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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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Mycothiol contents in M. tuberculosis mshA mutants and complemented strains. The strains were grown to stationary phase. Mycothiol contents were measured in triplicate for the M. tuberculosis mshA point mutants (A) and their complemented strains (B) as well as for the mshA  mutants (C) and their complemented strains (D) as described in Experimental procedures. c = pMV361::mshA.
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fig02: Mycothiol contents in M. tuberculosis mshA mutants and complemented strains. The strains were grown to stationary phase. Mycothiol contents were measured in triplicate for the M. tuberculosis mshA point mutants (A) and their complemented strains (B) as well as for the mshA mutants (C) and their complemented strains (D) as described in Experimental procedures. c = pMV361::mshA.

Mentions: The glycosyltransferase MshA is the first step in mycothiol biosynthesis that leads to the formation of N-acetyl-glucosamine inositol (Newton and Fahey, 2002; Newton et al., 2006). The biosynthesis of mycothiol requires five enzymes to form N-acetyl-cysteine glucosamine inositol or mycothiol from inositol-1-phosphate and UDP-N-acetyl-glucosamine: the glycosyltransferase MshA, the phosphatase MshA2, the deacetylase MshB, the cysteine ligase MshC and the acetyltransferase MshD (Fig. 1). To analyse the effects of the diverse mutations in mshA on the biosynthesis of mycothiol, the levels of mycothiol were measured in all the mutants using fluorescent high-performance liquid chromatography (HPLC) assay (Newton et al., 2000a). We found a dramatic reduction (83% to undetectable levels) in the concentration of mycothiol compared with wild type (Fig. 2A). As a control, we also measured the mycothiol level in an INH- and ETH-resistant M. tuberculosis inhA mutant, mc24911 (Vilcheze et al., 2006), and found that this mutant had a concentration of mycothiol similar to that in wild type. Complementation of the mutants with pMV361::mshA, an integrative plasmid containing only the mshA gene of M. tuberculosis driven by the hsp60 promoter, restored mycothiol biosynthesis in all the mutants (Fig. 2B). This confirms that the defect in mycothiol biosynthesis was due to the mutations in mshA. Although mycothiol has been suggested to be essential for the growth of M. tuberculosis (Sareen et al., 2003), our data show that M. tuberculosis strains that do not produce mycothiol are viable.


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)

Mycothiol contents in M. tuberculosis mshA mutants and complemented strains. The strains were grown to stationary phase. Mycothiol contents were measured in triplicate for the M. tuberculosis mshA point mutants (A) and their complemented strains (B) as well as for the mshA  mutants (C) and their complemented strains (D) as described in Experimental procedures. c = pMV361::mshA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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fig02: Mycothiol contents in M. tuberculosis mshA mutants and complemented strains. The strains were grown to stationary phase. Mycothiol contents were measured in triplicate for the M. tuberculosis mshA point mutants (A) and their complemented strains (B) as well as for the mshA mutants (C) and their complemented strains (D) as described in Experimental procedures. c = pMV361::mshA.
Mentions: The glycosyltransferase MshA is the first step in mycothiol biosynthesis that leads to the formation of N-acetyl-glucosamine inositol (Newton and Fahey, 2002; Newton et al., 2006). The biosynthesis of mycothiol requires five enzymes to form N-acetyl-cysteine glucosamine inositol or mycothiol from inositol-1-phosphate and UDP-N-acetyl-glucosamine: the glycosyltransferase MshA, the phosphatase MshA2, the deacetylase MshB, the cysteine ligase MshC and the acetyltransferase MshD (Fig. 1). To analyse the effects of the diverse mutations in mshA on the biosynthesis of mycothiol, the levels of mycothiol were measured in all the mutants using fluorescent high-performance liquid chromatography (HPLC) assay (Newton et al., 2000a). We found a dramatic reduction (83% to undetectable levels) in the concentration of mycothiol compared with wild type (Fig. 2A). As a control, we also measured the mycothiol level in an INH- and ETH-resistant M. tuberculosis inhA mutant, mc24911 (Vilcheze et al., 2006), and found that this mutant had a concentration of mycothiol similar to that in wild type. Complementation of the mutants with pMV361::mshA, an integrative plasmid containing only the mshA gene of M. tuberculosis driven by the hsp60 promoter, restored mycothiol biosynthesis in all the mutants (Fig. 2B). This confirms that the defect in mycothiol biosynthesis was due to the mutations in mshA. Although mycothiol has been suggested to be essential for the growth of M. tuberculosis (Sareen et al., 2003), our data show that M. tuberculosis strains that do not produce mycothiol are viable.

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