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Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria.

Alahari A, Trivelli X, Guérardel Y, Dover LG, Besra GS, Sacchettini JC, Reynolds RC, Coxon GD, Kremer L - PLoS ONE (2007)

Bottom Line: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation.Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope.The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier II et I, Centre National de Recherche Scientifique (CNRS), UMR 5235, Montpellier, France.

ABSTRACT

Background: Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets.

Methodology/principle findings: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the alpha- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs.

Conclusions/significance: This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

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Structural determination of lipids X and Y.Lipids X and Y were purified from cell wall extract of M. bovis BCG Pasteur culture, following treatment with SRI-224 (5 µg/ml) for 24 h. (A) Conventional TLC showing purity of the samples containing lipids X and Y, that were used for structural analyses, as seen by staining with phosphomolybdic acid and charring. (B) m/z values from MALDI-TOF-MS spectra correspond to [M+Na]+ adducts of a family of methylated keto-mycolates and α-mycolates for purified lipids X and Y, respectively. (C) For 1H-NMR analysis, protons are labelled (a to h) according to their respective positions in functional groups. Relative integrations of protons have been normalized according to the number of ethylenic protons (2 for X and 4 for Y) and are indicated in brackets. *stands for proton 1H signals of contaminant ethanol present in the NMR tubes.
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pone-0001343-g004: Structural determination of lipids X and Y.Lipids X and Y were purified from cell wall extract of M. bovis BCG Pasteur culture, following treatment with SRI-224 (5 µg/ml) for 24 h. (A) Conventional TLC showing purity of the samples containing lipids X and Y, that were used for structural analyses, as seen by staining with phosphomolybdic acid and charring. (B) m/z values from MALDI-TOF-MS spectra correspond to [M+Na]+ adducts of a family of methylated keto-mycolates and α-mycolates for purified lipids X and Y, respectively. (C) For 1H-NMR analysis, protons are labelled (a to h) according to their respective positions in functional groups. Relative integrations of protons have been normalized according to the number of ethylenic protons (2 for X and 4 for Y) and are indicated in brackets. *stands for proton 1H signals of contaminant ethanol present in the NMR tubes.

Mentions: We reasoned that a detailed structural analysis of the two products X and Y should help to determine which particular enzymatic step of the mycolic acid biosynthetic pathway is inhibited by these drugs. MAMEs were extracted from with either TAC- or SRI-224-treated cells (5 µg/ml) for 24 h and X and Y were isolated as described in Methods. Purity of the lipids was confirmed by conventional TLC analysis (Figure 4A).


Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria.

Alahari A, Trivelli X, Guérardel Y, Dover LG, Besra GS, Sacchettini JC, Reynolds RC, Coxon GD, Kremer L - PLoS ONE (2007)

Structural determination of lipids X and Y.Lipids X and Y were purified from cell wall extract of M. bovis BCG Pasteur culture, following treatment with SRI-224 (5 µg/ml) for 24 h. (A) Conventional TLC showing purity of the samples containing lipids X and Y, that were used for structural analyses, as seen by staining with phosphomolybdic acid and charring. (B) m/z values from MALDI-TOF-MS spectra correspond to [M+Na]+ adducts of a family of methylated keto-mycolates and α-mycolates for purified lipids X and Y, respectively. (C) For 1H-NMR analysis, protons are labelled (a to h) according to their respective positions in functional groups. Relative integrations of protons have been normalized according to the number of ethylenic protons (2 for X and 4 for Y) and are indicated in brackets. *stands for proton 1H signals of contaminant ethanol present in the NMR tubes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2147073&req=5

pone-0001343-g004: Structural determination of lipids X and Y.Lipids X and Y were purified from cell wall extract of M. bovis BCG Pasteur culture, following treatment with SRI-224 (5 µg/ml) for 24 h. (A) Conventional TLC showing purity of the samples containing lipids X and Y, that were used for structural analyses, as seen by staining with phosphomolybdic acid and charring. (B) m/z values from MALDI-TOF-MS spectra correspond to [M+Na]+ adducts of a family of methylated keto-mycolates and α-mycolates for purified lipids X and Y, respectively. (C) For 1H-NMR analysis, protons are labelled (a to h) according to their respective positions in functional groups. Relative integrations of protons have been normalized according to the number of ethylenic protons (2 for X and 4 for Y) and are indicated in brackets. *stands for proton 1H signals of contaminant ethanol present in the NMR tubes.
Mentions: We reasoned that a detailed structural analysis of the two products X and Y should help to determine which particular enzymatic step of the mycolic acid biosynthetic pathway is inhibited by these drugs. MAMEs were extracted from with either TAC- or SRI-224-treated cells (5 µg/ml) for 24 h and X and Y were isolated as described in Methods. Purity of the lipids was confirmed by conventional TLC analysis (Figure 4A).

Bottom Line: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation.Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope.The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier II et I, Centre National de Recherche Scientifique (CNRS), UMR 5235, Montpellier, France.

ABSTRACT

Background: Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets.

Methodology/principle findings: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the alpha- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs.

Conclusions/significance: This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

Show MeSH
Related in: MedlinePlus