First access to a mycolic acid-based bioorthogonal reporter for the study of the mycomembrane and mycoloyltransferases in Corynebacteria

2019 ◽  
Vol 55 (87) ◽  
pp. 13074-13077
Author(s):  
Emilie Lesur ◽  
Aurélie Baron ◽  
Christiane Dietrich ◽  
Marie Buchotte ◽  
Gilles Doisneau ◽  
...  
Keyword(s):  
Mycolic Acid ◽  
Complex Pattern ◽  
Mycolic Acids ◽  
Trehalose Monomycolate

In this study we describe the first synthesis of an alkyne-based trehalose monomycolate probe closely mimicking the complex pattern of mycolic acids and its utility for the study of mycomembrane and mycoloyltransferases in Corynebacteria.

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.

scholarly journals Two accessory proteins govern MmpL3 mycolic acid transport in mycobacteria

2019 ◽  
Author(s):  
Allison Fay ◽  
Nadine Czudnochowski ◽  
Jeremy Rock ◽  
Jeffrey R. Johnson ◽  
Nevan J. Krogan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Transport System ◽  
Cytoplasmic Membrane ◽  
Mycolic Acid ◽  
Accessory Proteins ◽  
Acid Transport ◽  
Mycolic Acids ◽  
Antibiotic Target ◽  
Transport Factor ◽  
Trehalose Monomycolate

AbstractMycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterial specific antibiotics, and a mediator of M. 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 M. smegmatis and M. tuberculosis, is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate 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 stabilizes the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosis.

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.

Purification and characterization of C28–55 fatty acids from Mycobacterium smegmatis

1985 ◽  
Vol 31 (3) ◽  
pp. 214-219 ◽  
Author(s):  
Neeraja Sathyamoorthy ◽  
Nilofer Qureshi ◽  
Kuni Takayama
Keyword(s):  
Fatty Acids ◽  
Mycobacterium Smegmatis ◽  
High Performance ◽  
Mycolic Acid ◽  
Argentation Thin Layer Chromatography ◽  
Mycolic Acids ◽  
Electron Impact Mass ◽  
Layer Chromatography ◽  
Impact Mass

The nonmycolic C16 to C55 fatty acids obtained from Mycobacterium smegmatis ATCC 356 by saponification were enriched with respect to the C28 to C55 acids by successive chromatography on silicic acid and Sephadex LH-20 columns. These partially purified fatty acids were then derivatized to the p-bromophenacyl ester and further fractionated by argentation thin-layer chromatography and reverse-phase high-performance liquid chromatography into their individual components.The esters were characterized by electron impact mass spectrometry. Two structural series of C28:1 to C42:1 and C45:2 to C55:2 fatty acids were identified as possible precursors of the monoenyl and dienyl mycolic acids, respectively. These acids were structurally related to the α-alkylhydroxyl group of the corresponding mycolic acid. The results suggest that these C28 to C55 fatty acids (meromycolic acids) of M. smegmatis might be precursors of mycolic acids.

scholarly journals Intact molecular characterization of cord factor (trehalose 6,6′-dimycolate) from nine species of mycobacteria by MALDI-TOF mass spectrometry

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3403-3416 ◽  
Author(s):  
Yukiko Fujita ◽  
Takashi Naka ◽  
Michael R. McNeil ◽  
Ikuya Yano
Keyword(s):  
Molecular Structure ◽  
Mass Spectrometry ◽  
Molecular Species ◽  
Mycolic Acid ◽  
Molecular Ions ◽  
Ion Distribution ◽  
Mycolic Acids ◽  
Maldi Tof ◽  
Cord Factor

Cord factor (trehalose 6,6′-dimycolate, TDM) is an unique glycolipid with a trehalose and two molecules of mycolic acids in the mycobacterial cell envelope. Since TDM consists of two molecules of very long branched-chain 3-hydroxy fatty acids, the molecular mass ranges widely and in a complex manner. To characterize the molecular structure of TDM precisely and simply, an attempt was made to determine the mycolic acid subclasses of TDM and the molecular species composition of intact TDM by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the first time. The results showed that less than 1 μg mycolic acid methyl ester of TDM from nine representative species of mycobacteria and TDM from the same species was sufficient to obtain well-resolved mass spectra composed of pseudomolecular ions [M+Na]+. Although the mass ion distribution was extremely diverse, the molecular species of each TDM was identified clearly by constructing a molecular ion matrix consisting of the combination of two molecules of mycolic acids. The results showed a marked difference in the molecular structure of TDM among mycobacterial species and subspecies. TDM from Mycobacterium tuberculosis (H37Rv and Aoyama B) showed a distinctive mass pattern and consisted of over 60 molecular ions with α-, methoxy- and ketomycolate. TDM from Mycobacterium bovis BCG Tokyo 172 similarly showed over 35 molecular ions, but that from M. bovis BCG Connaught showed simpler molecular ion clusters consisting of less than 35 molecular species due to a complete lack of methoxymycolate. Mass ions due to TDM from M. bovis BCG Connaught and Mycobacterium kansasii showed a biphasic distribution, but the two major peaks of TDM from M. kansasii were shifted up two or three carbon units higher compared with M. bovis BCG Connaught. Within the rapid grower group, in TDM consisting of α-, keto- and wax ester mycolate from Mycobacterium phlei and Mycobacterium flavescens, the mass ion distribution due to polar mycolates was shifted lower than that from the Mycobacterium avium–intracellulare group. Since the physico-chemical properties and antigenic structure of mycolic acid of TDM affect the host immune responses profoundly, the molecular characterization of TDM by MALDI-TOF mass analysis may give very useful information on the relationship of glycolipid structure to its biological activity.

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 MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine

2019 ◽  
Vol 116 (23) ◽  
pp. 11241-11246 ◽  
Author(s):  
Chih-Chia Su ◽  
Philip A. Klenotic ◽  
Jani Reddy Bolla ◽  
Georgiana E. Purdy ◽  
Carol V. Robinson ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Native Mass Spectrometry ◽  
Mycolic Acids ◽  
Mycobacterial Cell ◽  
Exact Function ◽  
Species Specific ◽  
Mycobacterial Cell Wall ◽  
Trehalose Monomycolate ◽  
Lipid Transporter

The cell envelope ofMycobacterium tuberculosisis notable for the abundance of mycolic acids (MAs), essential to mycobacterial viability, and of other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, which contributes to virulence and antibiotic resistance. However, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell-wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA-containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan, but the exact function of MmpL3 remains elusive. Here, we report a crystal structure ofMycobacterium smegmatisMmpL3 at a resolution of 2.59 Å, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins. A previously unknown MmpL3 ligand, phosphatidylethanolamine (PE), was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall.

scholarly journals Carbon Source-Induced Modifications in the Mycolic Acid Content and Cell Wall Permeability of Rhodococcus erythropolis E1

2003 ◽  
Vol 69 (12) ◽  
pp. 7019-7027 ◽  
Author(s):  
Ivana Sokolovská ◽  
Raoul Rozenberg ◽  
Christophe Riez ◽  
Paul G. Rouxhet ◽  
Spiros N. Agathos ◽  
...  
Keyword(s):  
Cell Wall ◽  
Carbon Source ◽  
Acid Content ◽  
Rhodococcus Erythropolis ◽  
Mycolic Acid ◽  
Mycolic Acids ◽  
Carbon Chains ◽  
Linear Alkanes ◽  
Wall Permeability ◽  
Cell Wall Permeability

ABSTRACT The influence of the carbon source on cell wall properties was analyzed in an efficient alkane-degrading strain of Rhodococcus erythropolis (strain E1), with particular focus on the mycolic acid content. A clear correlation was observed between the carbon source and the mycolic acid profiles as estimated by high-performance liquid chromatography and mass spectrometry. Two types of mycolic acid patterns were observed after growth either on saturated linear alkanes or on short-chain alkanoates. One type of pattern was characterized by the lack of odd-numbered carbon chains and resulted from growth on linear alkanes with even numbers of carbon atoms. The second type of pattern was characterized by mycolic acids with both even- and odd-numbered carbon chains and resulted from growth on compounds with odd-numbered carbon chains, on branched alkanes, or on mixtures of different compounds. Cellular short-chain fatty acids were twice as abundant during growth on a branched alkane (pristane) as during growth on acetate, while equal amounts of mycolic acids were found under both conditions. More hydrocarbon-like compounds and less polysaccharide were exposed at the cell wall surface during growth on alkanes. Whatever the substrate, the cells had the same affinity for aqueous-nonaqueous solvent interfaces. By contrast, bacteria displayed completely opposite susceptibilities to hydrophilic and hydrophobic antibiotics and were found to be strongly stained by hydrophobic dyes after growth on pristane but not after growth on acetate. Taken together, these data show that the cell wall composition of R. erythropolis E1 is influenced by the nutritional regimen and that the most marked effect is a radical change in cell wall permeability.

scholarly journals Mycobacterium shinjukuense sp. nov., a slowly growing, non-chromogenic species isolated from human clinical specimens

2011 ◽  
Vol 61 (8) ◽  
pp. 1927-1932 ◽  
Author(s):  
Hajime Saito ◽  
Tomotada Iwamoto ◽  
Kiyofumi Ohkusu ◽  
Yoshihito Otsuka ◽  
Yasushi Akiyama ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Novel Species ◽  
Sequence Similarity ◽  
Sputum Sample ◽  
Lavage Fluid ◽  
Mycolic Acid ◽  
Rrna Gene ◽  
Mycolic Acids ◽  
Negative Results

Seven isolates of a slowly growing, non-chromogenic Mycobacterium species were obtained from sputum and bronchial lavage fluid samples from elderly patients in different regions of Japan. These isolates were distinguished from related non-tuberculous species by colony morphology, positive results for Tween hydrolysis, catalase at 68 °C, nitrate reductase and pyrazinamidase and negative results for semi-quantitative catalase, urease and arylsulfatase. The mycolic acid pattern obtained by HPLC revealed a single cluster of late-eluting mycolic acids similar to but different from those of Mycobacterium malmoense ATCC 29571T. The 16S rRNA gene, 16S–23S internal transcribed spacer (ITS), rpoB and hsp65 sequences were unique in comparison with those of other mycobacteria. Comparison of 16S rRNA gene sequences showed that the isolates were most closely related to Mycobacterium tuberculosis H37RvT (21 base differences in 1508 bp; 98.6 % 16S rRNA gene sequence similarity). A representative strain, GTC 2738T, showed 91.9 % rpoB sequence similarity with Mycobacterium marinum strain M, 95 % hsp65 sequence similarity with Mycobacterium kansasii CIP 104589T and 81.1 % 16S–23S ITS sequence similarity with Mycobacterium gordonae ATCC 14470T. Phylogenetic analysis of concatenated sequences of the 16S rRNA, rpoB and hsp65 genes showed that strain GTC 2738T was located on a distinct clade adjacent to M. tuberculosis, M. ulcerans and M. marinum, with bootstrap values of 81 %. DNA–DNA hybridization demonstrated less than 70 % reassociation with type strains of genetically related species and supported the novel species status of the isolates. On the basis of this evidence, a novel species with the name Mycobacterium shinjukuense sp. nov. is proposed. The type strain, isolated from a sputum sample, is strain GTC 2738T( = JCM 14233T = CCUG 53584T).

scholarly journals Effect of an Oxadiazoline and a Lignan on Mycolic Acid Biosynthesis and Ultrastructural Changes ofMycobacterium tuberculosis

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Eduard Baquero ◽  
Wiston Quiñones ◽  
Wellman Ribon ◽  
Maria Leonor Caldas ◽  
Ladys Sarmiento ◽  
...  
Keyword(s):  
Antimycobacterial Activity ◽  
Mycolic Acid ◽  
Mass Spectrometry Analysis ◽  
Ultrastructural Changes ◽  
Acid Biosynthesis ◽  
Spectrometry Analysis ◽  
Mycolic Acids ◽  
The World ◽  
Bacterial Morphology ◽  
Important Disease

Tuberculosis (TB) is an important disease that causes thousands of deaths around the world. Resistance against antitubercular available drugs has been reported; so, research on new effective antimycobacterial molecules is needed. Antimycobacterial activity of three lignans and two synthetic hydrazones was assessed againstMycobacterium tuberculosisH37Rv by antimycobacterial microdilution assay (TEMA). An oxadiazoline (AC451) and a lignan (ethoxycubebin) were the most active compounds (MIC 6.09 and 62.4 μM, resp.). Several changes in mycolic acid profile of treated bacteria were detected with both compounds by mass spectrometry analysis. Additionally, the level of reduction of mycolic acids in ethoxycubebin treatment was correlated to disruption in bacterial morphology.

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