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Keywords:

  • mycothiol;
  • Mycobacterium tuberculosis;
  • essential gene;
  • mshA;
  • glycosyltransferase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mycothiol (MSH, AcCys-GlcN-Ins) is the predominant low-molecular-weight thiol in most actinomycetes, including Mycobacterium tuberculosis (Newton et al., 1996). The functions of mycothiol in mycobacteria are similar to those of glutathione in glutathione-producing organisms and include protection against oxidants and cellular toxins (Newton et al., 1999, 2000a, b; Newton & Fahey, 2002; Rawat et al., 2002; Buchmeier et al., 2003; Vogt et al., 2003). Four genes have been identified encoding enzymes involved in mycothiol biosynthesis (Fig. 1). Mycothiol synthase (MshD) catalyzes the acetylation of Cys-GlcN-Ins by acetyl-CoA to produce mycothiol (Koledin et al., 2002). The ligase MshC generates Cys-GlcN-Ins from Cys and GlcN-Ins energized by ATP (Sareen et al., 2002). GlcN-Ins is produced from GlcNAc-Ins by the deacetylase MshB (Newton et al., 2000a, b). Recent studies have elucidated the initial steps leading to GlcNAc-Ins (G.L. Newton, P. Ta, K. Bzymek and R.C. Fahey, submitted for publication). The previously identified (Newton et al., 2003) glycosyltransferase (MshA) catalyzes the reaction of UDP-GlcNAc with 1l-Ins-1-P to generate GlcNAc-(α1,3)-1l-Ins-1-P [alternatively designated GlcNAc-(α1,1)-1d-Ins-3-P], which is then dephosphorylated by an as yet unidentified phosphatase (MshA2) to generate GlcNAc-Ins. Because of the interest in using mycothiol biosynthesis as a target against M. tuberculosis, we have been determining which of the mycothiol biosynthetic genes is essential for the growth of M. tuberculosis.

image

Figure 1.  Pathway, identified genes and enzymes involved in mycothiol biosynthesis.

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Mycobacterium smegmatis has proven to be a useful model organism for studying mycothiol biosynthesis because it does not require mycothiol for growth, and mutants blocked at various steps in the biosynthetic pathway can be isolated and characterized. The mechanisms that allow M. smegmatis to survive without mycothiol are unknown, but presumably involve detoxification enzymes encoded in the larger genome of M. smegmatis (7 vs. 4.4 Mb) that are absent in M. tuberculosis. Studies of M. smegmatis mutants have shown that the mshA and mshC genes are required for mycothiol biosynthesis (Rawat et al., 2002; Newton et al., 2003) but that inactivation of the mshB or mshD genes does not fully prevent mycothiol formation (Rawat et al., 2003; Newton et al., 2005). Targeted mutagenesis of the mshC gene in M. tuberculosis established that this gene is essential for growth and indicated that mycothiol itself is essential for growth (Sareen et al., 2003). This is in accord with the results from high-density mutagenesis studies that reported that the mshC gene is essential for the growth of M. tuberculosis (Sassetti et al., 2003).

Because MshA is required for mycothiol biosynthesis in M. smegmatis, the mshA gene was expected to be essential for the growth of M. tuberculosis. However, this gene was not listed among the essential genes in the high-density mutagenesis study (Sassetti et al., 2003) because its essentiality in M. tuberculosis was not confirmed in studies with Mycobacterium bovis BCG (Christopher M. Sassetti, pers. commun.). To establish whether or not the mshA gene is essential in M. tuberculosis, we have conducted a targeted gene disruption of the mshA gene following protocols analogous to those used to establish essentiality of the mshC gene (Sareen et al., 2003).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains and growth conditions

Mycobacterium tuberculosis strain Erdman (ATCC 35801), M. smegmatis mc2155 and an M. smegmatis mshA mutant strain mshA::Tn5 (Newton et al., 2003) were cultured as described elsewhere (Newton et al., 2005; Buchmeier et al., 2006).

Mutagenic construct for mshA knockout in M. tuberculosis

For the production of a targeted gene disruption within the mshA gene of M. tuberculosis, the conditionally replicating mycobacteriophage phAE87 (Bardarov et al., 2002), previously used to construct mutations in the mshC gene of M. tuberculosis, was used to carry out the alleleic exchange (Sareen et al., 2003). The mutagenic construct used for allelic exchange deleted the highly conserved domains of the glycosyltransferase family present within the mshA gene Rv0486 and was constructed following a previously described cloning strategy (Sareen et al., 2003). The mutagenic construct included an ∼500-bp fragment containing the first 96 bp of the mshA gene plus 402 bp of the upstream sequence, the hygromycin resistance gene and a 500-bp fragment containing the last 260 bp of the mshA gene plus 240 bp of the downstream sequence. Mycobacterium tuberculosis cells were infected with the mutagenic phage at 39°C for 4 h, washed and plated on 7H9 Middlebrook plates containing hygromycin and incubated at 37°C until colonies appeared.

Construction of the 2X-mshA strain of M. tuberculosis

To construct an M. tuberculosis strain with two copies of the mshA gene, the mshA ORF plus ribosome-binding site (21 bp upstream of the ATG start codon) was amplified by PCR using the primers 5′-TTCGCGATTGATAAGTCACTTCGGTTCCTCGAAGG-3′ and 5′-GGACTAGCACGGTCGGCAAGGAGGAAGTC-3′. The product was sequenced to verify its fidelity and then directionally cloned into the integrative vector pCV125 that had been previously modified to include the streptomycin resistance gene (Buchmeier et al., 2000) following the strategy described for the mshC gene (Sareen et al., 2003) so that the mshA gene was expressed behind the aph promoter. To establish that a functional MshA protein was produced by this construct, pCV125::mshA was electroporated into M. smegmatis wild-type and into an M. smegmatis mshA::Tn5 mutant (Newton et al., 2003). Streptomycin-resistant colonies were selected on Middlebrook 7H9 plates containing OADC plus streptomycin (30 μg mL−1) and transformants were grown for mycothiol analysis.

Southern hybridization

Disruption of the mshA gene within the chromosome of M. tuberculosis was determined by Southern hybridization and followed the protocol for digoxigenin (DIG) nucleic acid detection (Roche). Chromosomal DNA from each of the hygromycin-resistant clones was digested with NotI, separated by electrophoresis and transferred onto a nylon positively charged membrane (Roche). The membrane was probed with a 500 bp digoxigenin-labeled fragment generated by PCR using DIG-dUTP (Roche) that contained the last 260 bases of the mshA gene plus 240 bases of downstream sequence. Washing conditions included two 5 min washes in 2 × SSC (sodium chloride, sodium citrate) plus 0.1% sodium dodecyl sulfate at room temperature, followed by two 15-min washes in 0.5 × SSC plus 0.1 % sodium dodecyl sulfate at 37°C.

Determination of mycothiol, GlcN-Ins and GlcNAc-Ins content

The levels of mycothiol, GlcN-Ins and GlcNAc-Ins were determined as described previously (Buchmeier et al., 2003). Samples were prepared from bacteria that had been grown in Middlebrook 7H9 plus ADS medium to a density corresponding to A600 nm between 0.3 and 0.5.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Construction of M. tuberculosis with two copies of mshA

Because of the essential nature of mycothiol for growth of M. tuberculosis (Sareen et al., 2003), there was a high probability that a functional MshA protein would be essential for growth of M. tuberculosis as M. smegmatis mutants in mshA produced undetectable levels of mycothiol (Newton et al., 1999, 2003). For this reason, we constructed an M. tuberculosis strain containing two functioning copies of the mshA gene to be used in parallel with wild-type M. tuberculosis during the mutagenesis procedures. The mshA ORF plus ribosome-binding site was amplified by PCR and directionally cloned into the integrative vector pCV125. In this construct, the mshA gene is integrated as a single copy into the chromosome and is constitutively expressed behind the aph promoter. Initially, pCV125::mshA was electroporated into M. smegmatis in order to establish that functional MshA activity was being expressed from the construct. Wild-type M. smegmatis containing pCV125::mshA produced 120% of wild-type levels of mycothiol, 29±1 nmol (109 cells)−1. The M. smegmatis mshA mutant, mshA::Tn5, generates less than 0.1% of the wild-type level of mycothiol but following incorporation of pCV125::mshA it was found to produce 79±4% (n=3) of the wild-type level. This confirmed that when integrated into the mycobacterial chromosome, pCV125::mshA expressed sufficient levels of MshA to produce near-normal levels of mycothiol.

To produce an M. tuberculosis strain containing two copies of mshA, pCV125::mshA DNA was electroporated into M. tuberculosis Erdman and streptomycin-resistant colonies were selected on Middlebrook 7H11 plates containing OADC and streptomycin (30 μg mL−1). A 2X-mshA strain of M. tuberculosis with two copies of the mshA gene was identified by Southern hybridization. The 2X-mshA strain generated 170% of the M. tuberculosis Erdman wild-type level of mycothiol. This is a greater enhancement in activity compared with the 2X-mshA M. smegmatis strain (120%) and may reflect a difference in MshA activity when the M. tuberculosis gene is expressed in a completely homologous system.

Inability to disrupt the mshA gene in M. tuberculosis containing a single copy of mshA

Disruption of the mshA gene within M. tuberculosis was carried out by allelic exchange using a mutagenic construct that deleted both of the highly conserved motifs of the glycosyltransferase family (Newton et al., 2003). This construct produced a deletion of amino acids 32 through 394 of the 480 amino acid MshA protein. Mycobacterium tuberculosis Erdman and the 2X-mshA strain of M. tuberculosis were infected as described previously (Sareen et al., 2003) with phAE87 containing the mshA mutagenic construct described above. Three independent infections were carried out and transductants were selected on Middlebrook 7H9 plates containing OADC plus hygromycin (50 μg mL−1) or for the 2X-mshA strain also containing streptomycin (30 μg mL−1). Transduction levels were lower than those observed for the mshC gene and less than 50 hygromycin-resistant clones were successfully grown out. These were screened for homologous recombination within the mshA gene using Southern hybridization (Sareen et al., 2003). Chromosomal DNA was digested with NotI and probed with a fragment that would detect a 5.4-kb fragment in DNA from wild-type M. tuberculosis. The predicted deletion produced in an mshA mutant would be missing the internal NotI site and would therefore produce a fragment that would be greater than 17 kb in size (Fig. 2a).

image

Figure 2.  Targeted gene disruption of mshA in Mycobacterium tuberculosis: (a) expected hybridization bands for wild type (wt) and mutated mshA after digestion with Not1; (b) Southern analysis of chromosomal DNA from wt, 2X-mshA and 2X-mshA mutant strains after digestion with Not1; (c) Southern analysis of chromosomal DNA from wt and wt mutant strains after digestion with Not1.

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Among the 34 hygromycin clones produced from the 2X-mshA strain, seven were missing the 5.4 kb fragment, indicating that the wild-type copy of mshA was disrupted. Examples of the wild-type mshA gene disrupted in the 2X-mshA strain are shown using clones 277, 275 and 171 in Fig. 2b. We also found 10 clones with a disruption in the second copy of mshA (e.g. clones 177, 74), and 17 clones with evidence of nonhomologous recombination (e.g. clone 267) or with no changes in their mshA hybridizing DNA (spontaneous hygromycin resistance). All the clones from the 2X-mshA strains that exhibited a disruption in their second copy of mshA acquired an additional mshA hybridizing fragment (large band at top of lane, Fig. 2b). The presence of three mshA fragments indicates that only a single crossover event had occurred at the recombination site. This resulted in a disrupted copy and a normal copy of mshA at the crossover site. The failure to generate a second crossover event in the second copy is likely due to the limited amount of homology (96 bp) between one side of the mutagenic construct and the second copy of mshA whose flanking DNA comes from pCV125.

All 31 of the hygromycin-resistant clones produced from M. tuberculosis carrying a single copy of the mshA gene contained the 5.4 kb mshA hybridizing fragment, indicating that their mshA gene was intact (Fig. 2c). In contrast, seven of the 34 transformants in the 2X-mshA strains had a disruption in their wild-type copy of mshA. A conservative estimate of the probability of finding no mshA mutants among the 31 clones resulting from transduction of the wild-type strain is (27/34)31, or 1/1000. A more realistic estimate, eliminating the second copy contributions, is (17/24)31, or 1 in 2 × 105. In either case, we are forced to conclude that the mshA gene is essential for growth.

Confirmation that transductants produce mycothiol and GlcN-Ins

We next established that the transformants that grew out from the mshA mutagenesis produced functional MshA activity. The concentration of mycothiol and its precursor GlcN-Ins were determined on a sample of the clones generated from either the wild-type or the 2X-mshA parent. Four hygromycin-resistant clones derived from the wild-type parent produced normal levels of mycothiol and GlcN-Ins, confirming that they were not mshA mutants (Table 1). Determination of GlcNAc-Ins from the difference in GlcN-Ins content before and after deacetylation of samples by MshB proved unreliable because the difference in values was comparable to the uncertainties in the GlcN-Ins determinations. The concentration of GlcN-Ins was substantially elevated in the 2X-mshA parent (∼8-fold) compared with mycothiol levels (∼1.7-fold), suggesting a rate-limiting step at MshC/MshD. The ∼8-fold increase in GlcN-Ins in the 2X-mshA strain also suggests that expression of mshA from the aph promoter within pCV125, rather than from its native promoter, results in substantially elevated MshA activity. All clones derived from the 2X-mshA parent continued to produce elevated levels of mycothiol and GlcN-Ins similar to their parent. This included both clones with a disruption in their wild-type copy of mshA (clones 171, 275, 277) and clones identified by Southern blot with a crossover in their second copy of mshA (clones 74, 186). Clones with a disruption in wild-type mshA exhibited no change in MshA activity because they retained the high levels of MshA activity expressed from the second copy using the nonnative promoter. Clones with a crossover in the second copy of mshA retain their high MshA activity because, as evidenced by the Southern blot, only a single crossover had occurred in the second copy of mshA and thus the gene remained active.

Table 1.   Concentration of mycothiol and GlcN-Ins in Mycobacterium tuberculosis
StrainNanomoles per 109 cells
MSHGlcN-Ins
Wild-type25.4 ± 0.71.8 ± 0.2
Clone 2924.2 ± 0.91.6 ± 0.2
Clone 13229.2 ± 3.11.7 ± 0.2
Clone 14423.8 ± 1.81.3 ± 0.08
Clone 24623.9 ± 0.91.4 ± 0.1
2X-mshA43.6 ± 1.215.2 ± 1.2
Clone 171 (Δwt)46.8 ± 2.712.1 ± 3.1
Clone 275 (Δwt)43.6 ± 2.214.7 ± 2.3
Clone 277 (Δwt)43.3 ± 2.613.7 ± 1.1
Clone 74 (Δ2nd)55.2 ± 1.611.5 ± 0.5
Clone 186 (Δ2nd)48.0 ± 5.013.3 ± 1.2

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The finding that the mshA gene is required for growth of M. tuberculosis confirms the earlier conclusion based upon targeted disruption of the mshC gene that mycothiol is essential in M. tuberculosis (Sareen et al., 2003). The pathway for mycothiol biosynthesis involves a number of intermediates (Fig. 1) and it is possible that one or more of these play an essential, but as yet unidentified, role in addition to the production of mycothiol. This possibility cannot be ruled out based upon the results with M. tuberculosis. However, if such an additional function occurs, it is not essential for growth of M. smegmatis or Streptomyces coelicolor, as inactivation of the mshA gene in these actinomycetes does not prevent growth (Newton et al., 2003; Park et al., 2006). It therefore seems highly unlikely that the intermediates in mycothiol biosynthesis serve essential functions in M. tuberculosis other than the biosynthesis of mycothiol.

This study completes our survey of the essentiality in M. tuberculosis of the genes that have been identified for mycothiol biosynthesis. One gene, mshA2, remains to be identified. The mshA gene is required for production of mycothiol in M. smegmatis (Newton et al., 2003) and is shown here to be essential for growth of M. tuberculosis. Inactivation of mshB in M. tuberculosis (Buchmeier et al., 2003) and in M. smegmatis (Rawat et al., 2003) reduces, but does not eliminate, mycothiol production and both mshB mutants exhibit normal growth. Disruption of the mshC gene in M. smegmatis blocks mycothiol production (Rawat et al., 2002) and is lethal in M. tuberculosis (Sareen et al., 2003). An M. smegmatis mutant in mshD produces a low level of mycothiol but higher levels of Cys-GlcN-Ins and N-formyl-Cys-GlcN-Ins (Newton et al., 2005). An M. tuberculosis mshD mutant produces a similar distribution of thiols but has a more disrupted redox status (Buchmeier et al., 2006) and disruption of the mshD gene severely limits survival of M. tuberculosis in the macrophage (Rengarajan et al., 2005). The results from targeted disruption of the genes for mycothiol biosynthesis are in accord with those from high-density mutagenesis studies (Sassetti et al., 2003), except for mshA where, as stated previously, the latter method did not provide a clear prediction.

The most promising targets for compounds that could block mycothiol biosynthesis are clearly MshA and MshC. MshD also shows some promise, although the N-formyl-Cys-GlcN-Ins produced by mshD mutants can substitute for mycothiol to some extent. MshB appears to be the least attractive target because of an alternative route for mycothiol production that may involve a compensating GlcNAc-Ins deacetylase activity (Steffek et al., 2003). The identification of inhibitors for MshC has been initiated (Newton et al., 2006) and recent studies identifying inhibitors of MurG (Hu et al., 2004), another UDP-GlcNAc-dependent glycosyltransferase, suggest that MshA is also likely to be a drugable target.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research was funded by National Institutes of Health Grant AI49174. We thank Gerald L. Newton and Philong Ta for conducting the HPLC analyses of labeled cell extracts, Christopher M. Sassetti and Eric J. Rubin for helpful discussions and Gerald L. Newton for critically reading the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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