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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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Related in: MedlinePlus

In vivo identification and relative quantification of cis-cyclopropanes by 1H HR-MAS NMR.(A) Detail of 1H HR-MAS spectrum of control whole cells of M. bovis BCG. (B) Unidimensional selective COSY spectrum after irradiation of Ha signal showing 3J and 2J connectivities of cis-cyclopropyl ring Hb and Hc protons to Ha as depicted in (C). (D) Relative quantification by 1H HR-MAS NMR of cis-cyclopropanes based on differential integration of the Ha signals in control untreated cells (c) or cells treated with TAC-treated (1 µg/ml) (TAC) or SRI-224-treated (1 µg/ml) (224). Results are representative of two independent experiments.
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pone-0001343-g006: In vivo identification and relative quantification of cis-cyclopropanes by 1H HR-MAS NMR.(A) Detail of 1H HR-MAS spectrum of control whole cells of M. bovis BCG. (B) Unidimensional selective COSY spectrum after irradiation of Ha signal showing 3J and 2J connectivities of cis-cyclopropyl ring Hb and Hc protons to Ha as depicted in (C). (D) Relative quantification by 1H HR-MAS NMR of cis-cyclopropanes based on differential integration of the Ha signals in control untreated cells (c) or cells treated with TAC-treated (1 µg/ml) (TAC) or SRI-224-treated (1 µg/ml) (224). Results are representative of two independent experiments.

Mentions: Relative quantification of mycolate cyclopropanation in whole cells of mycobacteria was assessed by High-Resolution Magic Angle Spinning (HR-MAS) Nuclear Magnetic Resonance (NMR). This technique was previously shown to be effective for directly demonstrating the effects of the antitubercular drug ethambutol on the cell wall polysaccharides of living mycobacteria [39]. Here, for the first time, we apply this method to observe modifications in the structures of mycolic acids directly on whole cells of mycobacteria. The 1H HR-MAS NMR spectrum of intact M. bovis BCG control cells in D2O showed the presence of a well-isolated but excessively broad signal, presumably resulting from the low diffusion rate of mycolates within the cell wall, centred at −0.33 ppm (data not shown). This was tentatively attributed to the upfield methylenic proton of the cis-cyclopropyl ring according to standard purified mycolates and literature [40]. As shown in Figure 6A, a well resolved signal (Ha) was obtained after partial disruption of mycobacterial cell wall by adding 5 µl of chloroform to cell pellets directly into the Zr rotor. Attribution of spin system of cyclopropyl ring was confirmed by irradiating Ha proton by a selective COSY experiment. This permitted observation of Hb and Hc protons of the cyclopropyl ring at δ 0.56 and 0.64 ppm through 3J and 2J connectivities with Ha, respectively (Figure 6B, C). Based on these data, we used Ha proton as a probe for the relative quantification of cyclopropanation by comparing its intensity in control and SRI-224-treated cells of M. bovis BCG, after normalizing the spectra on -CH2- signal at δ1.25. As shown in Figure 6D, treatment of cultures with 1 µg/ml of TAC or SRI-224 reduced the quantity of cis-cyclopropane rings by ∼75%.


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)

In vivo identification and relative quantification of cis-cyclopropanes by 1H HR-MAS NMR.(A) Detail of 1H HR-MAS spectrum of control whole cells of M. bovis BCG. (B) Unidimensional selective COSY spectrum after irradiation of Ha signal showing 3J and 2J connectivities of cis-cyclopropyl ring Hb and Hc protons to Ha as depicted in (C). (D) Relative quantification by 1H HR-MAS NMR of cis-cyclopropanes based on differential integration of the Ha signals in control untreated cells (c) or cells treated with TAC-treated (1 µg/ml) (TAC) or SRI-224-treated (1 µg/ml) (224). Results are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001343-g006: In vivo identification and relative quantification of cis-cyclopropanes by 1H HR-MAS NMR.(A) Detail of 1H HR-MAS spectrum of control whole cells of M. bovis BCG. (B) Unidimensional selective COSY spectrum after irradiation of Ha signal showing 3J and 2J connectivities of cis-cyclopropyl ring Hb and Hc protons to Ha as depicted in (C). (D) Relative quantification by 1H HR-MAS NMR of cis-cyclopropanes based on differential integration of the Ha signals in control untreated cells (c) or cells treated with TAC-treated (1 µg/ml) (TAC) or SRI-224-treated (1 µg/ml) (224). Results are representative of two independent experiments.
Mentions: Relative quantification of mycolate cyclopropanation in whole cells of mycobacteria was assessed by High-Resolution Magic Angle Spinning (HR-MAS) Nuclear Magnetic Resonance (NMR). This technique was previously shown to be effective for directly demonstrating the effects of the antitubercular drug ethambutol on the cell wall polysaccharides of living mycobacteria [39]. Here, for the first time, we apply this method to observe modifications in the structures of mycolic acids directly on whole cells of mycobacteria. The 1H HR-MAS NMR spectrum of intact M. bovis BCG control cells in D2O showed the presence of a well-isolated but excessively broad signal, presumably resulting from the low diffusion rate of mycolates within the cell wall, centred at −0.33 ppm (data not shown). This was tentatively attributed to the upfield methylenic proton of the cis-cyclopropyl ring according to standard purified mycolates and literature [40]. As shown in Figure 6A, a well resolved signal (Ha) was obtained after partial disruption of mycobacterial cell wall by adding 5 µl of chloroform to cell pellets directly into the Zr rotor. Attribution of spin system of cyclopropyl ring was confirmed by irradiating Ha proton by a selective COSY experiment. This permitted observation of Hb and Hc protons of the cyclopropyl ring at δ 0.56 and 0.64 ppm through 3J and 2J connectivities with Ha, respectively (Figure 6B, C). Based on these data, we used Ha proton as a probe for the relative quantification of cyclopropanation by comparing its intensity in control and SRI-224-treated cells of M. bovis BCG, after normalizing the spectra on -CH2- signal at δ1.25. As shown in Figure 6D, treatment of cultures with 1 µg/ml of TAC or SRI-224 reduced the quantity of cis-cyclopropane rings by ∼75%.

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