Inactivation of , a key gene in the mycothiol biosynthesis pathway in
Cellular levels of potential intermediates in the biosynthesis and degradation of mycothiol in Mycobacterium smegmatis
Biosynthesis of mycothiol: Elucidation of the sequence …
Although co-resistance to INH and ETH had been previously identified in M. smegmatis mshA transposon mutants (), no mechanism of resistance had been identified to link mycothiol biosynthesis with INH and ETH resistance (; ). Previously, co-resistance to INH and ETH has been shown to be conferred by three different mechanisms: (i) structural mutations in the inhA target (; ), (ii) inhA target overexpression (; ), or (iii) increased NADH/NAD+ ratios resulting in higher concentration of NADH, which competitively inhibits the binding of the INH-NAD or ETH-NAD adduct to InhA (; ). We reasoned that it was highly unlikely that the gene product of mshA directly interacted with InhA. However, it was possible that mutations in mshA caused overexpression of inhA or altered the NADH/NAD+ ratios inside the M. tuberculosis cells. To test these possibilities, we first measured the inhA mRNA levels in three mshA mutants and their complemented strains using a molecular beacon reverse transcription polymerase chain reaction (RT-PCR) assay (). In contrast to the P-15 mabA inhA mutation, which has been shown to confer 10-fold overexpression of the inhA mRNA (), all three of these mshA mutants revealed no increase in inhA mRNA levels () and so resistance to ETH and INH was not due to InhA overexpression. We also measured the NAD+ and NADH concentrations in each of the mutants and found that the mutants had mostly lower NADH concentrations compared with wild type (), demonstrating that the co-resistance to INH and ETH was not due to an increase in the NADH/NAD+ ratio. The sum of this work suggested that the mshA mutations must mediate a novel mechanism of resistance.
This entry represents the mycothiol biosynthesis protein MshB found in Mycobacterium tuberculosisand related species. This protein has been characterised as a 1D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase .
Mycothiol biosynthesis is essential for ethionamide ..
The glycosyltransferase MshA is the first step in mycothiol biosynthesis that leads to the formation of N-acetyl-glucosamine inositol (; ). 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 (). 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 (). We found a dramatic reduction (83% to undetectable levels) in the concentration of mycothiol compared with wild type (). As a control, we also measured the mycothiol level in an INH- and ETH-resistant M. tuberculosis inhA mutant, mc24911 (), 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 (). 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 (), our data show that M. tuberculosis strains that do not produce mycothiol are viable.
Numerous studies have demonstrated that there exist strains of M. tuberculosis that are resistant to INH and do not have mutations in the genes associated with INH resistance (katG, inhA structural gene and promoter, ndh) (; ; ; ; ). To eliminate the majority of spontaneous mutants of M. tuberculosis that are singly resistant to INH and map to katG, we chose to isolate mutants that were co-resistant to INH and its structural analogue ETH. Samples of three independent M. tuberculosis H37Rv cultures were plated on media containing low concentrations of both INH and ETH [≤ 4-fold the minimum inhibitory concentration (MIC)]. Seven mutants were isolated at low frequencies (1–4 × 10−8). DNA sequence analysis of targeted genes in these seven strains revealed the absence of mutations in the genes known to mediate co-resistance to INH and ETH, namely inhA (the gene or its promoter region) and ndh. This analysis provided the evidence that these strains possessed mutations that conferred INH and ETH resistance and had not been previously identified in M. tuberculosis. The mutants were transformed with a cosmid genomic library of the drug-susceptible M. tuberculosis parent. The frequency of transformation was extremely low for most of the mutants (less than 100 transformants per transformation), and only one mutant, mc24936, which had the lowest level of INH resistance, yielded more than 1000 transformants. The cosmid transformants were screened for restoration of INH and ETH susceptibility. One potential complementing cosmid was isolated, sequenced and shown to contain the mshA gene, a gene characterized as mediating the first step in the biosynthesis of mycothiol (; ), a key thiol in the family of Actinomycetes bacteria (). A link between mycothiol biosynthesis and resistance to INH and ETH had been previously established in Mycobacterium smegmatis when transposon mutants in mshA were found to be resistant to INH (more than 25-fold) and ETH (sixfold) (; ; ). Subsequent sequence analysis of mc24936 and the other mutants showed that all the M. tuberculosis H37Rv mutants had missense, nonsense or frameshift mutations in mshA (). The mshA mutants had various levels of resistance to INH (2- to 16-fold) and ETH (four- to eightfold) (). This is the first report that mshA mutations confer co-resistance to INH and ETH in M. tuberculosis.
Mycothiol biosynthesis is essential for ..
Methods: NTF1836 were the first MSH biosynthesis inhibitors identified. QSAR studies were performed on a novel series of NTF1836 as an inhibitor of MshC (enzyme involved in the synthesis of mycothiol). The series of molecules were subjected to CoMFA, CoMSIA and HQSAR studies.
Because of the unique phylogenetic distribution of mycothiol, and the enzymes responsible for its biosynthesis, these enzymes represent interesting potential targets for antimycobacterial agents.
Mycothiol: synthesis, biosynthesis and biological ..
The inhibition of the mycothiol biosynthesis is ..
Biosynthesis of mycothiol ..
Targeting Mycothiol Biosynthesis and Mycothiol …
Coresistance to isoniazid and ethionamide maps to mycothiol biosynthetic genes in Mycobacterium bovis
Mycothiol (MSH or AcCys-GlcN ..
QSAR Studies on NTF1836 Analogues as Mycothiol Biosynthesis Inhibitors Author(s): M
Marinomycins A−D, Antitumor-Antibiotics of a New …
Because of the unique phylogenetic distribution of mycothiol, and the enzymes responsible for its biosynthesis, these enzymes represent interesting potential targets for antimycobacterial agents.">
This is the official lab website for Dr. William R. Jacobs Jr.
Background: Mycothiol (MSH), a thiol compound found in Mycobacterium tuberculosis is required for the growth and survival of bacteria. This unusual compound is found only in actinomycetes. MSH biosynthesis involves many enzymes, one of them is MshC an essential enzyme for M. tuberculosis.
10.1021/ja0558948 - American Chemical Society
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 null 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.
The first total synthesis of mycothiol and ..
Isoniazid, the cornerstone of front-line TB treatment, shares a common target with the second-line TB drug ethionamide (ETH). Both INH and ETH are pro-drugs that require activation to form adducts with NAD to subsequently inhibit InhA, the NADH-dependent enoyl-ACP reductase (; ) of the fatty acid biosynthesis type II system (). However, activation of INH and of ETH occurs through different pathways. INH is activated by the katG-encoded catalase peroxidase (; ) to form the INH-NAD adduct (). ETH, on the other hand, is activated by the ethA-encoded mono-oxygenase (; ; ) to yield the ETH-NAD adduct (). Mutations in either activator confer resistance to INH or ETH respectively (; ; ; ). Co-resistance to INH and ETH can be mediated by mutations that alter the InhA target so as to prevent the INH-NAD or the ETH-NAD adduct from binding (), by mutations that cause InhA overexpression (; ) or by mutations in ndh that increase the intracellular NADH concentration, thereby competitively inhibiting the binding of the INH-NAD and ETH-NAD adducts to InhA (; ). While the majority of clinical isolates resistant to INH or ETH have been shown to map to the activator genes (katG, ethA) or the inhA target, current studies still show that up to 22% of the INH-resistant M. tuberculosis clinical isolates have no mutations in the genes known to be involved in INH or ETH resistance (). In this study, to identify novel mutations conferring INH and ETH resistance, we isolated spontaneous mutants of M. tuberculosis in vitro and found that they map to mshA, a gene encoding a glycosyltransferase involved in mycothiol biosynthesis, suggesting that mshA was non-essential. Additional genetic and biochemical studies demonstrated that mycothiol biosynthesis is required for ETH susceptibility in M. tuberculosis. Furthermore, in vivo studies showed that mycothiol is not required for growth in mice.
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