scholarly journals Discovery of a novel stereospecific β-hydroxyacyl-CoA lyase/thioesterase shared by three metabolic pathways in Mycobacterium tuberculosis

2018 ◽  
Author(s):  
Hua Wang ◽  
Alexander A. Fedorov ◽  
Elena V. Fedorov ◽  
Deborah M. Hunt ◽  
Angela Rodgers ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Metabolic Pathways ◽  
Resistance Mechanism ◽  
Physiological Role ◽  
Vast Number ◽  
X Ray Crystallography ◽  
Leucine Catabolism ◽  
Orphan Enzyme

AbstractThe vast number of poorly characterised enzymes in Mycobacterium tuberculosis (Mtb) is one of the key barriers precluding a better understanding of the biology that underpins pathogenesis. Here, we investigated the Mtb orphan enzyme Rv2498c to delineate its physiological role. Our results from in vitro enzymatic assays, phylogenetic analysis, X-ray crystallography and in vivo Mtb experiments, de-orphan Rv2498c as a multi-functional β-hydroxyacyl-CoA lyase/thioesterase (β-HAClyase/thioesterase) that participates in three different metabolic pathways: L-leucine catabolism, itaconate dissimilation, and glyoxylate shunt. Moreover, the deletion of the rv2498c gene from the Mtb genome resulted in attenuation in the mouse model compared to infection with the parent strain. To the best of our knowledge, this is the first report of an (R)-3-hydroxyl-3-methylglutaryl-CoA for leucine catabolism and an itaconate-specific resistance mechanism in Mtb.

scholarly journals An essential bifunctional enzyme in Mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism

2019 ◽  
Vol 116 (32) ◽  
pp. 15907-15913 ◽  
Author(s):  
Hua Wang ◽  
Alexander A. Fedorov ◽  
Elena V. Fedorov ◽  
Debbie M. Hunt ◽  
Angela Rodgers ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Resistance Mechanism ◽  
Infection Model ◽  
Enzymatic Assays ◽  
X Ray Crystallography ◽  
Mouse Infection Model ◽  
Leucine Catabolism ◽  
Global Population

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional β-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.

RNA and RNA–Protein Complex Crystallography and its Challenges

2014 ◽  
Vol 67 (12) ◽  
pp. 1741 ◽  
Author(s):  
Janine K. Flores ◽  
James L. Walshe ◽  
Sandro F. Ataide
Keyword(s):  
Protein Complex ◽  
Protein Complexes ◽  
Future Directions ◽  
Vast Number ◽  
X Ray Crystallography ◽  
Rna Biology ◽  
Non Coding Rnas ◽  
Rna Protein Complexes

RNA biology has changed completely in the past decade with the discovery of non-coding RNAs. Unfortunately, obtaining mechanistic information about these RNAs alone or in cellular complexes with proteins has been a major problem. X-ray crystallography of RNA and RNA–protein complexes has suffered from the major problems encountered in preparing and purifying them in large quantity. Here, we review the available techniques and methods in vitro and in vivo used to prepare and purify RNA and RNA–protein complex for crystallographic studies. We also discuss the future directions necessary to explore the vast number of RNA species waiting for their atomic-resolution structure to be determined.

scholarly journals The In Vivo Transcriptomic Blueprint of Mycobacterium tuberculosis in the Lung

2021 ◽  
Vol 12 ◽  
Author(s):  
Mariateresa Coppola ◽  
Rachel P-J. Lai ◽  
Robert J. Wilkinson ◽  
Tom H. M. Ottenhoff
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Metabolic Pathways ◽  
Large Scale ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Genes Encoding ◽  
High Concordance ◽  
Lower Correlation

Mycobacterium tuberculosis (Mtb) genes encoding proteins targeted by vaccines and drugs should be expressed in the lung, the main organ affected by Mtb, for these to be effective. However, the pulmonary expression of most Mtb genes and their proteins remains poorly characterized. The aim of this study is to fill this knowledge gap. We analyzed large scale transcriptomic datasets from specimens of Mtb-infected humans, TB-hypersusceptible (C3H/FeJ) and TB-resistant (C57BL/6J) mice and compared data to in vitro cultured Mtb gene-expression profiles. Results revealed high concordance in the most abundantly in vivo expressed genes between pulmonary Mtb transcriptomes from different datasets and different species. As expected, this contrasted with a lower correlation found with the highest expressed Mtb genes from in vitro datasets. Among the most consistently and highly in vivo expressed genes, 35 have not yet been explored as targets for vaccination or treatment. More than half of these genes are involved in protein synthesis or metabolic pathways. This first lung-oriented multi-study analysis of the in vivo expressed Mtb-transcriptome provides essential data that considerably increase our understanding of pulmonary TB infection biology, and identifies novel molecules for target-based TB-vaccine and drug development.

scholarly journals Characterization of proline utilization pathway in Mycobacterium tuberculosis

2014 ◽  
Vol 70 (a1) ◽  
pp. C822-C822
Author(s):  
Thomas Lagautriere ◽  
Ghader Bashiri ◽  
Gregory Cook ◽  
Edward Baker
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Three Dimensional ◽  
Selective Inhibition ◽  
Binding Mode ◽  
High Sequence Identity ◽  
X Ray Crystallography ◽  
Proline Utilization ◽  
Utilization Pathway

The proline utilization pathway in Mycobacterium tuberculosis (Mtb) has been recently identified as an important factor in Mtb persistence in vivo, suggesting that this pathway could be a valuable therapeutic target against tuberculosis (TB). In Mtb, two distinct enzymes perform the conversion of proline into glutamate; the first step is the oxidation of proline into Δ1-pyrroline-5-carboxylic acid (P5C) by the flavoenzyme proline dehydrogenase (PruB) and the second reaction involves converting the tautomeric form of P5C (glutamate-γ-semialdehyde) into glutamate using the NAD+-dependent Δ1-pyrroline-5-carboxylic dehydrogenase (PruA). Here we describe three-dimensional structures of Mtb-PruA, determined by X-ray crystallography both in its apo state and in complex with NAD+ at 2.5 and 2.1 Å resolution, respectively. The structure reveals a conserved NAD+ binding mode, common to other related enzymes. Conformational differences in the active site, however, linked to changes in the dimer interface, suggest possibilities for selective inhibition of Mtb-PruA despite reasonably high sequence identity with other PruA enzymes. Using recombinant PruA and PruB, the proline utilization pathway in Mtb has also been reconstituted in vitro. Functional validation using a novel NMR approach has demonstrated that the PruA and PruB enzymes are together sufficient to convert proline to glutamate, the first such demonstration for monofunctional proline utilization enzymes.

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 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.

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

scholarly journals The Scaffold Protein Cybr Is Required for Cytokine-Modulated Trafficking of Leukocytes In Vivo

2006 ◽  
Vol 26 (14) ◽  
pp. 5249-5258 ◽  
Author(s):  
Vincenzo Coppola ◽  
Colleen A. Barrick ◽  
Sara Bobisse ◽  
Maria Cecilia Rodriguez-Galan ◽  
Michela Pivetta ◽  
...  
Keyword(s):  
Immune System ◽  
Cytotoxic T Lymphocyte ◽  
Leukemia Virus ◽  
Physiological Role ◽  
Scaffold Protein ◽  
Nucleotide Exchange ◽  
Lymphocyte Migration ◽  
And Migration

ABSTRACT Trafficking and cell adhesion are key properties of cells of the immune system. However, the molecular pathways that control these cellular behaviors are still poorly understood. Cybr is a scaffold protein highly expressed in the hematopoietic/immune system whose physiological role is still unknown. In vitro studies have shown it regulates LFA-1, a crucial molecule in lymphocyte attachment and migration. Cybr also binds cytohesin-1, a guanine nucleotide exchange factor for the ARF GTPases, which affects actin cytoskeleton remodeling during cell migration. Here we show that expression of Cybr in vivo is differentially modulated by type 1 cytokines during lymphocyte maturation. In mice, Cybr deficiency negatively affects leukocytes circulating in blood and lymphocytes present in the lymph nodes. Moreover, in a Th1-polarized mouse model, lymphocyte trafficking is impaired by loss of Cybr, and Cybr-deficient mice with aseptic peritonitis have fewer cells than controls present in the peritoneal cavity, as well as fewer leukocytes leaving the bloodstream. Mutant mice injected with Moloney murine sarcoma/leukemia virus develop significantly larger tumors than wild-type mice and have reduced lymph node enlargement, suggesting reduced cytotoxic T-lymphocyte migration. Taken together, these data support a role for Cybr in leukocyte trafficking, especially in response to proinflammatory cytokines in stress conditions.

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