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Mycothiol biosynthesis is essential for ethionamide susceptibility in Mycobacterium tuberculosis.

Vilchèze C, Av-Gay Y, Attarian R, Liu Z, Hazbón MH, Colangeli R, Chen B, Liu W, Alland D, Sacchettini JC, Jacobs WR - Mol. Microbiol. (2008)

Bottom Line: Seven independent missense or frameshift mutations within mshA were identified and characterized.The mshA deletion mutants were defective in mycothiol biosynthesis, were only ethionamide-resistant and required catalase to grow.Biochemical studies suggested that the mechanism of ethionamide resistance in mshA mutants was likely due to a defect in ethionamide activation.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Spontaneous mutants of Mycobacterium tuberculosis that were resistant to the anti-tuberculosis drugs ethionamide and isoniazid were isolated and found to map to mshA, a gene encoding the first enzyme involved in the biosynthesis of mycothiol, a major low-molecular-weight thiol in M. tuberculosis. Seven independent missense or frameshift mutations within mshA were identified and characterized. Precise deletion mutations of the mshA gene were generated by specialized transduction in three different strains of M. tuberculosis. The mshA deletion mutants were defective in mycothiol biosynthesis, were only ethionamide-resistant and required catalase to grow. Biochemical studies suggested that the mechanism of ethionamide resistance in mshA mutants was likely due to a defect in ethionamide activation. In vivo, a mycothiol-deficient strain grew normally in immunodeficient mice, but was slightly defective for growth in immunocompetent mice. Mutations in mshA demonstrate the non-essentiality of mycothiol for growth in vitro and in vivo, and provide a novel mechanism of ethionamide resistance in M. tuberculosis.

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Fatty acid methyl ester (FAME) and mycolic acid methyl ester (MAME) analyses of ΔmshA mutants. M. tuberculosis wild-type strains, ΔmshA and complemented strains were treated with INH (0.5 mg l−1) or ETH (15 mg l−1) for 4 h, and then labelled with [1-14C]-acetate for 20 h. Fatty acids and mycolic acids were saponified, methylated, extracted and separated by thin-layer chromatography. 14C-labelled FAMEs and MAMEs were detected by autoradioragraphy after 36 h exposure at −80°C. c = pMV361::mshA.
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fig06: Fatty acid methyl ester (FAME) and mycolic acid methyl ester (MAME) analyses of ΔmshA mutants. M. tuberculosis wild-type strains, ΔmshA and complemented strains were treated with INH (0.5 mg l−1) or ETH (15 mg l−1) for 4 h, and then labelled with [1-14C]-acetate for 20 h. Fatty acids and mycolic acids were saponified, methylated, extracted and separated by thin-layer chromatography. 14C-labelled FAMEs and MAMEs were detected by autoradioragraphy after 36 h exposure at −80°C. c = pMV361::mshA.

Mentions: The death of the tubercle bacillus following treatment with INH correlates with inhibition of the biosynthesis of the long-chain α-alkyl β-hydroxy fatty acids (up to 90 carbons in length) called mycolic acids, which are a major constituent of the mycobacterial cell wall (Winder and Collins, 1970; Takayama et al., 1972). ETH, based on its similarity to INH, has also been predicted and shown to inhibit mycolic acid biosynthesis (Winder et al., 1971; Quemard et al., 1992; Baulard et al., 2000). As mycothiol is not known to be involved in the FASII pathway, the resistance mediated by mshA could suggest that the lethal event occurs in some redox function. If so, it may be possible that INH and ETH treatment of mshA mutants does not confer resistance to mycolic acid inhibition by ETH or INH. Fatty acids were extracted from the wild-type M. tuberculosis strains, the ΔmshA mutants and the ΔmshA-complemented strains following INH or ETH treatment, and derivatized to their methyl esters. Analysis by thin-layer chromatography (TLC) allowed for the separation between the short-chain fatty acid (up to 26 carbons in length) methyl esters (FAMEs) and the long-chain mycolic acid methyl esters (MAMEs) (Fig. 6). Treatment of the wild-type M. tuberculosis strains and the ΔmshA-complemented strains with INH or ETH resulted in inhibition of mycolic acid biosynthesis as shown by the absence of MAMEs on TLC. In contrast, the ΔmshA mutants were resistant to mycolic acid inhibition upon treatment with ETH, but not with INH (Fig. 6).


Mycothiol biosynthesis is essential for ethionamide susceptibility in Mycobacterium tuberculosis.

Vilchèze C, Av-Gay Y, Attarian R, Liu Z, Hazbón MH, Colangeli R, Chen B, Liu W, Alland D, Sacchettini JC, Jacobs WR - Mol. Microbiol. (2008)

Fatty acid methyl ester (FAME) and mycolic acid methyl ester (MAME) analyses of ΔmshA mutants. M. tuberculosis wild-type strains, ΔmshA and complemented strains were treated with INH (0.5 mg l−1) or ETH (15 mg l−1) for 4 h, and then labelled with [1-14C]-acetate for 20 h. Fatty acids and mycolic acids were saponified, methylated, extracted and separated by thin-layer chromatography. 14C-labelled FAMEs and MAMEs were detected by autoradioragraphy after 36 h exposure at −80°C. c = pMV361::mshA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2628429&req=5

fig06: Fatty acid methyl ester (FAME) and mycolic acid methyl ester (MAME) analyses of ΔmshA mutants. M. tuberculosis wild-type strains, ΔmshA and complemented strains were treated with INH (0.5 mg l−1) or ETH (15 mg l−1) for 4 h, and then labelled with [1-14C]-acetate for 20 h. Fatty acids and mycolic acids were saponified, methylated, extracted and separated by thin-layer chromatography. 14C-labelled FAMEs and MAMEs were detected by autoradioragraphy after 36 h exposure at −80°C. c = pMV361::mshA.
Mentions: The death of the tubercle bacillus following treatment with INH correlates with inhibition of the biosynthesis of the long-chain α-alkyl β-hydroxy fatty acids (up to 90 carbons in length) called mycolic acids, which are a major constituent of the mycobacterial cell wall (Winder and Collins, 1970; Takayama et al., 1972). ETH, based on its similarity to INH, has also been predicted and shown to inhibit mycolic acid biosynthesis (Winder et al., 1971; Quemard et al., 1992; Baulard et al., 2000). As mycothiol is not known to be involved in the FASII pathway, the resistance mediated by mshA could suggest that the lethal event occurs in some redox function. If so, it may be possible that INH and ETH treatment of mshA mutants does not confer resistance to mycolic acid inhibition by ETH or INH. Fatty acids were extracted from the wild-type M. tuberculosis strains, the ΔmshA mutants and the ΔmshA-complemented strains following INH or ETH treatment, and derivatized to their methyl esters. Analysis by thin-layer chromatography (TLC) allowed for the separation between the short-chain fatty acid (up to 26 carbons in length) methyl esters (FAMEs) and the long-chain mycolic acid methyl esters (MAMEs) (Fig. 6). Treatment of the wild-type M. tuberculosis strains and the ΔmshA-complemented strains with INH or ETH resulted in inhibition of mycolic acid biosynthesis as shown by the absence of MAMEs on TLC. In contrast, the ΔmshA mutants were resistant to mycolic acid inhibition upon treatment with ETH, but not with INH (Fig. 6).

Bottom Line: Seven independent missense or frameshift mutations within mshA were identified and characterized.The mshA deletion mutants were defective in mycothiol biosynthesis, were only ethionamide-resistant and required catalase to grow.Biochemical studies suggested that the mechanism of ethionamide resistance in mshA mutants was likely due to a defect in ethionamide activation.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Spontaneous mutants of Mycobacterium tuberculosis that were resistant to the anti-tuberculosis drugs ethionamide and isoniazid were isolated and found to map to mshA, a gene encoding the first enzyme involved in the biosynthesis of mycothiol, a major low-molecular-weight thiol in M. tuberculosis. Seven independent missense or frameshift mutations within mshA were identified and characterized. Precise deletion mutations of the mshA gene were generated by specialized transduction in three different strains of M. tuberculosis. The mshA deletion mutants were defective in mycothiol biosynthesis, were only ethionamide-resistant and required catalase to grow. Biochemical studies suggested that the mechanism of ethionamide resistance in mshA mutants was likely due to a defect in ethionamide activation. In vivo, a mycothiol-deficient strain grew normally in immunodeficient mice, but was slightly defective for growth in immunocompetent mice. Mutations in mshA demonstrate the non-essentiality of mycothiol for growth in vitro and in vivo, and provide a novel mechanism of ethionamide resistance in M. tuberculosis.

Show MeSH
Related in: MedlinePlus