Biomedical subjects

Robert C Fahey

Publications and source records attributed to Robert C Fahey.

At least 19 recordsLinked to original sources

PubMed2006

Biochemistry of the initial steps of mycothiol biosynthesis.

Mycothiol is the major thiol produced by mycobacteria and is required for growth of Mycobacterium tuberculosis. The final three steps in the biosynthesis of mycothiol have been fully elucidated but the initial steps have been unclear. A glycosyltransferase, MshA, is required for production of the mycothiol precursor, 1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol, but its substrates and immediate products were unknown. In this study, we show that the N-acetylglucosamine donor is UDP-N-acetylglucosamine and that the N-acetylglucosamine acceptor is 1L-myo-inositol 1-phosphate. The reaction generates UDP and 1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol 3-phosphate. Using cell-free extracts of M. smegmatis mc(2)155, little activity was obtained with myo-inositol, 1D-myo-inositol 1-phosphate, or myo-inositol 2-phosphate as the N-acetylglucosamine acceptor. A phosphatase, designated MshA2, is required to dephosphorylate 1-O-(2-acetamido-2-deoxy-alpha-glucopyranosyl)-D-myo-inositol 3-phosphate to produce 1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol. The latter is deacetylated, ligated with cysteine, and the cysteinyl amino group acetylated by acetyl-CoA to complete the mycothiol biosynthesis pathway. Uptake and concentration of myo-[14C]inositol is rapid in Mycobacterium smegmatis and leads to production of radiolabeled inositol 1-phosphate and mycothiol. This demonstrates the presence of a myo-inositol transporter and a kinase that generates 1L-myo-inositol 1-phosphate. The biochemical pathway of mycothiol biosynthesis is now fully elucidated.

Acetylation↗

PubMed2006

Cloning, expression and rapid purification of active recombinant mycothiol ligase as B1 immunoglobulin binding domain of streptococcal protein G, glutathione-S-transferase and maltose binding protein fusion proteins in Mycobacterium smegmatis.

Mycothiol ligase (MshC) is a key enzyme in the biosynthesis of mycothiol, a small molecular weight thiol found in Mycobacteria spp. and other actinomycetes. Mycothiol plays a fundamental role in these organisms by helping to provide protection from the effects of reactive oxygen species and electrophiles, including many antibiotics. It has recently been demonstrated that the MshC gene and more generally the production of mycothiol are essential to Mycobacterium tuberculosis, indicating that MshC may represent a novel target for new classes of antituberculars. Because MshC cannot be expressed heterologously in Escherichia coli and isolation from Mycobacterium smegmatis is impractical, we have optimized the E. coli-M. smegmatis shuttle vector pACE for cloning and recombinant expression of MshC (under control of an acetamidase-inducible promoter). To improve expression levels and simplify purification, we further constructed three N-terminal-MshC fusion proteins where N-terminal tags included the B1 domain of streptococcal protein G (to give GB1-MshC), glutathione-S-transferase (to give GST-MshC) and maltose binding protein (to give MBP-MshC), for expression in M. smegmatis. By expressing all three fusion proteins in a mutant strain of M. smegmatis mc(2)155, namely I64 L205P MshC M. smegmatis which lacks mycothiol ligase activity, we demonstrate in vivo mycothiol ligase activity for each construct. Recombinant GST-MshC and MBP-MshC were isolated in one step by affinity chromatography in a yield of 0.7 and 1.2 mg fusion protein/L and exhibited specific activities of 9 nmolmin(-1)mg(-1) and 25 nmolmin(-1)mg(-1), respectively.

Bacterial Proteins↗

PubMed2006

A coupled spectrophotometric assay for l-cysteine:1-D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase and its application for inhibitor screening.

Most actinomycetes, including Mycobacterium tuberculosis, do not produce glutathione but make an alternative thiol, mycothiol, which has functions similar to those of glutathione. A key step in mycothiol biosynthesis is the ATP-dependent ligation of Cys to GlcN-Ins catalyzed by MshC to produce Cys-GlcN-Ins, AMP, and PP(i). MshC is essential for growth of M. tuberculosis and is therefore a potential target for drugs directed against tuberculosis. A coupled-enzyme assay for MshC was developed using pyrophosphatase to convert pyrophosphate to phosphate and spectrophotometric detection of the latter via the phosphomolybdate complex with malachite green. The assay was readily adapted for use in a 96-well microtiter plate format. A secondary high-performance liquid chromatography assay measuring Cys-GlcN-Ins production was used to validate potential hits. Preliminary testing on a library of 2,024 compounds predicted to inhibit ATP-dependent enzymes identified many promiscuous and pyrophosphatase inhibitors of MshC and a single validated inhibitor with IC(50) approximately 100 microM.

Antitubercular Agents↗

PubMed2006

Purification and characterization of Mycobacterium tuberculosis 1D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase, MshB, a mycothiol biosynthetic enzyme.

Mycothiol (MSH, AcCys-GlcN-Ins) is the major low molecular weight thiol in actinomycetes and is essential for growth of Mycobacterium tuberculosis. MshB, the GlcNAc-Ins deacetylase, is a key enzyme in MSH biosynthesis. MshB from M. tuberculosis was cloned, expressed, purified, and its properties characterized. Values of k(cat) and K(m) for MshB were determined for the biological substrate, GlcNAc-Ins, and several other good substrates. The substrate specificity of MshB was compared to that of M. tuberculosis mycothiol S-conjugate amidase (Mca), a homologous enzyme having weak GlcNAc-Ins deacetylase activity. Both enzymes are metalloamidases with overlapping amidase activity toward mycothiol S-conjugates (AcCySR-GlcN-Ins). The Ins residue and hydrophobic R groups enhance the activity with both MshB and Mca, but changes in the acyl group attached to GlcN have opposite effects on the two enzymes.

Amidohydrolases↗

PubMed2006

The mshA gene encoding the glycosyltransferase of mycothiol biosynthesis is essential in Mycobacterium tuberculosis Erdman.

Mycothiol is the major low-molecular-weight thiol found in actinomycetes, including Mycobacterium tuberculosis, and has important antioxidant and detoxification functions. Gene disruption studies have shown that mycothiol is essential for the growth of M. tuberculosis. Because of mycothiol's unique characteristics, inhibitors directed against mycothiol biosynthesis have potential as drugs against M. tuberculosis. Four genes have been identified in mycobacteria that are involved in the biosynthesis of mycothiol. Two genes, mshB and mshD, are not essential for growth of M. tuberculosis. Mutants in these genes produce significant amounts of mycothiol or closely related thiol compounds. A targeted gene disruption in the mshC gene is lethal for M. tuberculosis, indicating that MshC is essential for growth. The remaining gene, mshA, encodes for a glycosyltransferase. In the present study, we attempted to produce a directed knock-out of the mshA gene in M. tuberculosis Erdman but found that this was only possible when a second copy of mshA was first incorporated into the chromosome. Bacteria with only a single copy of mshA that grew after mutagenesis produced normal levels of mycothiol. We therefore conclude that the mshA gene, like the mshC gene, is essential for the growth of M. tuberculosis.

Bacterial Proteins↗

PubMed2006

A mycothiol synthase mutant of Mycobacterium tuberculosis has an altered thiol-disulfide content and limited tolerance to stress.

Mycothiol (MSH) (acetyl-Cys-GlcN-Ins) is the major low-molecular-mass thiol in Mycobacterium tuberculosis. MSH has antioxidant activity, can detoxify a variety of toxic compounds, and helps to maintain the reducing environment of the cell. The production of MSH provides a potential novel target for tuberculosis treatment. Biosynthesis of MSH requires at least four genes. To determine which of these genes is essential in M. tuberculosis, we have been constructing targeted gene disruptions. Disruption in the mshC gene is lethal to M. tuberculosis, while disruption in the mshB gene results in MSH levels 20 to 100% of those of the wild type. For this study, we have constructed a targeted gene disruption in the mshD gene that encodes mycothiol synthase, the final enzyme in MSH biosynthesis. The mshD mutant produced approximately 1% of normal MSH levels but high levels of the MshD substrate Cys-GlcN-Ins and the novel thiol N-formyl-Cys-GlcN-Ins. Although N-formyl-Cys-GlcN-Ins was maintained in a highly reduced state, Cys-GlcN-Ins was substantially oxidized. In both the wild type and the mshD mutant, cysteine was predominantly oxidized. The M. tuberculosis mshD mutant grew poorly on agar plates lacking catalase and oleic acid and in low-pH media and had heightened sensitivity to hydrogen peroxide. The inability of the mshD mutant to survive and grow in macrophages may be associated with its altered thiol-disulfide status. It appears that N-formyl-Cys-GlcN-Ins serves as a weak surrogate for MSH but is not sufficient to support normal growth of M. tuberculosis under stress conditions such as those found within the macrophage.

Acetyltransferases↗

PubMed2005

A mycothiol synthase mutant of Mycobacterium smegmatis produces novel thiols and has an altered thiol redox status.

Mycobacteria and other actinomycetes do not produce glutathione but make mycothiol (MSH; AcCys-GlcN-Ins) that has functions similar to those of glutathione and is essential for growth of Mycobacterium tuberculosis. Mycothiol synthase (MshD) catalyzes N acetylation of Cys-GlcN-Ins to produce MSH in Mycobacterium smegmatis mc2155, and Cys-GlcN-Ins is maintained at a low level. The mycothiol synthase mutant, the mshD::Tn5 mutant, produces high levels of Cys-GlcN-Ins along with two novel thiols, N-formyl-Cys-GlcN-Ins and N-succinyl-Cys-GlcN-Ins, and a small amount of MSH. The nonenzymatic reaction of acyl-coenzyme A (CoA) with Cys-GlcN-Ins to produce acyl-Cys-GlcN-Ins is a facile reaction under physiologic conditions, with succinyl-CoA being an order of magnitude more reactive than acetyl-CoA. The uncatalyzed reaction rates are adequate to account for the observed production of N-succinyl-Cys-GlcN-Ins and MSH under physiologic conditions. It was shown that the N-acyl-Cys-GlcN-Ins compounds are maintained in a substantially reduced state in the mutant but that Cys-GlcN-Ins exists in disulfide forms at 5 to 40% at different stages of growth. MSH was able to facilitate reduction of N-succinyl-Cys-GlcN-Ins disulfide through thiol-disulfide exchange, but N-formyl-Cys-GlcN-Ins was ineffective. The oxidized state of Cys-GlcN-Ins in cells appears to result from a high susceptibility to autoxidation and a low capacity of the cell to reduce its disulfide forms. The mutant exhibited no enhanced sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, or cumene hydroperoxide relative to the parent strain, suggesting that the most abundant thiol, N-formyl-Cys-GlcN-Ins, functions as a substitute for MSH.

Acetyl Coenzyme A↗

PubMed2004

Targeted mutagenesis of the Mycobacterium smegmatis mca gene, encoding a mycothiol-dependent detoxification protein.

Mycothiol (MSH), a functional analogue of glutathione (GSH) that is found exclusively in actinomycetes, reacts with electrophiles and toxins to form MSH-toxin conjugates. Mycothiol S-conjugate amidase (Mca) then catalyzes the hydrolysis of an amide bond in the S conjugates, producing a mercapturic acid of the toxin, which is excreted from the bacterium, and glucosaminyl inositol, which is recycled back to MSH. In this study, we have generated and characterized an allelic exchange mutant of the mca gene of Mycobacterium smegmatis. The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S. Introduction of M. tuberculosis mca epichromosomally or introduction of M. smegmatis mca integratively resulted in complementation of Mca activity and reduced levels of S conjugates. The mutation in mca renders the mutant strain more susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin. Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin. Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycin in the mycobacterial cell in an MSH-dependent manner.

Amidohydrolases↗

PubMed2003

Characterization of Mycobacterium tuberculosis mycothiol S-conjugate amidase.

Mycothiol is comprised of N-acetylcysteine (AcCys) amide linked to 1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins) and is the predominant thiol found in most actinomycetes. Mycothiol S-conjugate amidase (Mca) cleaves the amide bond of mycothiol S-conjugates of a variety of alkylating agents and xenobiotics, producing GlcN-Ins and a mercapturic acid that can be excreted from the cell. Mca of Mycobacterium tuberculosis (Rv1082) was cloned and expressed as a soluble protein in Escherichia coli. The protein contained 1.4 +/- 0.1 equiv of zinc after purification, indicating that Mca is a metalloprotein with zinc as the native metal. Kinetic studies of Mca activity with 14 substrates demonstrated that Mca is highly specific for the mycothiol moiety of mycothiol S-conjugates and relatively nonspecific for the structure of the sulfur-linked conjugate. The deacetylase activity of Mca with GlcNAc-Ins is small but significant and failed to saturate at up to 2 mM GlcNAc-Ins, indicating that Mca may contribute modestly to the production of GlcN-Ins when GlcNAc-Ins levels are high. The versatility of Mca can be seen in its ability to react with a broad range of mycothiol S-conjugates, including two different classes of antibiotics. The mycothiol S-conjugate of rifamycin S was produced under physiologically relevant conditions and was shown to be a substrate for Mca in both oxidized and reduced forms. Significant activity was also seen with the mycothiol S-conjugate of the antibiotic cerulenin as a substrate for Mca.

Amidohydrolases↗

PubMed2003

The crystal structure of 1-D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) from Mycobacterium tuberculosis reveals a zinc hydrolase with a lactate dehydrogenase fold.

Mycothiol (1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside, MSH or AcCys-GlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn2+ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a C-terminal domain consisting of two beta-sheets and two alpha-helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD+ co-factor of lactate dehydrogenase. The Zn2+ is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn2+ providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol S-conjugate amidase based on the structure of MshB.

Amidohydrolases↗

PubMed2003

Inhibition and kinetics of mycobacterium tuberculosis and mycobacterium smegmatis mycothiol-S-conjugate amidase by natural product inhibitors.

The current rise in mycobacterial-related infections and disease, coupled with drug resistance, underlines the continuing need for new antimycobacterials. To this end, we have screened approximately 1500 extracts derived from marine plants and invertebrates and terrestrial fungi for their ability to inhibit a newly described mycobacterial detoxification enzyme mycothiol-S-conjugate amidase (MCA). As described in this paper, our screening and chemistry efforts thus far have led to the identification of 13 natural product inhibitors that represent six different structural classes. By conducting enzyme inhibition assays using varied inhibitor and substrate concentrations, we have determined the mode of inhibition of Mycobacterium tuberculosis MCA for four of these compounds. We show that two types of bromotyrosine-derived natural products are competitive inhibitors of MCA; while oceanapiside, an alpha,omega-bis-aminohydroxy glycosphingolipid, and the fungal metabolite gliotoxin, a dithiadiketopiperazine, are simple and mixed non-competitive inhibitors, respectively. Correlation of these results with the chemical structures suggests that MCA is a metalloenzyme and that the oximinoamide and spiro-isoxazoline amide groups present in the competitive inhibitors are substrate mimics.

Amidohydrolases↗

PubMed2003

Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics.

Mycothiol, MSH or 1D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside, is an unusual conjugate of N-acetylcysteine (AcCys) with 1D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins), and is the major low-molecular-mass thiol in mycobacteria. Mycothiol has antioxidant activity as well as the ability to detoxify a variety of toxic compounds. Because of these activities, MSH is a candidate for protecting Mycobacterium tuberculosis from inactivation by the host during infections as well as for resisting antituberculosis drugs. In order to define the protective role of MSH for M. tuberculosis, we have constructed an M. tuberculosis mutant in Rv1170, one of the candidate MSH biosynthetic genes. During exponential growth, the Rv1170 mutant bacteria produced approximately 20% of wild-type levels of MSH. Levels of the Rv1170 substrate, GlcNAc-Ins, were elevated, whereas those of the product, GlcN-Ins, were reduced. This establishes that the Rv1170 gene encodes for the major GlcNAc-Ins deacetylase activity (termed MshB) in the MSH biosynthetic pathway of M. tuberculosis. The Rv1170 mutant grew poorly on agar media lacking catalase and oleic acid, and had heightened sensitivities to the toxic oxidant cumene hydroperoxide and to the antibiotic rifampin. In addition, the mutant was more resistant to isoniazid, suggesting a role for MSH in activation of this prodrug. These data indicate that MSH contributes to the protection of M. tuberculosis from oxidants and influences resistance to two first-line antituberculosis drugs.

Amidohydrolases↗

PubMed2003

The glycosyltransferase gene encoding the enzyme catalyzing the first step of mycothiol biosynthesis (mshA).

Mycothiol is the major thiol present in most actinomycetes and is produced from the pseudodisaccharide 1D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins). A transposon mutant of Mycobacterium smegmatis shown to be GlcNAc-Ins and mycothiol deficient was sequenced to identify a putative glycosyltransferase gene designated mshA. The ortholog in Mycobacterium tuberculosis, Rv0486, was used to complement the mutant phenotype.

Amino Acid Sequence↗

PubMed2003

Mycothiol is essential for growth of Mycobacterium tuberculosis Erdman.

Mycothiol (MSH) is the major low-molecular-mass thiol in mycobacteria and is associated with the protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. The biosynthesis of MSH is a multistep process, with the enzymatic reaction designated MshC being the ligase step in MSH production. A targeted disruption of the native mshC gene in M. tuberculosis Erdman produced no viable clones possessing either a disrupted mshC gene or reduced levels of MSH. However, when a second copy of the mshC gene was incorporated into the chromosome prior to the targeted disruption, multiple clones having the native gene disrupted and the second copy of mshC intact were obtained. These clones produced normal levels of MSH. These results demonstrate that the mshC gene and, more generally, the production of MSH are essential for the growth of M. tuberculosis Erdman under laboratory conditions.

Adenosine Triphosphate↗

PubMed2002

Mycothiol biochemistry.

Mycothiol (MSH) is a novel thiol comprised of N-acetylcysteine amide-linked to GlcN-alpha(1-1)-Ins. It is the major thiol in most actinomycetes and is produced at millimolar levels in mycobacteria and streptomycetes. MSH biosynthesis occurs by linkage of GlcNAc to Ins, deacetylation to GlcN-Ins, ligation of the latter to L-cysteine, and transacetylation of the cysteinyl residue by CoASAc to produce MSH. The genes encoding the respective enzymes have been designated mshA, mshB, mshC, and mshD; all but mshA have been identified. Mycobacterium smegmatis mutants deficient in mshA, mshC, and mshD have been characterized. MSH plays a significant role in the detoxification of thiol-reactive substances, including formaldehyde, various electrophiles, and antibiotics. Mycothiol S-conjugates derived from electrophiles and antibiotics are cleaved by mycothiol S-conjugate amidase to release GlcN-Ins, used to resynthesize MSH, and a mercapturic acid which is excreted from the cell. A mycothiol-disulfide-selective reductase has been identified and likely helps to maintain cellular MSH in the reduced state. Mycothiol biochemistry has characteristics similar to those of glutathione but also has a variety of unique features.

Aldehyde Oxidoreductases↗

PubMed2002

Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes.

Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis. In this work we describe the isolation and characterization of a Tn 5 transposon mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its precursor, 1 D-myo-inosityl 2- L-cysteinylamido-2-deoxy-alpha-D-glucopyranoside (Cys-GlcN-Ins). Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys-GlcN-Ins acetyltransferase (mycothiol synthase, MshD) activity, which was found in wild-type cells, but not in the mutant. Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M. smegmatis genome, as well as the orthologous gene Rv0819 in the M. tuberculosis genome. Cloning and expression of mshD from M. tuberculosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene.

Acetyltransferases↗

PubMed2002

ATP-dependent L-cysteine:1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase, mycothiol biosynthesis enzyme MshC, is related to class I cysteinyl-tRNA synthetases.

Mycothiol is a novel thiol produced only by actinomycetes and is the major low molecular weight thiol in mycobacteria. The mycothiol biosynthetic pathway has been postulated to involve ATP-dependent ligation of L-cysteine (Cys) with 1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside; GlcN-Ins) catalyzed by MshC to produce Cys-GlcN-Ins. The ligase activity was purified approximately 2400-fold from Mycobacterium smegmatis and two proteins of slightly different M(r) approximately 47000 were identified with MshC activity. The N-terminal sequence of the smaller protein revealed that it was coded by a gene in the databases for M. smegmatis and M. tuberculosis previously designated as cysS2. The larger protein was coded by the same gene in M. smegmatis but included an eight amino acid N-terminal extension involving a different start codon. The ligase was found to have K(m) values of 40 +/- 3 and 72 +/- 9 microM for Cys and GlcN-Ins, respectively. The cysS2 gene was thought to encode a second cysteinyl-tRNA synthetase in addition to cysS but the present results indicate that cysS2 is actually the mshC gene encoding ATP-dependent Cys:GlcN-Ins ligase.

Adenosine Triphosphate↗

PubMed2002

Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to alkylating agents, free radicals, and antibiotics.

Mycothiol (MSH; 1D-myo-inosityl 2-[N-acetyl-L-cysteinyl]amido-2-deoxy-alpha-D-glucopyranoside) is the major low-molecular-weight thiol produced by mycobacteria. Mutants of Mycobacterium smegmatis mc(2)155 deficient in MSH production were produced by chemical mutagenesis as well as by transposon mutagenesis. One chemical mutant (mutant I64) and two transposon mutants (mutants Tn1 and Tn2) stably deficient in MSH production were isolated by screening for reduced levels of MSH content. The MSH contents of transposon mutants Tn1 and Tn2 were found to be less than 0.1% that of the parent strain, and the MSH content of I64 was found to be 1 to 5% that of the parent strain. All three strains accumulated 1D-myo-inosityl 2-deoxy-alpha-D-glucopyranoside to levels 20- to 25-fold the level found in the parent strain. The cysteine:1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase (MshC) activities of the three mutant strains were < or =2% that of the parent strain. Phenotypic analysis revealed that these MSH-deficient mutants possess increased susceptibilities to free radicals and alkylating agents and to a wide range of antibiotics including erythromycin, azithromycin, vancomycin, penicillin G, rifamycin, and rifampin. Conversely, the mutants possess at least 200-fold higher levels of resistance to isoniazid than the wild type. We mapped the mutation in the chemical mutant by sequencing the mshC gene and showed that a single amino acid substitution (L205P) is responsible for reduced MSH production and its associated phenotype. Our results demonstrate that there is a direct correlation between MSH depletion and enhanced sensitivity to toxins and antibiotics.

Alkylating Agents↗