scholarly journals INVESTIGATION OF ACCD3 GENE OF MYCOBACTERIUM TUBERCULOSIS IRAQI ISOLATES

2018 ◽  
Vol 11 (8) ◽  
pp. 208
Author(s):  
Asra'a A Abdul Jalil ◽  
Zahra M Al-khafaji ◽  
Mushtak T Al-ouqaili
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Conventional Polymerase Chain Reaction ◽  
Mycolic Acid ◽  
Human Pathogens ◽  
Mycolic Acids ◽  
Pcr Product ◽  
Malonyl Coa ◽  
Polymerase Chain ◽  
Acetyl Coenzyme A Carboxylase

Objective: Mycobacterium tuberculosis, one of the deadliest human pathogens, causes several million new infections and about 2 million fatalities annually. The cell wall of M. tuberculosis is endowed with a highly impermeable, complex array of diverse lipids such as mycolic acids, which bestow the bacterium with not only virulence but also resistance to host immunity and antibiotics.Methods: Mycobacterial lipid metabolism has thus emerged as an attractive target for the design and development of novel antimycobacterial therapeutics. The first committed step in the biosynthesis of mycolic acid is the carboxylation of acetyl-CoA to malonyl-CoA which is catalyzed by acetyl-coenzyme A carboxylase carboxyl transferase beta subunit (accD3), a primer pairs were designed computationally and used for the amplification of accD3 gene using conventional polymerase chain reaction (PCR) and sequencing the PCR product and analyze the results.Results: Two sequences of the detection gene (LprM gene) and eight sequences of accD3 gene under study were deposited at NCBI – GenBank database with accession numbers (LC009881, LC009880.1, LC006979, LC008196, LC009412, LC009414, LC034168, LC038020, LC041163, and LC041368) and primer pairs deposited at Probe database/NCBI with accession number Pr032816836.Conclusion: AccD3 gene is a good drug target in MDR M. tuberculosis strains.

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 Redundant Function of cmaA2 and mmaA2 in Mycobacterium tuberculosis cis Cyclopropanation of Oxygenated Mycolates

2010 ◽  
Vol 192 (14) ◽  
pp. 3661-3668 ◽  
Author(s):  
Daniel Barkan ◽  
Vivek Rao ◽  
George D. Sukenick ◽  
Michael S. Glickman
Keyword(s):  
Fatty Acids ◽  
Mycobacterium Tuberculosis ◽  
Genetic Analysis ◽  
Biosynthetic Pathway ◽  
Cell Envelope ◽  
Mycolic Acid ◽  
Acid Modification ◽  
Mycolic Acids ◽  
Powerful Approach ◽  
Long Chain

ABSTRACT The Mycobacterium tuberculosis cell envelope contains a wide variety of lipids and glycolipids, including mycolic acids, long-chain branched fatty acids that are decorated by cyclopropane rings. Genetic analysis of the mycolate methyltransferase family has been a powerful approach to assign functions to each of these enzymes but has failed to reveal the origin of cis cyclopropanation of the oxygenated mycolates. Here we examine potential redundancy between mycolic acid methyltransferases by generating and analyzing M. tuberculosis strains lacking mmaA2 and cmaA2, mmaA2 and cmaA1, or mmaA1 alone. M. tuberculosis lacking both cmaA2 and mmaA2 cannot cis cyclopropanate methoxymycolates or ketomycolates, phenotypes not shared by the mmaA2 and cmaA2 single mutants. In contrast, a combined loss of cmaA1 and mmaA2 had no effect on mycolic acid modification compared to results with a loss of mmaA2 alone. Deletion of mmaA1 from M. tuberculosis abolishes trans cyclopropanation without accumulation of trans-unsaturated oxygenated mycolates, placing MmaA1 in the biosynthetic pathway for trans-cyclopropanated oxygenated mycolates before CmaA2. These results define new functions for the mycolic acid methyltransferases of M. tuberculosis and indicate a substantial redundancy of function for MmaA2 and CmaA2, the latter of which can function as both a cis and trans cyclopropane synthase for the oxygenated mycolates.

scholarly journals Two Accessory Proteins Govern MmpL3 Mycolic Acid Transport in Mycobacteria

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Allison Fay ◽  
Nadine Czudnochowski ◽  
Jeremy M. Rock ◽  
Jeffrey R. Johnson ◽  
Nevan J. Krogan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Complex Structure ◽  
Mycolic Acid ◽  
Accessory Proteins ◽  
Acid Transport ◽  
Content Type ◽  
Mycolic Acids ◽  
Antibiotic Target

ABSTRACT Mycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterium-specific antibiotics and a mediator of Mycobacterium tuberculosis pathogenesis. Mycolic acids are synthesized in the cytoplasm and are thought to be transported to the cell wall as a trehalose ester by the MmpL3 transporter, an antibiotic target for M. tuberculosis. However, the mechanism by which mycolate synthesis is coupled to transport, and the full MmpL3 transport machinery, is unknown. Here, we identify two new components of the MmpL3 transport machinery in mycobacteria. The protein encoded by MSMEG_0736/Rv0383c is essential for growth of Mycobacterium smegmatis and M. tuberculosis and is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate (TMM) transport to the cell wall. In light of these findings, we propose MSMEG_0736/Rv0383c be named “TMM transport factor A”, TtfA. The protein encoded by MSMEG_5308 also interacts with the MmpL3 complex but is nonessential for growth or TMM transport. However, MSMEG_5308 accumulates with inhibition of MmpL3-mediated TMM transport and stabilizes the MmpL3/TtfA complex, indicating that it may stabilize the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosis. IMPORTANCE The cell envelope of Mycobacterium tuberculosis, the bacterium that causes the disease tuberculosis, is a complex structure composed of abundant lipids and glycolipids, including the signature lipid of these bacteria, mycolic acids. In this study, we identified two new components of the transport machinery that constructs this complex cell wall. These two accessory proteins are in a complex with the MmpL3 transporter. One of these proteins, TtfA, is required for mycolic acid transport and cell viability, whereas the other stabilizes the MmpL3 complex. These studies identify two new components of the essential cell envelope biosynthetic machinery in mycobacteria.

scholarly journals The Assessment of Viability of M. Tuberculosis after Exposure to CPC Using Different Methods

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Gomathi Sekar ◽  
R. Lakshmi ◽  
N. Selvakumar
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Metabolic Activity ◽  
Acid Content ◽  
Reference Strain ◽  
Vital Staining ◽  
Mycolic Acid ◽  
Viable Count ◽  
Mycolic Acids ◽  
Minimal Reduction

Settings. National Institute for Research in Tuberculosis, Chennai. Objective. To assess the proportion of metabolically active cells of Mycobacterium tuberculosis after exposed to CPC using FDA-EB vital staining and viable counts on LJ medium. Mycolic acid content in M. tuberculosis after exposure to CPC was estimated using HPLC. Methods. Clinical isolates of M. tuberculosis and standard reference strain M. tuberculosis H37Rv were used for FDA-EB, viable count, and HPLC. Results. FDA/EB consistently stained 70–90% of log phase cells as green and the remaining cells as red-orange. After CPC treatment, 65–70% of the cells stained red-orange. The viability counts were comparable to 0-day controls. Synthesis of mycolic acids in mycobacteria was reduced when exposed to CPC using HPLC due to the decreased metabolic activity of the organisms. Conclusion. The cells are metabolically inactive during storage with CPC but these cells grew well on LJ medium after removal of CPC. The viability of M. tuberculosis was maintained in CPC with minimal reduction. Mycolic acid content was reduced if the cells of M. tuberculosis were treated with CPC for 7 days. All the above findings provide yet another evidence for the damage of cell wall of M. tuberculosis.

scholarly journals Isoxyl Activation Is Required for Bacteriostatic Activity against Mycobacterium tuberculosis

2007 ◽  
Vol 51 (11) ◽  
pp. 3824-3829 ◽  
Author(s):  
Jana Korduláková ◽  
Yves L. Janin ◽  
Avraham Liav ◽  
Nathalie Barilone ◽  
Tiago Dos Vultos ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Chemical Transformations ◽  
Mycolic Acids ◽  
Monooxygenase Gene ◽  
Acid Pathway ◽  
The 1960S ◽  
First Time

ABSTRACT Isoxyl (ISO), a thiourea derivative that was successfully used for the clinical treatment of tuberculosis during the 1960s, is an inhibitor of the synthesis of oleic and mycolic acids in Mycobacterium tuberculosis. Its effect on oleic acid synthesis has been shown to be attributable to its inhibitory activity on the stearoyl-coenzyme A desaturase DesA3, but its enzymatic target(s) in the mycolic acid pathway remains to be identified. With the goal of elucidating the mode of action of ISO, we have isolated a number of spontaneous ISO-resistant mutants of M. tuberculosis and undertaken their genotypic characterization. We report here the characterization of a subset of these strains carrying mutations in the monooxygenase gene ethA. Through complementation studies, we demonstrate for the first time that the EthA-mediated oxidation of ISO is absolutely required for this prodrug to inhibit its lethal enzymatic target(s) in M. tuberculosis. An analysis of the metabolites resulting from the in vitro transformation of ISO by purified EthA revealed the occurrence of a formimidamide allowing the formulation of an activation pathway in which the oxidation of ISO catalyzed by EthA is followed by chemical transformations involving extrusion or elimination and, finally, hydrolysis.

scholarly journals The Synthesis of Single Enantiomers of α-Mycolic Acids of Mycobacterium­ tuberculosis and Related Organisms, with Alternative­ Cyclopropane Stereochemistries

SynOpen ◽  
2017 ◽  
Vol 01 (01) ◽  
pp. 0103-0116 ◽  
Author(s):  
Chioma Don Lawson ◽  
Max Maza-Iglesias ◽  
Muthana Sirhan ◽  
Jumaa Al Dulayymi ◽  
Mark Baird
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycolic Acid ◽  
Mycolic Acids

We report the synthesis of three stereoisomers of a mycolic acid from Mycobacterium tuberculosis containing a di-cis-cyclopropane and of two stereoisomers of a mycolic acid containing a proximal trans-cyclopropane and a distal cis-cyclopropane.

scholarly journals Molecular Dynamics of Mycolic Acid Monolayers

2019 ◽  
Author(s):  
Wilma Groenewald ◽  
Monica Bulacu ◽  
Anna Croft ◽  
Siewert-Jan Marrink
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Dynamics ◽  
Mycolic Acid ◽  
Phase Changes ◽  
Coarse Grained ◽  
Head Group ◽  
Mycolic Acids ◽  
Coarse Grained Model ◽  
Surface Areas ◽  
Martini Force Field

<p><i>Mycobacterium tuberculosis</i>, the organism responsible for TB infection in humans, is inherently resilient against host defences and anti-TB drugs. This persistence is attributed partly to the presence of lipids, such as mycolic acids (MAs), which make the cell wall impermeable. To study the conformational dynamics of MAs, we present a coarse-grained model for a representative α-MA (AMA) from <i>Mycobacterium tuberculosis</i>using the MARTINI force field. The model is used to simulate monolayers of different sizes; a small monolayer consisting of 220 MAs and a large monolayer consisting of 1972 MAs. The model could replicate key features of experimental monolayers such as phase changes and the collapse point. By studying the conformation of MAs in the simulated monolayers, it was found that AMA did not fold into the W-conformation at large surface areas but was only folded at the head group to give a wide U-shape. On monolayer compression, the MA chains came closer together, into a narrower U-shape, and an ordered monolayer was formed before it collapsed.</p>

scholarly journals Molecular Dynamics of Mycolic Acid Monolayers

2019 ◽  
Author(s):  
Wilma Groenewald ◽  
Monica Bulacu ◽  
Anna Croft ◽  
Siewert-Jan Marrink
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Dynamics ◽  
Mycolic Acid ◽  
Phase Changes ◽  
Coarse Grained ◽  
Head Group ◽  
Mycolic Acids ◽  
Coarse Grained Model ◽  
Surface Areas ◽  
Martini Force Field

<p><i>Mycobacterium tuberculosis</i>, the organism responsible for TB infection in humans, is inherently resilient against host defences and anti-TB drugs. This persistence is attributed partly to the presence of lipids, such as mycolic acids (MAs), which make the cell wall impermeable. To study the conformational dynamics of MAs, we present a coarse-grained model for a representative α-MA (AMA) from <i>Mycobacterium tuberculosis</i>using the MARTINI force field. The model is used to simulate monolayers of different sizes; a small monolayer consisting of 220 MAs and a large monolayer consisting of 1972 MAs. The model could replicate key features of experimental monolayers such as phase changes and the collapse point. By studying the conformation of MAs in the simulated monolayers, it was found that AMA did not fold into the W-conformation at large surface areas but was only folded at the head group to give a wide U-shape. On monolayer compression, the MA chains came closer together, into a narrower U-shape, and an ordered monolayer was formed before it collapsed.</p>

scholarly journals OPTIMASI DETEKSI GEN PADA Stelechocarpus burahol (Bl.) Hook.f. & Th. MENGGUNAKAN DIRECT KIR PCR

2020 ◽  
Vol 14 (2) ◽  
pp. 93-99
Author(s):  
Tri Suwarni Wahyudiningsih ◽  
◽  
Dian Sartika ◽  
Keyword(s):  
Dna Isolation ◽  
Conventional Polymerase Chain Reaction ◽  
Gene Detection ◽  
Direct Pcr ◽  
Isolation And Purification ◽  
Botanical Gardens ◽  
Pcr Product ◽  
Large Populations ◽  
Polymerase Chain ◽  
Time And Energy

DNA isolation and purification in the conventional Polymerase Chain Reaction (PCR) process require reagents that are toxic, more costly and time consuming, and contamination. S. burahol leaves contain phenolics, flavonoids, and terpenoids which can interfere with DNA isolation. The use of direct PCR kits can detect genes without DNA extraction.The objective of study was to determinethe method of gene detection ofStelechocarpus buraholusing directPCRkit.In each location, one tree was taken as a source of leaf samples from Garut, Purwodadi Botanical Gardens, Kyai Langgeng Gardens, Yogyakarta Palace, Turi Sleman, Wanagama, Karanganyar, and South Kalimantan, except Bogor Botanical Gardens, two trees were taken. The primers used for the trials were ITS 1F primers and 4R primers. In the sequencing stage, PCR product samples of 40 -50 μl that showed positive results were detected by electrophoresis. The PCR product was measuredat ± 750 bp from ten samples. Direct PCR kits can be used for S. buraholgene detection, time and energy efficient, only requires a small amount of tissue, and reduces contamination due to DNA extraction. Direct PCR kits can be an effective method that can be utilized to detect target genesfor large populations.

scholarly journals SQ109 Targets MmpL3, a Membrane Transporter of Trehalose Monomycolate Involved in Mycolic Acid Donation to the Cell Wall Core of Mycobacterium tuberculosis

2012 ◽  
Vol 56 (4) ◽  
pp. 1797-1809 ◽  
Author(s):  
Kapil Tahlan ◽  
Regina Wilson ◽  
David B. Kastrinsky ◽  
Kriti Arora ◽  
Vinod Nair ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Mycolic Acid ◽  
Spontaneous Generation ◽  
Content Type ◽  
Mycolic Acids ◽  
Cell Wall Assembly ◽  
Resistant Mutants ◽  
Trehalose Monomycolate

ABSTRACTSQ109, a 1,2-diamine related to ethambutol, is currently in clinical trials for the treatment of tuberculosis, but its mode of action remains unclear. Here, we demonstrate that SQ109 disrupts cell wall assembly, as evidenced by macromolecular incorporation assays and ultrastructural analyses. SQ109 interferes with the assembly of mycolic acids into the cell wall core ofMycobacterium tuberculosis, as bacilli exposed to SQ109 show immediate inhibition of trehalose dimycolate (TDM) production and fail to attach mycolates to the cell wall arabinogalactan. These effects were not due to inhibition of mycolate synthesis, since total mycolate levels were unaffected, but instead resulted in the accumulation of trehalose monomycolate (TMM), the precursor of TDM and cell wall mycolates.In vitroassays using purified enzymes showed that this was not due to inhibition of the secreted Ag85 mycolyltransferases. We were unable to achieve spontaneous generation of SQ109-resistant mutants; however, analogs of this compound that resulted in similar shutdown of TDM synthesis with concomitant TMM accumulation were used to spontaneously generate resistant mutants that were also cross-resistant to SQ109. Whole-genome sequencing of these mutants showed that these all had mutations in the essentialmmpL3gene, which encodes a transmembrane transporter. Our results suggest that MmpL3 is the target of SQ109 and that MmpL3 is a transporter of mycobacterial TMM.

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