scholarly journals A vitamin B 12 transporter in Mycobacterium tuberculosis

Open Biology ◽  
2013 ◽  
Vol 3 (2) ◽  
pp. 120175 ◽  
Author(s):  
Krishnamoorthy Gopinath ◽  
Česlovas Venclovas ◽  
Thomas R. Ioerger ◽  
James C. Sacchettini ◽  
John D. McKinney ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type System ◽  
Mycobacterial Infection ◽  
Vitamin B ◽  
Abc Protein ◽  
Genome Wide ◽  
Host Infection ◽  
Obligate Pathogen

Vitamin B 12 -dependent enzymes function in core biochemical pathways in Mycobacterium tuberculosis , an obligate pathogen whose metabolism in vivo is poorly understood. Although M. tuberculosis can access vitamin B 12 in vitro , it is uncertain whether the organism is able to scavenge B 12 during host infection. This question is crucial to predictions of metabolic function, but its resolution is complicated by the absence in the M. tuberculosis genome of a direct homologue of BtuFCD, the only bacterial B 12 transport system described to date. We applied genome-wide transposon mutagenesis to identify M. tuberculosis mutants defective in their ability to use exogenous B 12 . A small proportion of these mapped to Rv1314c , identifying the putative PduO-type ATP : co(I)rrinoid adenosyltransferase as essential for B 12 assimilation. Most notably, however, insertions in Rv1819c dominated the mutant pool, revealing an unexpected function in B 12 acquisition for an ATP-binding cassette (ABC)-type protein previously investigated as the mycobacterial BacA homologue. Moreover, targeted deletion of Rv1819c eliminated the ability of M. tuberculosis to transport B 12 and related corrinoids in vitro . Our results establish an alternative to the canonical BtuCD-type system for B 12 uptake in M. tuberculosis , and elucidate a role in B 12 metabolism for an ABC protein implicated in chronic mycobacterial infection.

scholarly journals Functional Characterization of a Vitamin B12-Dependent Methylmalonyl Pathway in Mycobacterium tuberculosis: Implications for Propionate Metabolism during Growth on Fatty Acids

2008 ◽  
Vol 190 (11) ◽  
pp. 3886-3895 ◽  
Author(s):  
Suzana Savvi ◽  
Digby F. Warner ◽  
Bavesh D. Kana ◽  
John D. McKinney ◽  
Valerie Mizrahi ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Mycobacterium Tuberculosis ◽  
Isocitrate Lyase ◽  
Glyoxylate Cycle ◽  
Functional Characterization ◽  
Vitamin B ◽  
Branched Chain ◽  
The Glyoxylate Cycle

ABSTRACT Mycobacterium tuberculosis is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd- and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C3) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in M. tuberculosis pathogenesis. In addition to the well-characterized methylcitrate cycle, the M. tuberculosis genome contains a complete methylmalonyl pathway, including a mutAB-encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B12-derived cofactor for activity. Here, we demonstrate the ability of M. tuberculosis to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B12 is supplied exogenously. We show that this ability is dependent on mutAB and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Importantly, although the glyoxylate and methylcitrate cycles supported robust growth of M. tuberculosis on the C17 fatty acid heptadecanoate, growth on valerate (C5) was significantly enhanced through vitamin B12 supplementation. Moreover, both wild-type and methylcitrate cycle mutant strains grew on B12-supplemented valerate in the presence of 3-nitropropionate, an inhibitor of the glyoxylate cycle enzyme isocitrate lyase, indicating an anaplerotic role for the methylmalonyl pathway. The demonstrated functionality of MCM reinforces the potential relevance of vitamin B12 to mycobacterial pathogenesis and suggests that vitamin B12 availability in vivo might resolve the paradoxical dispensability of the methylcitrate cycle for the growth and persistence of M. tuberculosis in mice.

scholarly journals The promises and limitations of N-acetylcysteine as a potentiator of first-line and second-line tuberculosis drugs

2021 ◽  
Author(s):  
Catherine Vilchèze ◽  
William R. Jacobs
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Liver Injury ◽  
Multidrug Resistant ◽  
Human Macrophage ◽  
Second Line ◽  
Macrophage Cell ◽  
First Line ◽  
Host Infection

N-acetylcysteine (NAC) is most commonly used for the treatment of acetaminophen overdose and acetaminophen-induced liver injury. In patients infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), NAC is given to treat hepatotoxicity induced by TB drugs. We had previously shown that cysteine, a derivative of NAC, potentiated the activity of isoniazid, a first-line TB drug, by preventing the emergence of INH resistance and persistence in M. tuberculosis in vitro. Herein, we demonstrate that in vitro, NAC has the same boosting activity with various combinations of first- and second-line TB drugs against drug-susceptible and multidrug-resistant M. tuberculosis strains. Similar to cysteine, NAC increased M. tuberculosis respiration. However, in M. tuberculosis-infected mice, the addition of NAC did not augment the activity of first- or second-line TB drugs. A comparison of the activity of NAC combined with TB drugs in murine and human macrophage cell lines revealed that studies in mice might not be recapitulated during host infection in vivo.

scholarly journals The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
E. F. Perkowski ◽  
K. E. Zulauf ◽  
D. Weerakoon ◽  
J. D. Hayden ◽  
T. R. Ioerger ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Host Environment ◽  
Bacterial Proteins ◽  
Novel Method ◽  
Host Infection ◽  
Membrane Cell ◽  
Significant Effort

ABSTRACT Exported proteins of bacterial pathogens function both in essential physiological processes and in virulence. Past efforts to identify exported proteins were limited by the use of bacteria growing under laboratory (in vitro) conditions. Thus, exported proteins that are exported only or preferentially in the context of infection may be overlooked. To solve this problem, we developed a genome-wide method, named EXIT (exported in vivo technology), to identify proteins that are exported by bacteria during infection and applied it to Mycobacterium tuberculosis during murine infection. Our studies validate the power of EXIT to identify proteins exported during infection on an unprecedented scale (593 proteins) and to reveal in vivo induced exported proteins (i.e., proteins exported significantly more during in vivo infection than in vitro). Our EXIT data also provide an unmatched resource for mapping the topology of M. tuberculosis membrane proteins. As a new approach for identifying exported proteins, EXIT has potential applicability to other pathogens and experimental conditions. IMPORTANCE There is long-standing interest in identifying exported proteins of bacteria as they play critical roles in physiology and virulence and are commonly immunogenic antigens and targets of antibiotics. While significant effort has been made to identify the bacterial proteins that are exported beyond the cytoplasm to the membrane, cell wall, or host environment, current methods to identify exported proteins are limited by their use of bacteria growing under laboratory (in vitro) conditions. Because in vitro conditions do not mimic the complexity of the host environment, critical exported proteins that are preferentially exported in the context of infection may be overlooked. We developed a novel method to identify proteins that are exported by bacteria during host infection and applied it to identify Mycobacterium tuberculosis proteins exported in a mouse model of tuberculosis. IMPORTANCE There is long-standing interest in identifying exported proteins of bacteria as they play critical roles in physiology and virulence and are commonly immunogenic antigens and targets of antibiotics. While significant effort has been made to identify the bacterial proteins that are exported beyond the cytoplasm to the membrane, cell wall, or host environment, current methods to identify exported proteins are limited by their use of bacteria growing under laboratory (in vitro) conditions. Because in vitro conditions do not mimic the complexity of the host environment, critical exported proteins that are preferentially exported in the context of infection may be overlooked. We developed a novel method to identify proteins that are exported by bacteria during host infection and applied it to identify Mycobacterium tuberculosis proteins exported in a mouse model of tuberculosis.

scholarly journals Mycobacterium tuberculosis Rv3194c efficiently facilitates mycobacterial-lung epithelial interaction through its HA-binding site

2020 ◽  
Author(s):  
Dongyue Zhao ◽  
Chen Xu ◽  
Danfeng Lin
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Binding Site ◽  
A549 Cells ◽  
Mycobacterial Infection ◽  
Lung Epithelial Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Surface Plasmon Resonance Analysis ◽  
Lung Epithelial

AbstractMycobacterium tuberculosis adhesins are surface-exposed molecules that mediate pathogen-host interaction, a fundamental step towards host infection. Here we show that serine protease (Rv3194c) promotes mycobacterial infection to lung epithelial through its hyaluronic acid (HA)-binding site. Both enzyme-linked immunosorbent assay and surface plasmon resonance analysis revealed that Rv3194c bound to HA. Utilizing synthetic peptides, we next defined HA-binding site of 20 amino acids from 91 to 110 of Rv3194c (P91-110). Immunofluorescence assay and an FACScan showed that Rv3194c was interacted with A549 cells (human lung epithelial cells), and its interaction was abolished by the addition of hyaluronidase or P91-110. Experimental infection in Vitro revealed that Rv3194c participates in attachment of recombinant Mycobacterium smegmatis (Rv3194c/MS) to A549 cells, and P91-110 treatment of A549 cells almost inhibited Rv3194c/MS-A549 cells interaction. To provide in vivo evidence, we constructed a reporter strain of M. smegmatis expressed a derivative of the firefly luciferase that is shifted to red (FFlucRT) in combination with Rv3194c (Rv3194c+FFlucRT/MS) to infect the rodents and monitor the progression of the disease. Using bioluminescence imaging and bacterial counts in lung tissue confirmed that Rv3194c dramatically enhanced the persistence of M. smegmatis. In addition, treatment of intratracheal Rv3194c+FFlucRT/MS-infected mice with P91-110 significantly suppressed the growth of Rv3194c+FFlucRT/MS in vivo. Taken together, these results demonstrate that Rv3194c was identified as a HA-binding adhesin, and P91-110 as anti-adhesion agents has potential for therapeutic and prophylactic interventions in mycobacterial infection.

Tuberkulose - Screening

2011 ◽  
Vol 68 (7) ◽  
pp. 381-387
Author(s):  
Otto Schoch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Interferon Gamma ◽  
Wird Eine ◽  
Interferon Gamma Release Assays

Das primäre Ziel der Aktivitäten zur bevölkerungsbezogenen Tuberkulosekontrolle ist die Identifizierung von Patienten mit sputummikroskopisch positiver Lungentuberkulose. Wenn diese Patienten umgehend therapiert werden, haben sie nicht nur eine optimale Heilungschance, sondern übertragen auch den Krankheitserreger nicht weiter auf andere Personen. Das Screening, die systematische Suche nach Tuberkulose, erfolgt in der Regel radiologisch bei der Suche nach Erkrankten, während immunologische Teste bei der Suche nach einer Infektion mit Mycobacterium tuberculosis zur Anwendung kommen. Diese Infektion, die ein erhöhtes Risiko für die Entwicklung einer Tuberkulose-Erkrankung mit sich bringt, wird im Rahmen der Umgebungsuntersuchungen oder bei Hochrisikogruppen gesucht. Neben dem traditionellen in vivo Mantoux Hauttest stehen heute die neueren in vitro Blutteste, die sogenannten Interferon Gamma Release Assays (IGRA) zur Verfügung, die unter anderem den Vorteil einer höheren Spezifität mit sich bringen, weil die verwendeten Antigene der Mykobakterien-Wand beim Impfstamm Bacille Calmitte Guerin (BCG) und bei den meisten atypischen Mykobakterien nicht vorhanden sind. Zudem kann bei Immunsupprimierten dank einer mitgeführten Positivkontrolle eine Aussage über die Wahrscheinlichkeit eines falsch negativen Testresultates gemacht werden. Bei neu diagnostizierter Infektion mit Mycobacterium tuberculosis wird eine präventive Chemotherapie mit Isoniazid während 9 Monaten durchgeführt.

Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

2019 ◽  
Vol 12 (1) ◽  
pp. 27-49 ◽  
Author(s):  
Shahinda S.R. Alsayed ◽  
Chau C. Beh ◽  
Neil R. Foster ◽  
Alan D. Payne ◽  
Yu Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Enzymatic Activity ◽  
Bacterial Pathogen ◽  
Building Blocks ◽  
Mycolic Acid ◽  
Adaptive Responses ◽  
Mycolic Acids ◽  
Hydroxylated Fatty Acids

Background:Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human’s, there have been some early drug discovery efforts towards developing potent and selective inhibitors.Objective:Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors.Conclusion:Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

scholarly journals Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 776
Author(s):  
Shipra Kumari ◽  
Bashistha Kumar Kanth ◽  
Ju young Ahn ◽  
Jong Hwa Kim ◽  
Geung-Joo Lee
Keyword(s):  
Abiotic Stress ◽  
Biotic Stress ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Lilium Longiflorum ◽  
Biotic And Abiotic Stress ◽  
Biotic Stresses ◽  
Genome Wide

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes—LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12—were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.

scholarly journals Intracellular localisation of Mycobacterium tuberculosis affects efficacy of the antibiotic pyrazinamide

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pierre Santucci ◽  
Daniel J. Greenwood ◽  
Antony Fearns ◽  
Kai Chen ◽  
Haibo Jiang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Combination Chemotherapy ◽  
In Vitro Activity ◽  
In Vivo Efficacy ◽  
Human Macrophages ◽  
Ion Microscopy ◽  
Intracellular Localisation

AbstractTo be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of the pathogen, Mycobacterium tuberculosis. However, how host cell microenvironments affect antibiotic accumulation and efficacy remains unclear. Here, we use correlative light, electron, and ion microscopy to investigate how various microenvironments within human macrophages affect the activity of pyrazinamide (PZA), a key antibiotic against TB. We show that PZA accumulates heterogeneously among individual bacteria in multiple host cell environments. Crucially, PZA accumulation and efficacy is maximal within acidified phagosomes. Bedaquiline, another antibiotic commonly used in combined TB therapy, enhances PZA accumulation via a host cell-mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy. Our results may explain the potent in vivo efficacy of PZA, compared to its modest in vitro activity, and its critical contribution to TB combination chemotherapy.

scholarly journals Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Poushali Chakraborty ◽  
Sapna Bajeli ◽  
Deepak Kaushal ◽  
Bishan Dass Radotra ◽  
Ashwani Kumar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Immune System ◽  
Biofilm Formation ◽  
Antimycobacterial Activity ◽  
Drug Tolerance ◽  
Host Immune System ◽  
Tissue Sections ◽  
Slow Growing

AbstractTuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.

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