis. We showed that obesity increased the susceptibility, the pulmonary inflammation and IFN-γ levels in M. tuberculosis-infected mice. During the comorbidity obesity and tuberculosis, there is an increase of Bacteroidetes and Firmicutes in the lungs, and an increase of Firmicutes and butyrate in the feces. Depletion of gut microbiota by antibiotic treatment in the obese infected mice reduced the frequencies of CD4+IFN-γ+IL-17− cells and IFN-γ levels in the lungs, associated with an increase of Lactobacillus. Our findings reinforce the role of the gut–lung axis in chronic infections and suggest that the gut microbiota modulation may be a potential host-directed therapy as an adjuvant to treat TB in the context of IFN-γ-mediated immunopathology.

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Neither Primary nor Memory Immunity to Mycobacterium tuberculosis Infection Is Compromised in Mice with Chronic Enteric Helminth Infection

Infection and Immunity ◽

10.1128/iai.03004-14 ◽

2015 ◽

Vol 83 (3) ◽

pp. 1217-1223 ◽

Cited By ~ 15

Author(s):

Wasiulla Rafi ◽

Kamlesh Bhatt ◽

William C. Gause ◽

Padmini Salgame

Keyword(s):

Mycobacterium Tuberculosis ◽

T Regulatory Cells ◽

Helminth Infection ◽

Treg Cells ◽

Helminth Species ◽

Nippostrongylus Brasiliensis ◽

T Regulatory Cell ◽

Chronic Infections ◽

Mycobacterium Tuberculosis Infection ◽

Content Type

Previously we had reported thatNippostrongylus brasiliensis, a helminth with a lung migratory phase, affected host resistance againstMycobacterium tuberculosisinfection through the induction of alternatively activated (M2) macrophages. Several helminth species do not have an obligatory lung migratory phase but establish chronic infections in the host that include potent immune downregulatory effects, in part mediated through induction of a FoxP3+T regulatory cell (Treg) response. Treg cells exhibit duality in their functions in host defense againstM. tuberculosisinfection since their depletion leads to enhanced priming of T cells in the lymph nodes and attendant improved control ofM. tuberculosisinfection, while their presence in the lung granuloma protects against excessive inflammation.Heligmosomoides polygyrusis a strictly murine enteric nematode that induces a strong FoxP3 Treg response in the host. Therefore, in this study we investigated whether host immunity toM. tuberculosisinfection would be modulated in mice with chronicH. polygyrusinfection. We report that neither primary nor memory immunity conferred byMycobacterium bovisBCG vaccination was affected in mice with chronic enteric helminth infection, despite a systemic increase in FoxP3+T regulatory cells. The findings indicate that anti-M. tuberculosisimmunity is not similarly affected by all helminth species and highlight the need to consider this inequality in human coinfection studies.

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BacA, an ABC Transporter Involved in Maintenance of Chronic Murine Infections with Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.01132-08 ◽

2008 ◽

Vol 191 (2) ◽

pp. 477-485 ◽

Cited By ~ 53

Author(s):

Pilar Domenech ◽

Hajime Kobayashi ◽

Kristin LeVier ◽

Graham C. Walker ◽

Clifton E. Barry

Keyword(s):

Mycobacterium Tuberculosis ◽

Comprehensive Evaluation ◽

Cell Envelope ◽

Membrane Integrity ◽

Chronic Infections ◽

Host Pathogen Interaction ◽

Host Pathogen ◽

Ultimate Outcome ◽

Inner Membrane Protein ◽

Related Proteins

ABSTRACT BacA is an inner membrane protein associated with maintenance of chronic infections in several diverse host-pathogen interactions. To understand the function of the bacA gene in Mycobacterium tuberculosis (Rv1819c), we insertionally inactivated this gene and analyzed the resulting mutant for a variety of phenotypes. BacA deficiency in M. tuberculosis did not affect sensitivity to detergents, acidic pH, and zinc, indicating that there was no global compromise in membrane integrity, and a comprehensive evaluation of the major lipid constituents of the cell envelope failed to reveal any significant differences. Infection of mice with this mutant revealed no impact on establishment of infection but a profound effect on maintenance of extended chronic infection and ultimate outcome. As in alphaproteobacteria, deletion of BacA in M. tuberculosis led to increased bleomycin resistance, and heterologous expression of the M. tuberculosis BacA homolog in Escherichia coli conferred sensitivity to antimicrobial peptides. These results suggest a striking conservation of function for BacA-related proteins in transport of a critical molecule that determines the outcome of the host-pathogen interaction.

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Arginine-deprivation–induced oxidative damage sterilizes Mycobacterium tuberculosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808874115 ◽

2018 ◽

Vol 115 (39) ◽

pp. 9779-9784 ◽

Cited By ~ 22

Author(s):

Sangeeta Tiwari ◽

Andries J. van Tonder ◽

Catherine Vilchèze ◽

Vitor Mendes ◽

Sherine E. Thomas ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Mycobacterium Tuberculosis ◽

De Novo ◽

Target Space ◽

Biosynthesis Pathway ◽

Arginine Biosynthesis ◽

Arginine Deprivation

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

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Oxidative Phosphorylation—an Update on a New, Essential Target Space for Drug Discovery in Mycobacterium tuberculosis

Applied Sciences ◽

10.3390/app10072339 ◽

2020 ◽

Vol 10 (7) ◽

pp. 2339 ◽

Cited By ~ 2

Author(s):

Caroline Shi-Yan Foo ◽

Kevin Pethe ◽

Andréanne Lupien

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Discovery ◽

Oxidative Phosphorylation ◽

New Drugs ◽

Target Space ◽

Future Directions ◽

Tb Treatment ◽

Fda Approval ◽

The Subject ◽

Synthase Inhibitor

New drugs with new mechanisms of action are urgently required to tackle the global tuberculosis epidemic. Following the FDA-approval of the ATP synthase inhibitor bedaquiline (Sirturo®), energy metabolism has become the subject of intense focus as a novel pathway to exploit for tuberculosis drug development. This enthusiasm stems from the fact that oxidative phosphorylation (OxPhos) and the maintenance of the transmembrane electrochemical gradient are essential for the viability of replicating and non-replicating Mycobacterium tuberculosis (M. tb), the etiological agent of human tuberculosis (TB). Therefore, new drugs targeting this pathway have the potential to shorten TB treatment, which is one of the major goals of TB drug discovery. This review summarises the latest and key findings regarding the OxPhos pathway in M. tb and provides an overview of the inhibitors targeting various components. We also discuss the potential of new regimens containing these inhibitors, the flexibility of this pathway and, consequently, the complexity in targeting it. Lastly, we discuss opportunities and future directions of this drug target space.

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Mycobacterium tuberculosis Diverts Alpha Interferon-Induced Monocyte Differentiation from Dendritic Cells into Immunoprivileged Macrophage-Like Host Cells

Infection and Immunity ◽

10.1128/iai.72.8.4385-4392.2004 ◽

2004 ◽

Vol 72 (8) ◽

pp. 4385-4392 ◽

Cited By ~ 36

Author(s):

Sabrina Mariotti ◽

Raffaela Teloni ◽

Elisabetta Iona ◽

Lanfranco Fattorini ◽

Giulia Romagnoli ◽

...

Keyword(s):

Dendritic Cells ◽

Mycobacterium Tuberculosis ◽

Immune Surveillance ◽

Interleukin 10 ◽

T Cell Response ◽

Host Cells ◽

Alpha Interferon ◽

Chronic Infections ◽

Necrosis Factor Alpha ◽

Monocyte Differentiation

ABSTRACT Dendritic cells (DCs) are critical for initiating a pathogen-specific T-cell response. During chronic infections the pool of tissue DCs must be renewed by recruitment of both circulating DC progenitors and in loco differentiating monocytes. However, the interaction of monocytes with pathogens could affect their differentiation. Mycobacterium tuberculosis has been shown to variably interfere with the generation and function of antigen-presenting cells (APCs). In this study we found that when alpha interferon (IFN-α) is used as an inductor of monocyte differentiation, M. tuberculosis inhibits the generation of DCs, forcing the generation of immunoprivileged macrophage-like cells instead. Cells derived from M. tuberculosis-infected monocyte-derived macrophages (M. tuberculosis-infected MoMφ) retained CD14 without acquiring CD1 molecules and partially expressed B7.2 but did not up-regulate B7.1 and major histocompatibility complex (MHC) class I and II molecules. They synthesized tumor necrosis factor alpha and interleukin-10 (IL-10) but not IL-12. They also showed a reduced ability to induce proliferation and functional polarization of allogeneic T lymphocytes. Thus, in the presence of IFN-α, M. tuberculosis may hamper the renewal of potent APCs, such as DCs, generating a safe habitat for intracellular growth. M. tuberculosis-infected MoMφ, in fact, showed reduced expression of both signal 1 (CD1, MHC classes I and II) and signal 2 (B7.1 and B7.2), which are essential for mycobacterium-specific T-lymphocyte priming and/or activation. These data further suggest that M. tuberculosis has the ability to specifically interfere with monocyte differentiation. This ability may represent an effective M. tuberculosis strategy for eluding immune surveillance and persisting in the host.

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Deletion of the Mycobacterium tuberculosis Resuscitation-Promoting Factor Rv1009 Gene Results in Delayed Reactivation from Chronic Tuberculosis

Infection and Immunity ◽

10.1128/iai.74.5.2985-2995.2006 ◽

2006 ◽

Vol 74 (5) ◽

pp. 2985-2995 ◽

Cited By ~ 89

Author(s):

JoAnn M. Tufariello ◽

Kaixia Mi ◽

Jiayong Xu ◽

Yukari C. Manabe ◽

Anup K. Kesavan ◽

...

Keyword(s):

Gene Expression ◽

Mycobacterium Tuberculosis ◽

Human Population ◽

Acute Infection ◽

Dependent Manner ◽

Survival Times ◽

Chronic Infections ◽

Synthase Inhibitor

ABSTRACT Approximately one-third of the human population is latently infected with Mycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintaining M. tuberculosis in the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five related M. tuberculosis proteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role of M. tuberculosis rpf genes in an established mouse model of M. tuberculosis persistence and reactivation. After an aerosol infection with the M. tuberculosis Erdman wild type (Erdman) or single-deletion rpf mutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the five rpf deletion mutants tested, one (ΔRv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman- and ΔRv1009-infected mice at advanced stages of reactivation. Analysis of rpf gene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the five rpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting that rpf gene expression may be regulated in a growth phase-dependent manner. To our knowledge, ΔRv1009 is the first M. tuberculosis mutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.

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Higher order Rab programming in phagolysosome biogenesis

The Journal of Cell Biology ◽

10.1083/jcb.200603026 ◽

2006 ◽

Vol 174 (7) ◽

pp. 923-929 ◽

Cited By ~ 86

Author(s):

Esteban A. Roberts ◽

Jennifer Chua ◽

George B. Kyei ◽

Vojo Deretic

Keyword(s):

Mycobacterium Tuberculosis ◽

Higher Order ◽

Intracellular Pathogen ◽

Control Mechanisms ◽

Rab Gtpases ◽

Endosomal Compartment ◽

Phagosomal Maturation ◽

Naturally Occurring ◽

Latex Beads

Phagosomes offer kinetically and morphologically tractable organelles to dissect the control of phagolysosome biogenesis by Rab GTPases. Model phagosomes harboring latex beads undergo a coordinated Rab5–Rab7 exchange, which is akin to the process of endosomal Rab conversion, the control mechanisms of which are unknown. In the process of blocking phagosomal maturation, the intracellular pathogen Mycobacterium tuberculosis prevents Rab7 acquisition, thus, providing a naturally occurring tool to study Rab conversion. We show that M. tuberculosis inhibition of Rab7 acquisition and arrest of phagosomal maturation depends on Rab22a. Four-dimensional microscopy revealed that phagosomes harboring live mycobacteria recruited and retained increasing amounts of Rab22a. Rab22a knockdown in macrophages via siRNA enhanced the maturation of phagosomes with live mycobacteria. Conversely, overexpression of the GTP-locked mutant Rab22aQ64L prevented maturation of phagosomes containing heat-killed mycobacteria, which normally progress into phagolysosomes. Moreover, Rab22a knockdown led to Rab7 acquisition by phagosomes harboring live mycobacteria. Our findings show that Rab22a defines the critical checkpoint for Rab7 conversion on phagosomes, allowing or disallowing organellar transition into a late endosomal compartment. M. tuberculosis parasitizes this process by actively recruiting and maintaining Rab22a on its phagosome, thus, preventing Rab7 acquisition and blocking phagolysosomal biogenesis.

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Cathepsins and Their Endogenous Inhibitors in Host Defense During Mycobacterium tuberculosis and HIV Infection

Frontiers in Immunology ◽

10.3389/fimmu.2021.726984 ◽

2021 ◽

Vol 12 ◽

Author(s):

Elsa Anes ◽

José Miguel Azevedo-Pereira ◽

David Pires

Keyword(s):

Mycobacterium Tuberculosis ◽

Bacterial Pathogen ◽

Hiv Infections ◽

Pathogen Transmission ◽

Host Cells ◽

Chronic Infections ◽

Host Immune Responses ◽

Endogenous Inhibitors ◽

Innovative Therapies ◽

The Moment

The moment a very old bacterial pathogen met a young virus from the 80’s defined the beginning of a tragic syndemic for humanity. Such is the case for the causative agent of tuberculosis and the human immunodeficiency virus (HIV). Syndemic is by definition a convergence of more than one disease resulting in magnification of their burden. Both pathogens work synergistically contributing to speed up the replication of each other. Mycobacterium tuberculosis (Mtb) and HIV infections are in the 21st century among the leaders of morbidity and mortality of humankind. There is an urgent need for development of new approaches for prevention, better diagnosis, and new therapies for both infections. Moreover, these approaches should consider Mtb and HIV as a co-infection, rather than just as separate problems, to prevent further aggravation of the HIV-TB syndemic. Both pathogens manipulate the host immune responses to establish chronic infections in intracellular niches of their host cells. This includes manipulation of host relevant antimicrobial proteases such as cathepsins or their endogenous inhibitors. Here we discuss recent understanding on how Mtb and HIV interact with cathepsins and their inhibitors in their multifactorial functions during the pathogenesis of both infections. Particularly we will address the role on pathogen transmission, during establishment of intracellular chronic niches and in granuloma clinical outcome and tuberculosis diagnosis. This area of research will open new avenues for the design of innovative therapies and diagnostic interventions so urgently needed to fight this threat to humanity.

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Isolation of enzymes involved in threonine biosynthesis from sorghum seeds

Brazilian Journal of Plant Physiology ◽

10.1590/s1677-04202004000200005 ◽

2004 ◽

Vol 16 (2) ◽

pp. 95-104 ◽

Cited By ~ 2

Author(s):

Renato Rodrigues Ferreira ◽

Ariane Vendemiatti ◽

Lyndel Wayne Meinhardt ◽

Peter John Lea ◽

Ricardo Antunes Azevedo

Keyword(s):

Amino Acids ◽

Essential Amino Acids ◽

Aspartate Kinase ◽

Homoserine Dehydrogenase ◽

Higher Plant ◽

Level Of Activity ◽

Immature Seeds ◽

Aspartate Pathway ◽

Threonine Biosynthesis

Cereal seeds are poor in essential amino acids, particularly lysine, tryptophan and threonine. The amino acids lysine and threonine are synthesized in the aspartate pathway. Although most of the enzymes of the aspartate pathway have been isolated and characterized in higher plant species, the metabolism of lysine and threonine is totally unknown in sorghum. We have isolated two enzymes, aspartate kinase (AK) and homoserine dehydrogenase (HSDH) from sorghum. Optimum assay conditions were established for the determination of AK and HSDH activities. The highest level of activity was observed in immature seeds. AK was shown to be inhibited by threonine and lysine indicating the existence of at least two isoenzymes, one sensitive to threonine inhibition and the other sensitive to lysine inhibition with the latter being predominant in sorghum seeds. HSDH was shown to be inhibited by threonine indicating the existence of a threonine-sensitive HSDH, however, most of the activity was not inhibited by threonine, suggesting the existence of a second predominant isoenzyme of HSDH resistant to threonine inhibition.

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