Department of Infectious Diseases and Microbiology, Imperial College School of Medicine, St Marys Campus, Norfolk Place, London W2 1PG, UK1
Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage SG1 2NY, UK2
Author for correspondence: Douglas B. Young. Tel: +44 171 594 3956. Fax: +44 171 262 6299. e-mail: d.young{at}ic.ac.uk
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ABSTRACT |
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Keywords: acetamidase, fosfomycin, MurA, mycobacteria, tuberculosis
The EMBL accession number for the sequence in this paper is X96711.
a Present address: Research Division, Innogenetics NV, 9052 Ghent, Belgium.
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INTRODUCTION |
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Fosfomycin is a broad-spectrum antibiotic produced by some strains of Streptomyces (Christensen et al., 1969 ; Hendlin et al., 1969
). Its mechanism of action was first described by Kahan et al. (1974)
, who showed that it inhibits the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which transfers the enolpyruvyl moiety of phosphoenolpyruvate to the 3' hydroxyl group of UDP-N-acetylglucosamine, the first committed step in bacterial peptidoglycan biosynthesis. Investigation of the detailed mechanism of Escherichia coli MurA revealed that fosfomycin irreversibly inactivates MurA by forming a covalent adduct with a critical cysteine residue in the active site of the enzyme (Kahan et al., 1974
; Skarzynski et al., 1996
).
Resistance to fosfomycin is commonly by decreased uptake of the drug, although drug inactivation by a plasmid-encoded glutathione transferase has also been reported (Arca et al., 1997 ). Fosfomycin has no activity against M. tuberculosis, and Kim et al. (1996)
noted that, in the sequence of the M. tuberculosis murA homologue, the key active site cysteine (amino acid 117 in the M. tuberculosis sequence) is replaced by an aspartic acid residue. They went on to show that replacement of cysteine by aspartate in E. coli MurA resulted in retention of enzyme activity, but conferred resistance to fosfomycin. They postulated that the presence of aspartate in place of cysteine in the MurA enzyme might account for the innate fosfomycin resistance of M. tuberculosis.
The aim of the present study was firstly to determine whether or not M. tuberculosis MurA is inhibited by fosfomycin, and secondly to assess the effect of substitution of cysteine for aspartate-117 on fosfomycin susceptibility of M. tuberculosis MurA. In addressing these questions, we encountered difficulties in expression of recombinant M. tuberculosis MurA in a functionally active form. It formed insoluble products in a range of E. coli expression systems and, although it was soluble in mycobacterial systems, expression was rapidly lost during subculture. We report on the use of an inducible expression system in mycobacteria to overcome problems associated with stable expression of M. tuberculosis MurA, and demonstrate that, as in E. coli MurA, residue 117 is a critical determinant of fosfomycin susceptibility.
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METHODS |
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To analyse mutations occurring during subculture, plasmids were isolated from M. smegmatis cells incubated for 1 h with 10 mg lysozyme ml-1 in Tris/EDTA buffer followed by plasmid purification using a commercial kit (Qiagen).
A mutant form of M. tuberculosis murA with cysteine in place of aspartate in position 117 (D117C) was prepared by using the complementary primers MUR-3 and MUR-4, covering codon 117. Two independent amplifications using primer sets MUR-1 + MUR-4 and MUR-2 + MUR-3 produced two fragments of the murA gene. After removal of remaining primers, the two PCR products were pooled and the full-length D117C murA gene was amplified by PCR using the outer primers. The PCR product was cloned in pCR-Script and then subcloned in pACE. Sequence determination using fluorescent dideoxynucleotides on an ABI 310 analyser confirmed that the construct encoded murA with the desired single change at position 117.
Mycobacterial expression vectors.
MycobacteriaE. coli shuttle vectors were constructed by modification of the previously described hygromycin-resistance vector p16R1 (Garbe et al., 1994 ). pOLYG, a p16R1 derivative with a multiple cloning site, and pSMT3, an expression plasmid containing the promoter region from M. tuberculosis hsp60, have been described previously (OGaora et al., 1997
). To generate p19Kpro, a 195 bp region upstream of the 19 kDa antigen gene (Ashbridge et al., 1989
) was amplified from M. tuberculosis H37Rv chromosomal DNA by PCR using Deep Vent Polymerase (New England Biolabs) with oligonucleotide primers 19-1 and 19-2 (Table 1
). The PCR product was digested with XbaI and BamHI, and cloned in pOLYG digested with the same enzymes. Similarly, pSODIT-2 was constructed by PCR amplification of a 193 bp fragment from M. tuberculosis sodA (Zhang et al., 1991
) using primers SOD-1 and SOD-2, followed by cloning in pOLYG. pACE was constructed by PCR amplification of a 3 kb region upstream of the acetamidase gene of M. smegmatis (Mahenthiralingam et al., 1993
; Parish et al., 1997
) using primers ACE-1 and ACE-2.
Expression of MurA in M. smegmatis.
Single hygromycin-resistant colonies obtained after electroporation of M. smegmatis were inoculated into 5 ml LuriaBertani (LB) medium, or minimal medium containing 0·2% (w/v) succinate as carbon source, and cultured for 13 d with shaking at 37 °C. Minimal medium was prepared by dissolving the following in 1 l distilled water: 4 g NaCl, 0·2 g MgSO4, 2 g KH2PO4, 2 g (NH4)2HPO4, 2 g Na-succinate, 0·05% Tween 80, pH 7·2. After autoclaving, 2 ml filter-sterilized trace element solution was added, containing (l-1) 40 mg ZnCl2, 200 mg (NH4)Fe(SO4)2, 10 mg CuSO4, 10 mg MnCl2, 10 mg borax and 10 mg ammonium molybdate. Samples were then checked for expression (see below) or mixed 1:1 with 50% glycerol and frozen at -80 °C. To analyse the effect of subculture, 10 ml LB medium (pSOD-murA) or minimal medium (pACE-murA) was inoculated from frozen stock and incubated with shaking at 37 °C till the cultures reached stationary phase (23 d). Ten microlitres was used to inoculate 10 ml fresh medium, and incubation and subculture steps were repeated. To analyse MurA expression in the inducible vector system, pACE constructs were grown to stationary phase in minimal medium, diluted 1/100 in fresh medium and grown overnight. Acetamide was added to a final concentration of 0·2% (w/v), and cultures were grown for a further 24 h with shaking at 37 °C.
Production of antiserum and Western blotting.
M. tuberculosis MurA was expressed in E. coli using the pQE31 vector (Qiagen). Cultures were induced by IPTG according to the manufacturers recommended procedures, generating insoluble protein which was denatured in the presence of urea and purified by nickel affinity chromatography (Qiagen). Subsequent removal of the urea by dialysis resulted in precipitation of the recombinant protein, which was emulsified with Freunds incomplete adjuvant and used to immunize BALB/c mice. Mice received two intraperitoneal injections of 50 µg protein 2 weeks apart, with subsequent boosting with protein without adjuvant.
To prepare M. smegmatis samples for analysis of MurA expression, bacteria from 10 ml cultures were harvested by centrifugation, suspended in PBS and disrupted by sonication [Soniprep 150 (MSE) for 10 s at an amplitude of 8 microns]. Cell debris was removed by centrifugation for 5 min in a microfuge and protein levels in soluble extracts were determined using the Coomassie Plus Protein Assay (Pierce). Soluble extracts (5 µg protein per lane) were fractionated by SDS-PAGE and transferred to nitrocellulose membranes for Western blotting. Blots were developed using the monovalent antiserum raised against M. tuberculosis MurA at a dilution of 1:1000. Antibody binding was visualized using peroxidase-conjugated anti-mouse antibody, with subsequent incubation with SuperSignal (Pierce) and autoradiography.
Assay of MurA activity.
To assay MurA activity, pACE constructs in M. smegmatis were grown with or without acetamide induction as described above. After the final incubation, cells were harvested by centrifugation, washed in 50 mM Tris/Cl (pH 7·5) with 2 mM DTT, and lysed by sonication as above. Cell debris was removed by centrifugation for 5 min in a microfuge, and the resulting supernatant was subjected to gel filtration using a PD10 column (Pharmacia) to remove small molecules that may interfere with enzyme assays.
The assay mixture (total volume 50 µl) contained buffer (50 mM Tris/Cl, pH 7·5, 2 mM DTT), UDP-N-acetylglucosamine (10 mM) and bacterial extract (515 µg protein). After 15 min at 37 °C, the reaction was started by addition of 5 µl phosphoenolpyruvate (to a final concentration of 1 mM), and was stopped after 1 h by addition of 200 µl colour reagent [1% ammonium molybdate, 12·5% HCl, 0·15% malachite green (Itaya & Ui, 1966 )]. Results are expressed as the A630, corrected for the background reading observed in the absence of UDP-N-acetylglucosamine. Where appropriate, fosfomycin (Sigma) was added during the initial incubation period at concentrations ranging from 30 µM to 10 mM. Appropriate dilutions were added to the reactions instead of water to reach the required concentration without altering the final volume.
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RESULTS |
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Expression of M. tuberculosis MurA protein from clone pMurA2T6. All attempts to express native soluble MurA in E. coli transformed with pMurA2T6 were unsuccessful. A range of growth conditions, rich and minimal media, and IPTG induction conditions were tested. In each case, although expression of MurA protein was visible on SDS-PAGE, the protein was in the insoluble fraction, indicating that it had formed inclusion bodies (data not shown). A small amount of protein was detectable in the soluble fraction by Western blotting, but we were unable to isolate the native enzyme from this starting material.
Cloning and expression of M. tuberculosis murA in M. smegmatis
Mycobacterial expression vectors. To test for expression in the rapid growing mycobacterial host strain M. smegmatis, the M. tuberculosis murA gene was cloned under the control of different promoters in a set of three expression vectors (Fig. 1). All three vectors were derived from p16R1 (Garbe et al., 1994
), which encodes resistance to hygromycin, and has replication origins suitable for maintenance in E. coli and in mycobacteria. In addition, pSMT3 contains a 389 bp fragment corresponding to the promoter and first six amino acids of M. tuberculosis groEL (hsp60) (OGaora et al., 1997
). p19Kpro contains a 195 bp fragment from the promoter region of the M. tuberculosis 19 kDa antigen (Ashbridge et al., 1989
), and pSODIT-2 has a 193 bp promoter region from the M. tuberculosis superoxide dismutase (sodA) gene (Zhang et al., 1991
), engineered to include a start codon and two additional amino acids (i.e. Met-Gly-Ser) fused to the N-terminus of expressed proteins. Clones containing the M. tuberculosis murA gene in the three different expression vectors were named pSM-murA, p19-murA and pSOD-murA, respectively.
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DISCUSSION |
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Rapid loss of expression of genes cloned under control of constitutively active promoters in mycobacterial plasmid vectors has previously been reported by several authors (Boshoff & Mizrahi, 1998 ; Kumar et al., 1998
; Prammananan et al., 1999
). Possible approaches to overcome this problem include the use of recombination-deficient host strains (Prammananan et al., 1999
) and the use of regulated promoters. Inducible promoters have been widely used in development of recombinant expression systems in E. coli. In mycobacteria, emphasis on use of expression technology to construct recombinant BCG vaccines has stimulated a search for promoters that are induced during the process of infection in vivo, but less effort has been invested in development of inducible systems suitable for work with mycobacteria in vitro. The potential utility of such systems is illustrated in the present study using the acetamidase promoter; to our knowledge, the only system available for convenient in vitro induction of gene expression in mycobacteria (Mahenthiralingam et al., 1993
; Parish et al., 1997
; Triccas et al., 1998
). With the availability of the complete genome sequence of M. tuberculosis (Cole et al., 1998
) it is possible to identify a series of potential drug targets, and there is a need for simple expression systems to allow experimental testing of functional activities predicted on the basis of homology searches. It is likely that other key enzymes will resemble MurA in posing problems when they are constitutively expressed in a functionally active form; inducible expression systems provide an important solution to these problems.
Analysis of the functional activity of M. tuberculosis MurA fully supports the hypothesis put forward by Kim et al. (1996) , in that the wild-type enzyme is completely resistant to fosfomycin, and substitution of cysteine at position 117 confers susceptibility. The evolutionary pathway leading to a difference in sequence at position 117 between different bacteria is unknown. M. smegmatis murA resembles the M. tuberculosis gene in having aspartate at position 117 (M. J. Everett & K. Duncan, unpublished data), and it will be of interest to analyse the sequence in other bacterial genera. While results from the present study clearly demonstrate resistance of M. tuberculosis MurA to fosfomycin in cell-free assays, they do not exclude the possibility that other factors including permeability could contribute to drug resistance in whole bacilli. Application of gene replacement techniques (Bardarov et al., 1997
; Pelicic et al., 1997
) to exchange the mutant D117C gene for the wild-type murA in intact mycobacteria provides an experimental strategy to evaluate this possibility. If the alteration in MurA structure is confirmed as the key factor in innate resistance, fosfomycin derivatives designed to bind in a MurA active site containing an aspartate rather than a cysteine residue might represent a route to production of novel drugs active against bacteria, such as the mycobacteria, which express an aspartyl-MurA.
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ACKNOWLEDGEMENTS |
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Received 26 May 1999;
revised 29 July 1999;
accepted 2 August 1999.