Mycobacterial Epoxide Hydrolase EphD Is Inhibited by Urea and Thiourea Derivatives

The genome of the human intracellular pathogen Mycobacterium tuberculosis encodes an unusually large number of epoxide hydrolases, which are thought to be involved in lipid metabolism and detoxification reactions needed to endure the hostile environment of host macrophages. These enzymes therefore represent suitable targets for compounds such as urea derivatives, which are known inhibitors of soluble epoxide hydrolases. In this work, we studied in vitro the effect of the thiourea drug isoxyl on six epoxide hydrolases of M. tuberculosis using a fatty acid substrate. We show that one of the proteins inhibited by isoxyl is EphD, an enzyme involved in the metabolism of mycolic acids, key components of the mycobacterial cell wall. By analyzing mycolic acid profiles, we demonstrate the inhibition of EphD epoxide hydrolase activity by isoxyl and two other urea-based inhibitors, thiacetazone and AU1235, inside the mycobacterial cell.

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TREM2 is a receptor for non-glycosylated mycolic acids of mycobacteria that limits anti-mycobacterial macrophage activation

Nature Communications ◽

10.1038/s41467-021-22620-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ei’ichi Iizasa ◽

Yasushi Chuma ◽

Takayuki Uematsu ◽

Mio Kubota ◽

Hiroaki Kawaguchi ◽

...

Keyword(s):

Cell Wall ◽

Macrophage Activation ◽

Mycolic Acid ◽

Dependent Manner ◽

Independent Manner ◽

Lectin Receptors ◽

Mycobacterial Cell ◽

Mycobacterial Cell Wall

AbstractMycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, a mechanism of action is unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages respond to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages respond to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhances Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerates the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.

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Mycobacteriophage Ms6 LysB specifically targets the outer membrane of Mycobacterium smegmatis

Microbiology ◽

10.1099/mic.0.032821-0 ◽

2010 ◽

Vol 156 (5) ◽

pp. 1497-1504 ◽

Cited By ~ 36

Author(s):

Filipa Gil ◽

Anna E. Grzegorzewicz ◽

Maria João Catalão ◽

João Vital ◽

Michael R. McNeil ◽

...

Keyword(s):

Cell Wall ◽

Outer Membrane ◽

Mycobacterium Smegmatis ◽

Mycolic Acid ◽

Head Group ◽

Lipolytic Enzyme ◽

Ester Bond ◽

Mycolic Acids ◽

Mycobacterial Cell ◽

Mycobacterial Cell Wall

LysB, a mycobacteriophage Ms6-encoded protein, was previously identified as a lipolytic enzyme able to hydrolyse the ester bond in lipase and esterase substrates. In the present work, we show that LysB can hydrolyse lipids containing mycolic acids from the outer membrane of the mycobacterial cell wall. LysB was shown to hydrolyse the mycolic acids from the mycolyl-arabinogalactan–peptidoglycan complex where the mycolates of the inner leaflet of the outer membrane are covalently attached to an arabinosyl head group. In addition, treatment of the extractable lipids from Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with LysB showed that trehalose 6,6′-dimycolate (TDM), a trehalose diester of two mycolic acid molecules, was hydrolysed by the enzyme. We have also determined the structures of the mycolic acid molecules that form the M. smegmatis TDM. The identification of a phage-encoded enzyme that targets the outer membrane of the mycobacterial cell wall enhances our understanding of the mechanism of mycobacteriophage lysis.

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Contrasting roles of the innate receptors TREM2 versus Mincle in the recognition and response of macrophages to mycolic acid-containing lipids in mycobacterial cell walls

10.21203/rs.3.rs-37581/v1 ◽

2020 ◽

Author(s):

Ei'ichi Iizasa ◽

Yasushi Chuma ◽

Takayuki Uematsu ◽

Mio Kubota ◽

Hiroaki Kawaguchi ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterial Infection ◽

Mycolic Acid ◽

Dependent Manner ◽

Independent Manner ◽

Lectin Receptors ◽

Mycobacterial Cell ◽

Mycobacterial Cell Wall

Abstract Mycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, their mechanism of action remains unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages responded to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages responded to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhanced Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerated the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.

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Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

Current Molecular Pharmacology ◽

10.2174/1874467211666181025141114 ◽

2019 ◽

Vol 12 (1) ◽

pp. 27-49 ◽

Cited By ~ 6

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.

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In vitro effects of lipopolysaccharides and mycobacterial cell wall components on swine alveolar macrophages

Research in Veterinary Science ◽

10.1016/s0034-5288(18)32262-8 ◽

1983 ◽

Vol 34 (2) ◽

pp. 212-217 ◽

Cited By ~ 5

Author(s):

B. Charley ◽

C. Leclerc ◽

E. Petit ◽

L. Chedid

Keyword(s):

Cell Wall ◽

Alveolar Macrophages ◽

Cell Wall Components ◽

Mycobacterial Cell ◽

In Vitro Effects ◽

Mycobacterial Cell Wall ◽

Wall Components

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MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1901346116 ◽

2019 ◽

Vol 116 (23) ◽

pp. 11241-11246 ◽

Cited By ~ 28

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.

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Synthesis of a Di-Mycoloyl Tri-Arabinofuranosyl Glycerol Fragment of the Mycobacterial Cell Wall, Based on Synthetic Mycolic Acids

Molecules ◽

10.3390/molecules24193596 ◽

2019 ◽

Vol 24 (19) ◽

pp. 3596

Author(s):

Ali ◽

Mohammed ◽

Dulayymi ◽

Baird

Keyword(s):

Cell Wall ◽

Cell Walls ◽

Stereoselective Synthesis ◽

Complex Mixtures ◽

Mycolic Acids ◽

Mycobacterial Cell ◽

Antigenic Properties ◽

Mycobacterial Cell Wall ◽

Structural Classes

Fragments of mycobacterial cell walls such as arabinoglycerol mycolate and dimycoloyl diarabinoglycerol, comprising complex mixtures of mycolic acids, have immunostimulatory and antigenic properties. A related di-mycoloyl tri-arabinofuranosyl glycerol fragment has been isolated from cell wall hydrolysates. An effective stereoselective synthesis of tri-arabinofuranosyl glycerol, followed by coupling with stereochemically defined mycolic acids of different structural classes, to provide unique di-mycoloyl tri-arabinofuranosyl glycerols is now described.

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