1 Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, British Columbia, Canada V5Z 3J5
2 Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia
3 Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia
Correspondence
Yossef Av-Gay
yossi{at}interchange.ubc.ca
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ABSTRACT |
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INTRODUCTION |
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Mycothiol biosynthesis is proposed to proceed through a four-step pathway (Bornemann et al., 1997; Anderberg et al., 1998
; Newton & Fahey, 2002
). First the formation GlcNAc-Ins is catalysed by N-acetylglucosamine transferase (MshA), followed by deacetylation by MshB deacetylase. The resulting GlcN-Ins is ligated with a cysteine in a reaction catalysed by a ligase, MshC (Sareen et al., 2002
). The Cys-GlcN-Ins is then acetylated to form mycothiol in a reaction catalysed by MshD acetyltransferase (Koledin et al., 2002
). Recently, the enzymes MshB from M. tuberculosis (mtMshB), MshC from M. smegmatis (msMshC) and MshD from M. smegmatis (msMshD) have been characterized and M. smegmatis mutants in mshA (Newton et. al., 2003
), mshC (Rawat et al., 2002
) and mshD (Koledin et al., 2002
) have been isolated. The mshB gene was identified based upon its homology to the mycothiol-dependent amidase MCA, which is encoded by the M. tuberculosis ORF Rv1082 (Cole et al., 1998
). M. tuberculosis MshB was cloned and expressed in Escherichia coli and the recombinant mtMshB was shown to possess GlcNAc-Ins deacetylase activity in vitro (Newton et al., 2000b
). Although mtMshB was the first enzyme to be identified and characterized we were not able to identify mshB-defective mutants by screening transposon and chemical mutant libraries of M. smegmatis. To test whether it is possible to generate such mutants, and to determine the role of MshB within living mycobacterial cells, we constructed an mshB-deficient strain of M. smegmatis. In this study we describe the creation and characterization of the mshB mutant.
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METHODS |
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Molecular biology techniques.
Strains, plasmids and oligonucleotides used in this study are described in Table 1. Genomic DNA was isolated from M. smegmatis cultures according to Larsen (2000)
and as previously described (Billman-Jacobe et al., 1999
). M. smegmatis transformations were carried out with a Bio-Rad Gene Pulser using mycobacterial cells prepared as described by Snapper et al. (1988)
. Standard recombinant DNA techniques such as restriction digests, ligations and transformations were carried out as described by Sambrook et al. (1989)
. Probes for Southern blotting were labelled with digoxigenin (DIG)-labelled dNTPs using a Roche DIG labelling kit, and membranes developed according to the manufacturer's instructions.
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Toxicity and antibiotic sensitivity studies.
The minimal inhibitory concentrations (MICs) of ethionamide and isoniazid (Sigma) were determined for the M. smegmatis wild-type (WT) and derivatives. Exponentially growing cultures were serially diluted and applied to Middlebrook 7H10 plates containing various concentrations of antibiotics using a replicator. An inoculum of 5001000 c.f.u. per spot was used and the MICs were read as the lowest concentration that inhibited at least 98·8 % of growth. All MICs were tested in duplicate at least twice.
Disk diffusion assays, as described by Rawat et al. (2002), were also used to determine the sensitivity of M. smegmatis wild-type and mutants to ethionamide, cerulenin and vancomycin and to various oxidative and alkylating stress generators: diamide (1·010·0 µmol), cumene hydroperoxide (0·15·0 µmol), menadione (0·011·0 µmol), nitrofurantoin (0·110·0 µmol), plumbagin (0·011 µmol), DTNB (0·011·0 µmol), CDNB (0·011·0 µmol), mBBr (0·55·0 µmol) and iodoacetamide (0·010·1 µmol). The disk diffusion assays were performed in triplicate at least three times.
Determination of mycothiol levels.
Derivatization of cell extracts with mBBr to determine the thiol content was performed essentially as described by Anderberg et al. (1998). Briefly, cell pellets were suspended in 50 % aqueous acetonitrile, 2 mM mBBr, 20 mM HEPES (pH 8·0). The suspensions were then incubated in the dark at 60 °C for 15 min and after the incubation period, 2 µl concentrated 12 M HCl was added to acidify the suspensions. The suspensions were then centrifuged to collect the cell debris and the supernatant was diluted with 10 mM HCl and subjected to HPLC analysis. Control samples were extracted with 50 % aqueous acetonitrile, 5 mM N-ethylmaleimide and 20 mM HEPES (pH 8·0). The suspensions were incubated for 15 min at 60 °C. After addition of 2 mM mBBr, the suspensions were incubated again for 15 min at 60 °C. The suspensions were then centrifuged to collect the cell debris and the supernatant was diluted with 10 mM HCl and subjected to HPLC analysis on a Beckman Ultrasphere C18 ion-pair HPLC column. The thiols were eluted with 0·25 % glacial acetic acid pH 3·6 (buffer A) and 100 % methanol (buffer B) using the following gradient: initial conditions, 10 % buffer B; at 15 min, 18 % buffer B; at 30 min, 27 % buffer B; at 33 min, 100 % buffer B; at 36 min, 10 % buffer B; and at 52 min, 10 % buffer B and reinjection. The flow rate was 1 ml min-1 and fluorescence detection was as described previously (Anderberg et al., 1998
).
Complementation of Myco504 with M. smegmatis mshB homologue.
The M. smegmatis mshB ORF was PCR amplified from M. smegmatis mc2155 genomic DNA using primers 1 and 2 (Table 1). The PCR product included 300 bp upstream of the ORF and was presumed to contain the mshB promoter. Sites were included in the primers to facilitate cloning into pGINT, an integrative M. smegmatisE. coli shuttle vector derived from the pHINT vector (O'Gaora et al., 1997
). The M. smegmatis mshB gene was PCR amplified and the PCR product was cloned into pCR2.1. The mshB gene was then excised using HindIII and XhoI and ligated with HindIII- and XhoI-digested pGINT to create pM3. Myco504, the MshB- mutant, was transformed with pM3 and kanamycin/gentamicin-resistant transformants were selected. The mycothiol content of the transformants was measured and one transformant, named Myco504mshB, was selected for further study. We cannot eliminate the possiblity that some transcriptional readthrough from a vector-encoded promoter could occur and regarded the expression as uncontrolled expression.
Expression of M. tuberculosis mca in Myco504.
The M. tuberculosis mca gene was PCR amplified using primers 3 and 4 (Table 1) and the PCR product was cloned into pCR2.1 vector. The cloned mca gene was then excised by BamHI and NdeI digestion and ligated with pALACE (De Smet et al., 1999
) previously digested with BamHI and NdeI to create the plasmid pM4. Myco504 and wild-type M. smegmatis were transformed with pM4 and hygromycin/kanamycin-resistant transformants were selected. The transformants were screened by SDS-PAGE analysis and one transformant from each transformation was selected for further study; the transformants were named Myco504mca and WTmca.
To confirm that the expressed protein was the mca gene product, mycothiol amidase, MCA was purified from the wild-type mca strain which had been induced for 48 h with 1 % acetamide. The cells were harvested and the cell pellet was sonicated and purified using a TALON affinity resin (Clonetech) according to the manufacturer's instructions. The amidase activity of the purified protein was determined as described by Newton et al. (2000a).
Determination of mycothiol content during the growth cycle of mc2155, Myco504 and Myco504mshB.
Stock bacterial cultures of mc2155, Myco504, Myco504mshB grown in TSB medium were diluted to OD600 0·05 with TSB medium supplemented with appropriate antibiotics. The cultures were diluted in 1 l Erlenmeyer flasks and incubated at 37 °C. The OD600 was measured at various time intervals with a Beckman DU 640 spectrophotometer using a cuvette with a pathlength of 1 cm. Samples of the cultures were harvested at various time points. Cells were collected by centrifugation and frozen for later mycothiol analysis. This experiment was repeated three times.
Determination of mycothiol content of Myco504 mca.
Stock bacterial cultures of mc2155, Myco504 and Myco504mca grown in Middlebrook medium supplemented with OADC were diluted to OD600 0·5 and then washed twice with Middlebrook medium without OADC to remove the OADC. The cultures were divided into two flasks, one containing Middlebrook 7H9 medium with 1 % glucose and one containing Middlebrook 7H9 medium with 1 % acetamide for the induction of the acetamidase promoter in the pALACE vector. Samples were taken at 0, 24 and 48 h from both flasks and the mycothiol content of the cells was determined as above.
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RESULTS |
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Myco504 contains low levels of mycothiol
Myco504 was analysed for mycothiol content to determine whether disruption of mshB affected mycothiol synthesis. In the exponential phase of growth, Myco504 contained 510 % of the wild-type levels of mycothiol: 1·00±0·21 µmol (g dry weight)-1 as compared with 11·07±0·58 µmol (g dry weight)-1 for M. smegmatis wild-type mc2155. As seen in Fig. 2, the amount of mycothiol in the parent strain was dependent on the phase of growth of the cultures and ranged from 1·00 to 3·7 nmol per OD600 unit ml-1, whereas Myco504 consistently had low levels of mycothiol (0·15 to 0·249 nmol per OD600 unit ml-1). The parent strain and Myco504 grew at similar rates in TSB and Middlebrook 7H9 media, indicating that disruption of mshB, which causes a decrease in mycothiol levels, still permits normal growth under these conditions (Fig. 3
).
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Expression of M. tuberculosis mca in Myco504 does not increase mycothiol levels
MshB was shown to have weak mycothiol amidase (MCA) activity (Newton et al., 2000b). However, whether MCA has mycothiol deacetylase activity has not been conclusively verified in vivo. MCA and MshB have similar amino acid sequences (36 % identity in 299 aa overlap) and thus we hypothesized that MCA may have some deacetylase activity that is responsible for the basal level of mycothiol present in the MshB- mutant. To examine this hypothesis, we cloned the M. tuberculosis mca gene in the M. smegmatisE. coli shuttle vector pALACE and named it pM4. In this vector, the cloned gene was under the control of an inducible acetamidase promoter and also had the added advantage of a hexahistidine tag allowing easy purification of the expressed protein. Both Myco504 and the wild-type parent M. smegmatis were transformed with pM4 and the transformants screened by SDS-PAGE analysis after induction with acetamide to overexpress mca in the MshB- mutant as well as the parental strain. SDS-PAGE clearly showed that mca is overexpressed in Myco504mca, the mutant strain with the overexpressed mca, and in WTmca, the parent strain with the overexpressed mca (Fig. 4
). To confirm that the induced protein was enzymically active, recombinant MCA was purified from the induced WTmca cell extract using affinity chromatography as above (Fig. 4
) and checked for amidase activity as described by Newton et al. (2000a)
. The purified MCA had activity levels comparable to the purified native M. tuberculosis protein (G. Newton, personal communication), indicating that the recombinant MCA is indeed active. Mycothiol levels were then determined for induced and uninduced Myco504mca. We found no difference in the levels of mycothiol in Myco504 and Myco504mca, indicating that under the growth conditions we have tested, MCA is not capable of increasing mycothiol levels in the mutant (data not shown).
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DISCUSSION |
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That a mutant in mycothiol biosynthesis still contains observable levels of mycothiol is not unprecedented. Previously, we reported a chemical missense mutant in mshC that retained 15 % of the mycothiol of the parental strain (Rawat et al., 2002). Nevertheless, the levels of mycothiol in the MshB- mutant are the highest reported so far for a mutant missing a gene in mycothiol biosynthesis. Indeed, the other mutants in mshA and mshD have virtually no mycothiol (mshA: Newton et al., 1999
, 2003
; mshD: Koledin et al., 2002
).
Earlier we reported that MshB can act in vitro as both a deacetylatse and an amidase like MCA (Newton et al., 2000b). However, the deacetylation activity of MCA has not been checked in vivo and it could be that it is MCA that is responsible for the basal levels of mycothiol in the mshB- mutant. Overexpression of mca in the MshB- mutant did not result in an increase in mycothiol levels. It is entirely possible that MCA is not responsible for the mycothiol present in the MshB- mutant. Alternatively, it is also possible that even a small amount of mycothiol as in the MshB- mutant may downregulate or block MCA activity. In that case, overexpressing MCA may not have any observable effect on the mycothiol content of the mutant. A double mutant, MshB- Mca-, may reveal whether it is indeed the MCA that is responsible for the 510 % mycothiol that is present in Myco504.
Recently, Vetting et al. (2002) reported the crystal structure of an M. tuberculosis aminoglycoside 2'-N-acetyltransferase, AAC(2'), that catalyses the coenzyme A (CoA)-dependent acetylation of the 2'-hydroxyl or amino group of a wide range of aminoglycosides. The authors hypothesize that this enzyme may acetylate GlcN-Ins to form GlcNAc-Ins in a reverse reaction to MshB deacetylase. It is unlikely that this enzyme catalyses the reverse reaction for two main reasons: (i) if GlcN-Ins acetylation activity was present in M. smegmatis we would not been able to isolate the mshC mutants with high levels of GlcN-Ins as described in our previous publication (Rawat et al., 2002
); and (ii) AAC(2') lacks any metal ion, which is required for hydrolytic activity of MshB.
In our recent publication we reported that mutants lacking mycothiol and MshC ligase activity are more sensitive to antibiotic, oxidative and alkylating stress (Rawat et. al., 2002). Interestingly, the mshB mutant, Myco504, did not display increased sensitivity to oxidative stresses and to most alkylating agents with the exception of mBBr and CDNB. Most surprising was the finding that the mshB mutant is not resistant to the prodrug isoniazid. M. smegmatis mc2155 and the MshB- mutant derived from it are equally sensitive to isoniazid (MIC 112·5 µg) whereas MshA- mutants and the MshD- mutant are extremely resistant to isoniazid (MIC
256 µg) (Newton et al., 1999
, 2003
; Koledin et al., 2002
). Even mutants with mycothiol levels that are 15 % of the wild-type amount, such as the MshC- mutant I64, are isoniazid resistant, having a MIC of 32 µg that is significantly higher than the MIC of the wild-type M. smegmatis. Mycothiol is normally present in millimolar levels in the wild-type cell; however, the amount of mycothiol present in Myco504 appears to be sufficient to maintain isoniazid sensitivity and protect the cells against oxidative stress, some alkylating stress, and antibiotic stress under otherwise normal growth conditions.
Possibly the most exciting finding from this work is the drug resistance of the mutants. Ethionamide and isoniazid are both specific antimycobacterial drugs that share at least one site of action in mycolic acid biosynthesis. Isoniazid is a prodrug which is oxidized by the bacterial catalase-peroxidase, KatG, to form a reactive toxic species. Mutations in the KatG gene that confer isoniazid resistance do not result in ethionamide resistance. Despite the common site of action, ethionamide and isoniazid are activated by different enzymes. The first step of activation of ethionamide is an NADPH- and O2-dependent reaction that yields the corresponding S-oxide metabolite which requires further activation to a final cytotoxic species (Vannelli et al., 2002; De Barber et al., 2000
). We have compared the ethionamide sensitivity of M. smegmatis mc2155 and MshA-, MshB- and MshC- mutants derived from it. The MshA- and MshB- mutants were resistant to ethionamide. Complementation of the MshB- mutant restored wild-type sensitivity to ethionamide; however, after mshA complementation of the MshA- mutants the strains were still resistant to low levels of ethionamide (Table 4
). These results are in contrast to the MshC- mutant, I64, which was as sensitive to ethionamide as wild-type M. smegmatis. Because resistance to ethionamide is not a characteristic of all mutants that lack mycothiol, it is most likely that an intermediate in the mycothiol biosynthetic pathway but not mycothiol itself is involved in ethionamide resistance. This effect could be indirect, occurring through the regulation or activation of enzymes that partcipate in ethionamide activation.
Baulard et al. (2000) have demonstrated that expression of EthA, the flavin monooxygenase which activates ethionamide in mycobacteria, is controlled by a regulator, EthR, which itself may be regulated. When EthR is expressed at high levels, EthA is repressed, resulting in ethionamide resistance. The EthA/EthR system does not modulate resistance to isoniazid. The mechanism of EthR regulation is unknown; however, it is possible that EthA is repressed in the MshA- and MshB- mutants and not in the MshC- mutant. Futher investigation to test this hypothesis is under way.
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ACKNOWLEDGEMENTS |
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Received 31 October 2002;
revised 10 February 2003;
accepted 13 February 2003.