scholarly journals A mixed nitrogen diet and compartmentalized utilization for Mycobacterium tuberculosis replicating in host cells: results of a systems-based analysis

2019 ◽  
Author(s):  
Khushboo Borah ◽  
Martin Beyss ◽  
Axel Theorell ◽  
Huihai Wu ◽  
Piyali Basu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Nitrogen Source ◽  
Nitrogen Metabolism ◽  
Nitrogen Sources ◽  
Spectral Ratio ◽  
Host Cells ◽  
Ratio Analysis ◽  
Nitrogen Donor

Nitrogen metabolism of Mycobacterium tuberculosis (Mtb) is crucial for the survival and virulence of this pathogen inside host macrophages but little is known about the nitrogen sources acquired from the host or their route of assimilation. Here we performed a systems-based analysis of nitrogen metabolism in intracellullar Mtb and developed 15N-Flux Spectral Ratio Analysis (FSRA) to probe the metabolic cross-talk between the host cell and Mtb. We demonstrate that intracellular Mtb acquires nitrogen from multiple amino acids in the macrophage including glutamate, glutamine, aspartate, alanine, glycine and valine, with glutamine being the predominant nitrogen donor. Each nitrogen source is uniquely assimilated into specific intracellular pools indicating compartmentalised metabolism. This was not observed for in vitro-grown Mtb indicating that there is a switch in nitrogen metabolism when the pathogen enters the intracellular environment. These results provide clues about the potential metabolic targets for development of innovative anti-tuberculosis therapies.

scholarly journals Flexible nitrogen utilisation by the metabolic generalist pathogen Mycobacterium tuberculosis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Aleksandra Agapova ◽  
Agnese Serafini ◽  
Michael Petridis ◽  
Debbie M Hunt ◽  
Acely Garza-Garcia ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Nitrogen Source ◽  
Metabolic Network ◽  
Nitrogen Sources ◽  
Ammonium Assimilation ◽  
Bacterial Metabolism ◽  
Bacterial Physiology ◽  
Depth Analysis ◽  
Nitrogen Utilisation

Bacterial metabolism is fundamental to survival and pathogenesis. We explore how Mycobacterium tuberculosis utilises amino acids as nitrogen sources, using a combination of bacterial physiology and stable isotope tracing coupled to mass spectrometry metabolomics methods. Our results define core properties of the nitrogen metabolic network from M. tuberculosis, such as: (i) the lack of homeostatic control of certain amino acid pool sizes; (ii) similar rates of utilisation of different amino acids as sole nitrogen sources; (iii) improved nitrogen utilisation from amino acids compared to ammonium; and (iv) co-metabolism of nitrogen sources. Finally, we discover that alanine dehydrogenase is involved in ammonium assimilation in M. tuberculosis, in addition to its essential role in alanine utilisation as a nitrogen source. This study represents the first in-depth analysis of nitrogen source utilisation by M. tuberculosis and reveals a flexible metabolic network with characteristics that are likely a product of evolution in the human host.

scholarly journals Urease Activity Represents an Alternative Pathway for Mycobacterium tuberculosis Nitrogen Metabolism

2012 ◽  
Vol 80 (8) ◽  
pp. 2771-2779 ◽  
Author(s):  
Wenwei Lin ◽  
Vanessa Mathys ◽  
Emily Lei Yin Ang ◽  
Vanessa Hui Qi Koh ◽  
Julia María Martínez Gómez ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitrogen Source ◽  
Nitrogen Metabolism ◽  
Urease Activity ◽  
Alternative Pathway ◽  
Bacterial Species ◽  
Deficient Mutant ◽  
Content Type ◽  
Metabolic Versatility

ABSTRACTUrease represents a critical virulence factor for some bacterial species through its alkalizing effect, which helps neutralize the acidic microenvironment of the pathogen. In addition, urease serves as a nitrogen source provider for bacterial growth. Pathogenic mycobacteria express a functional urease, but its role during infection has yet to be characterized. In this study, we constructed a urease-deficientMycobacterium tuberculosisstrain and confirmed the alkalizing effect of the urease activity within the mycobacterium-containing vacuole in resting macrophages but not in the more acidic phagolysosomal compartment of activated macrophages. However, the urease-mediated alkalizing effect did not confer any growth advantage onM. tuberculosisin macrophages, as evidenced by comparable growth profiles for the mutant, wild-type (WT), and complemented strains. In contrast, the urease-deficient mutant exhibited impairedin vitrogrowth compared to the WT and complemented strains when urea was the sole source of nitrogen. Substantial amounts of ammonia were produced by the WT and complemented strains, but not with the urease-deficient mutant, which represents the actual nitrogen source for mycobacterial growth. However, the urease-deficient mutant displayed parental colonization profiles in the lungs, spleen, and liver in mice. Together, our data demonstrate a role for the urease activity inM. tuberculosisnitrogen metabolism that could be crucial for the pathogen's survival in nutrient-limited microenvironments where urea is the sole nitrogen source. Our work supports the notion thatM. tuberculosisvirulence correlates with its unique metabolic versatility and ability to utilize virtually any carbon and nitrogen sources available in its environment.

scholarly journals Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo

2016 ◽  
Vol 213 (5) ◽  
pp. 809-825 ◽  
Author(s):  
Yancheng Liu ◽  
Shumin Tan ◽  
Lu Huang ◽  
Robert B. Abramovitch ◽  
Kyle H. Rohde ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Stress Responses ◽  
Drug Action ◽  
Host Cells ◽  
Transcriptional Analysis ◽  
Drug Pressure ◽  
Cytokine Activation ◽  
The Impact

Successful chemotherapy against Mycobacterium tuberculosis (Mtb) must eradicate the bacterium within the context of its host cell. However, our understanding of the impact of this environment on antimycobacterial drug action remains incomplete. Intriguingly, we find that Mtb in myeloid cells isolated from the lungs of experimentally infected mice exhibit tolerance to both isoniazid and rifampin to a degree proportional to the activation status of the host cells. These data are confirmed by in vitro infections of resting versus activated macrophages where cytokine-mediated activation renders Mtb tolerant to four frontline drugs. Transcriptional analysis of intracellular Mtb exposed to drugs identified a set of genes common to all four drugs. The data imply a causal linkage between a loss of fitness caused by drug action and Mtb’s sensitivity to host-derived stresses. Interestingly, the environmental context exerts a more dominant impact on Mtb gene expression than the pressure on the drugs’ primary targets. Mtb’s stress responses to drugs resemble those mobilized after cytokine activation of the host cell. Although host-derived stresses are antimicrobial in nature, they negatively affect drug efficacy. Together, our findings demonstrate that the macrophage environment dominates Mtb’s response to drug pressure and suggest novel routes for future drug discovery programs.

scholarly journals Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Javeed Ahmad ◽  
Aisha Farhana ◽  
Rita Pancsa ◽  
Simran Kaur Arora ◽  
Alagiri Srinivasan ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Terminal Segment ◽  
Host Protein ◽  
Host Cells ◽  
Productive Infection ◽  
Disordered Proteins ◽  
Intrinsically Disordered

ABSTRACT Pathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium-specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion—an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis. IMPORTANCE To hijack the human host cell machinery to enable survival inside macrophages, the pathogen Mycobacterium tuberculosis requires a repertoire of proteins that can mimic host protein function and modulate host cell machinery. Here, we have shown how a single protein can play multiple functions and hijack the host cell for the benefit of the pathogen. Full-length membrane-anchored PPE37 protein is cleaved into N- and C-terminal domains under iron-depleted conditions. The N-terminal domain facilitates the propathogen semimature tolerogenic state of dendritic cells, whereas the C-terminal segment is localized into host cell nucleus and induces apoptosis. The immune implications of these in vitro observations were assessed and validated in mice and also human TB patients. This study presents novel mechanistic insight adopted by M. tuberculosis to survive inside host cells.

Effects of explant position and orientation, medium ph and nitrogen sources on micropropagation of blackberry

2021 ◽  
Vol 12 (4) ◽  
pp. 84-92
Author(s):  
Thábata Cristina Faxina de Aguiar ◽  
Ana Paola Negri ◽  
Pedro Boff ◽  
Murilo Dalla Costa ◽  
Mari Inês Carissimi Boff ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Nitrogen Sources ◽  
Original Position ◽  
Multiplication Rate ◽  
Highest Weight ◽  
Explant Position ◽  
Rubus Species ◽  
Position And Orientation ◽  
Ph Level

Composition of the medium and the explant origin are factors that interfere on success of micropropagation of Rubus species. For blackberry cultivar LochNess it was not investigated yet how the position and orientation of explant, pH levels and nitrogen source interfere on micropropagation. In this work, focused on the establishment of in vitro culture, variables were studied on R. fruticosus cv Loch Ness, such as the choice of the explants depending on their original position on the mother plant, pH level and nitrogen sources of the culture medium. For the first time in vitro on Rubus, the downward orientation (capogatto) of shoot tips explants was compared with the normal upward orientation. The highest weight and length values were recorded for the shoots proliferated from basal and nodal explants. For the initiation medium, the best multiplication rate were obtained in pH adjusted to 4.5. Shoot length was influenced by the nitrogen source; when associated with an increased light intensity, the complete substitution of ammonium by nitrate allowed results comparable with those obtained with the control medium containing both sources. The use of aminoacids did not improve the results. Apex orientation did not affect anatomical parameters or rooting rates of wild Rubus, but more efforts should be devoted on in vitro capogatto technique considering that advantages like reduction of plant growth regulators, cultivation on the same medium culture for more time and easily rooting can be established.

THE IN VITRO RELEASE OF AMINO ACIDS BY RUMEN PAPILLAE

1980 ◽  
Vol 60 (2) ◽  
pp. 281-291 ◽  
Author(s):  
R. J. BOILA ◽  
L. P. MILLIGAN
Keyword(s):  
Amino Acids ◽  
Carbon Sources ◽  
In Vitro Release ◽  
Nitrogen Sources ◽  
Chromatographic Technique ◽  
Salts Of Organic Acids ◽  
Liquid Chromatographic ◽  
Vitro Release ◽  
Effective Source

Rumen papillae from cattle were incubated aerobically with combinations of NH4Cl, amino acids and salts of organic acids, the latter including propionate, pyruvate, α-ketoglutarate and glyoxylate. Amino acids in the incubation media were analyzed using a gas-liquid chromatographic technique entailing separation of the isobutyl-N(0)-heptafluorobutyryl esters: glutamine was recovered with glutamate, asparagine with aspartate, and citrulline with ornithine. Rumen papillae incubated with pyruvate or propionate released alanine, but with the latter substrate only glutamate was effective as a nitrogen source. Glycine and glutamate plus glutamine were released in the presence of glyoxylate and α-ketoglutarate, respectively. Serine and aspartate plus asparagine were not quantitatively major products released by rumen papillae. Glutamate was an effective source of nitrogen for the release of alanine and glycine with pyruvate and glyoxylate, respectively, as carbon sources. When rumen papillae were incubated with pyruvate or glyoxylate as the added carbon source, glutamine nitrogen disappeared and was not accounted for by the amino acids measured. With arginine as a substrate, there was a release of ornithine by rumen papillae indicating urea production. The tissues of rumen papillae appear to synthesize amino acids from expected carbon sources with ammonia or glutamate as nitrogen sources and to catabolize glutamine and arginine. The metabolism of amino acids by rumen papillae would contribute to the interchange of nitrogen between the rumen and the host.

scholarly journals An In Vitro TORC1 Kinase Assay That Recapitulates the Gtr-Independent Glutamine-Responsive TORC1 Activation Mechanism on Yeast Vacuoles

2017 ◽  
Vol 37 (14) ◽  
Author(s):  
Mirai Tanigawa ◽  
Tatsuya Maeda
Keyword(s):  
Amino Acids ◽  
Nitrogen Sources ◽  
Small Gtpases ◽  
Activation Mechanism ◽  
Kinase Assay ◽  
Content Type ◽  
Vacuolar Protein ◽  
Vacuolar Membranes ◽  
First Time

ABSTRACT Evolutionarily conserved target of rapamycin (TOR) complex 1 (TORC1) responds to nutrients, especially amino acids, to promote cell growth. In the yeast Saccharomyces cerevisiae, various nitrogen sources activate TORC1 with different efficiencies, although the mechanism remains elusive. Leucine, and perhaps other amino acids, was reported to activate TORC1 via the heterodimeric small GTPases Gtr1-Gtr2, the orthologues of the mammalian Rag GTPases. More recently, an alternative Gtr-independent TORC1 activation mechanism that may respond to glutamine was reported, although its molecular mechanism is not clear. In studying the nutrient-responsive TORC1 activation mechanism, the lack of an in vitro assay hinders associating particular nutrient compounds with the TORC1 activation status, whereas no in vitro assay that shows nutrient responsiveness has been reported. In this study, we have developed a new in vitro TORC1 kinase assay that reproduces, for the first time, the nutrient-responsive TORC1 activation. This in vitro TORC1 assay recapitulates the previously predicted Gtr-independent glutamine-responsive TORC1 activation mechanism. Using this system, we found that this mechanism specifically responds to l-glutamine, resides on the vacuolar membranes, and involves a previously uncharacterized Vps34-Vps15 phosphatidylinositol (PI) 3-kinase complex and the PI-3-phosphate [PI(3)P]-binding FYVE domain-containing vacuolar protein Pib2. Thus, this system was proved to be useful for dissecting the glutamine-responsive TORC1 activation mechanism.

scholarly journals The Escherichia coli gabDTPC Operon: Specific γ-Aminobutyrate Catabolism and Nonspecific Induction

2002 ◽  
Vol 184 (24) ◽  
pp. 6976-6986 ◽  
Author(s):  
Barbara L. Schneider ◽  
Stephen Ruback ◽  
Alexandros K. Kiupakis ◽  
Hillary Kasbarian ◽  
Christine Pybus ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Nitrogen Limitation ◽  
Nitrogen Sources ◽  
Wild Type ◽  
Gaba A ◽  
Specific Induction ◽  
Related Compounds ◽  
Type Strains

ABSTRACT Nitrogen limitation induces the nitrogen-regulated (Ntr) response, which includes proteins that assimilate ammonia and scavenge nitrogen. Nitrogen limitation also induces catabolic pathways that degrade four metabolically related compounds: putrescine, arginine, ornithine, and γ-aminobutyrate (GABA). We analyzed the structure, function, and regulation of the gab operon, whose products degrade GABA, a proposed intermediate in putrescine catabolism. We showed that the gabDTPC gene cluster constitutes an operon based partially on coregulation of GabT and GabD activities and the polarity of an insertion in gabT on gabC. A ΔgabDT mutant grew normally on all of the nitrogen sources tested except GABA. The unexpected growth with putrescine resulted from specific induction of gab-independent enzymes. Nac was required for gab transcription in vivo and in vitro. Ntr induction did not require GABA, but various nitrogen sources did not induce enzyme activity equally. A gabC (formerly ygaE) mutant grew faster with GABA and had elevated levels of gab operon products, which suggests that GabC is a repressor. GabC is proposed to reduce nitrogen source-specific modulation of expression. Unlike a wild-type strain, a gabC mutant utilized GABA as a carbon source and such growth required σS. Previous studies showing σS-dependent gab expression in stationary phase involved gabC mutants, which suggests that such expression does not occur in wild-type strains. The seemingly narrow catabolic function of the gab operon is contrasted with the nonspecific (nitrogen source-independent) induction. We propose that the gab operon and the Ntr response itself contribute to putrescine and polyamine homeostasis.

scholarly journals Mycobacterium tuberculosis (Mtb) lipid mediated lysosomal rewiring in infected macrophages modulates intracellular Mtb trafficking and survival

2020 ◽  
Vol 295 (27) ◽  
pp. 9192-9210 ◽  
Author(s):  
Kuldeep Sachdeva ◽  
Manisha Goel ◽  
Malvika Sudhakar ◽  
Mansi Mehta ◽  
Rajmani Raju ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cells ◽  
Bacterial Survival ◽  
Cell Integrity ◽  
Quantitative Image ◽  
Transcription Factor Eb ◽  
Lysosomal System ◽  
Mtor Complex 1

Intracellular pathogens commonly manipulate the host lysosomal system for their survival. However, whether this pathogen-induced alteration affects the organization and functioning of the lysosomal system itself is not known. Here, using in vitro and in vivo infections and quantitative image analysis, we show that the lysosomal content and activity are globally elevated in Mycobacterium tuberculosis (Mtb)-infected macrophages. We observed that this enhanced lysosomal state is sustained over time and defines an adaptive homeostasis in the infected macrophage. Lysosomal alterations are caused by mycobacterial surface components, notably the cell wall-associated lipid sulfolipid-1 (SL-1), which functions through the mTOR complex 1 (mTORC1)–transcription factor EB (TFEB) axis in the host cells. An Mtb mutant lacking SL-1, MtbΔpks2, shows attenuated lysosomal rewiring compared with the WT Mtb in both in vitro and in vivo infections. Exposing macrophages to purified SL-1 enhanced the trafficking of phagocytic cargo to lysosomes. Correspondingly, MtbΔpks2 exhibited a further reduction in lysosomal delivery compared with the WT. Reduced trafficking of this mutant Mtb strain to lysosomes correlated with enhanced intracellular bacterial survival. Our results reveal that global alteration of the host lysosomal system is a defining feature of Mtb-infected macrophages and suggest that this altered lysosomal state protects host cell integrity and contributes to the containment of the pathogen.

Hemolytic phospholipaseRv0183of Mycobacterium tuberculosis induces inflammatory response and apoptosis in alveolar macrophage RAW264.7 cells

2010 ◽  
Vol 56 (11) ◽  
pp. 916-924 ◽  
Author(s):  
Guangxian Xu ◽  
Hao Jia ◽  
Yong Li ◽  
Xiaoming Liu ◽  
Min Li ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inflammatory Response ◽  
Alveolar Macrophage ◽  
Cell Apoptosis ◽  
Time Course ◽  
Ectopic Expression ◽  
Host Cells ◽  
Raw264.7 Cells ◽  
Raw264.7 Cell

The metabolic pathway of phospholipids is one of the most important physiologic pathways in Mycobacterium tuberculosis , a typical intracellular bacterium. The hemolytic phospholipase lip gene (Rv0183) is one of 24 phospholipase genes that have been demonstrated to play critical roles in the metabolism of phospholipids in M. tuberculosis. Quantitative RT–PCR and flow cytometry were used to elucidate the immunological and pathogenic implications of the Rv0183 gene on the inflammatory response following persistent expression of Rv0183 in mouse alveolar macrophage RAW264.7 cells. Our results demonstrate that a time-course-dependent ectopic expression of Rv0183 significantly elevated the expression of IL-6, NF-κB, TLR-2, TLR-6, TNFα, and MyD88 in these alveolar macrophage cells. Furthermore, the persistent expression of Rv0183 induced RAW264.7 cell apoptosis in vitro. These findings demonstrate that the expression of Rv0183 induces an inflammatory response and cell apoptosis in the host cells, suggesting that Rv0183 may play an important role in the virulence and pathogenesis of M. tuberculosis infection.

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