University Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK1
Author for correspondence: Edith Sim. Tel: +44 1865 271850. Fax: +44 1865 271853. e-mail: esim{at}molbiol.ox.ac.uk
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ABSTRACT |
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Keywords: mycobacteria, knockout, immunogenic, antibacterial, tuberculosis
Abbreviations: NAT, arylamine N-acetyltransferase; INH, isoniazid
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INTRODUCTION |
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The distribution of NATs in prokaryotes has recently been described based on activity studies (Delomenie et al., 2001 ) and genome analysis (Payton et al., 2001
). In contrast to eukaryotes, where two or three isoenzymes are often found, only a single nat gene is present in prokaryotes. The substrate specificity of the prokaryotic NATs appears to differ from that of any eukaryotic isoenzyme. A human NAT1 specific substrate, 5-amino salicylate (Delomenie et al., 2001
), and a human NAT2 specific substrate, INH, are both acetylated by the same enzyme (Watanabe et al., 1992
; Sinclair et al., 1998
; Payton et al., 1999
).
Mycobacteria are known to infect many animal species (Balasubramanian et al., 1994 ; Thorel et al., 1997
; OReilly & Daborn, 1995
). Moreover, much interest is focused on this genus as it includes major human pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae, and also Mycobacterium avium, the major opportunistic pathogenic agent affecting HIV-infected people in the developed world (Schutt-Gerowitt, 1995
). Tuberculosis, caused by M. tuberculosis, has been declared a global emergency and is the most frequent infectious cause of mortality in the world. Incidence of this disease is now reaching epidemic proportions in developing countries, and in some regions of the USA and the UK (World Health Organization, 1999
). The emergence of many strains of M. tuberculosis resistant to the currently available chemotherapeutics, particularly INH, is an additional cause of alarm. INH has been used since 1952, but despite extensive research, the molecular basis underlying this resistance has not been fully deduced.
We have recently identified genes homologous to nat in M. smegmatis and M. tuberculosis, and have shown that heterologous expression of M. tuberculosis nat in M. smegmatis results in a concurrent increase in resistance to INH (Payton et al., 1999 ). It is possible that increased expression of the gene or certain mutations resulting in the increase of NAT activity in some strains of M. tuberculosis may contribute to their resistance to INH.
The endogenous function of both eukaryotic and prokaryotic NATs remains to be determined. One approach to investigating their role is to knock out the gene of an organism and to identify the subsequent effects on its phenotype. As a model for M. tuberculosis, the non-pathogenic Mycobacterium smegmatis has been much studied (Parish & Stoker, 1998a ). In this report, we extend these studies by knocking out the nat gene of M. smegmatis to identify whether this strategy affects the sensitivity of M. smegmatis to INH. The knockout was also created with a view to the elucidation of the endogenous function of the mycobacterial NATs.
The nat gene in M. tuberculosis and in Mycobacterium bovis has been predicted to be part of a gene cluster (Payton et al., 2001 ). Therefore we have also investigated whether the nat gene is transcribed as part of a larger message in M. smegmatis.
To extend these studies to M. tuberculosis, it is necessary to have a specific tool to identify NAT production in these bacteria. We have therefore raised an antiserum against recombinant NAT protein from M. tuberculosis, and have identified a major immunogenic peptide in the protein. The antiserum uniquely recognizes M. tuberculosis NAT.
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METHODS |
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Genetically modified M. smegmatis containing a chromosomal copy of the kanamycin-resistance transposon Tn903 (Oka et al., 1981 ) was generated by using an E. coliM. smegmatis kanr/hygr shuttle vector (pAGAN 40, a gift from Dr T. Parish).
M. smegmatis was grown in Middlebrook 7H9 medium enriched with 10% (v/v) albumin-dextrose-catalase (ADC; Difco) and 0·05% (v/v) Tween 80 (Sigma) until they reached the exponential phase (2535 h) at 37 °C. E. coli was grown in standard LB medium (Sambrook et al., 1989
) supplemented with the required antibiotics.
Where appropriate, M. smegmatis was cultured in medium supplemented with 20 µg kanamycin ml-1 and 50 µg hygromycin ml-1. For E. coli, 30 µg kanamycin ml-1 and 100 µg hygromycin ml-1 were used. Growth was determined by measuring turbidity at 600 nm.
Transformation of E. coli and M. smegmatis.
E. coli cells were transformed by electroporation according to the manufacturers recommendations (Easyject, Equibio). Transformation of M. smegmatis with the shuttle vector pAGAN 40 was carried out as previously described (Parish & Stoker, 1998b ). The knockout construct pGEMTnatKO is capable of replicating in E. coli but not in M. smegmatis, making the construct a candidate for integration via a suicide vector approach (Husson, 1998
). Plasmid DNA was prepared from transformed E. coli and UV treated at 100 mJ cm-2 to enhance homologous recombination (Hinds et al., 1999
). Competent cells of M. smegmatis were electroporated with 1 µg plasmid DNA. Transformants were selected on 7H11 agar (Difco) supplemented with 10% (v/v) oleic acid-ADC (OADC; Difco) and 20 µg kanamycin ml-1.
Purification of chromosomal DNA.
Chromosomal DNA from mycobacteria was extracted as described by Wilson et al. (1993) . DNA concentration was estimated following agarose gel electrophoresis and also from the A260 (Sambrook et al., 1989
).
Construction of the suicide vector.
A chromosomal clone containing the nat gene with flanking sequences at both ends has been isolated from M. smegmatis (accession no. AJ006588; Payton et al., 1999 ). The 2·5 kb insert was excised from this plasmid (pBS, Stratagene) with ClaI and XbaI. It has a single EcoRV site within the nat coding sequence and two more EcoRV sites at each end outside the coding sequence.
A 1·2 kb fragment containing the kanamycin resistance gene, Tn903, was amplified by PCR from pUC4K (Pharmacia). EcoRV sites were also introduced during the reaction. We used Pfu DNA polymerase and the primers 5'KanR/EcoRV 5'-AGGATATCCGTCGACCTGCAGG-3' and 3'KanR/EcoRV 5'-AGGATATCCCCGGATCCGTCGAC-3'. Cycles were 94 °C for 30 s, 56 °C for 30 s and 72 °C for 2·5 min, for 25 cycles.
The 1·2 kb fragment was digested with EcoRV then purified from the agarose gel, and ligated between the 1·6 and 0·6 kb EcoRV fragments from the 2·5 kb M. smegmatis chromosomal XbaI/ClaI fragment which make up the left and right homology arms. The final product (3·5 kb) was then purified, adenosine-tagged and ligated into pGEM-T (Promega) to create the suicide construct, pGEMTnatKO in which the ORF of nat is disrupted. To confirm the orientation of the ligated segments of the nat gene and the introduced kanamycin resistance gene, PCR using the transformants as templates, followed by the appropriate restriction digestions was carried out.
Screening for chromosomal integration by PCR.
Cells from single colonies were disrupted by heating (5 min at 95 °C) and used as template. As controls, 100 ng plasmid DNA or 1 µg M. smegmatis genomic DNA were used. The nat ORF was amplified using Taq DNA polymerase (Promega) with the primers SMNAT/5'PET28 (5'-CGAGTGCCATATGGCGATGGACCTCGGC-3') and SMNAT/3'1PET28 (5'-GGAATTCTCAGGTGTCGAGCACCTC-3'). DMSO at 6% (v/v) was added to all reactions. Primers were annealed at 58 °C for 30 s and the elongation was done either for 1 min to amplify the uninterrupted gene (840 bp) or for 2 min to amplify the nat ORF, now containing the Tn903 kanamycin resistance gene (2·1 kb). The products were denatured at 94 °C for 30 s and the cycle was repeated 30 times.
The correct 3' integration site was also confirmed by PCR amplification. One primer annealed to the 3' end of the Tn903 insert (5'KanrF1600, 5'-ATCTTGTGCAATGTAACATCAG-3') and the other annealed to the chromosomal DNA in the 3' flanking segment of the knockout construct (SMEG/KO-MINUS, 5'-CACTCGTAGGTGCTCGCAC-3'). Primers were annealed at 56 °C for 30 s and the elongation was done for 2·5 min. The product was then denatured at 94 °C for 30 s and the cycle was repeated 30 times. Following correct integration, a 2 kb fragment was amplified.
RNA preparation and Northern analysis.
RNA was prepared from exponential phase cultures of M. smegmatis using the detergent Catrimox 14 as previously described (Payton & Pinter, 1999 ). Approximately 10 µg total RNA was separated in a 1% agarose gel containing formaldehyde (Sambrook et al., 1989
). RNA was transferred onto a nylon membrane by capillary action and cross-linked to the membrane by UV. Hybridization was carried out with digoxigenin (DIG)-labelled DNA probes for detection by chemiluminescence (Roche). Labelled probes were generated by PCR, incorporating the DIG-labelled dUTP into the reaction mix, and then into the final product. The probe is the full coding sequence of M. smegmatis nat. To probe for the kanamycin resistance gene Tn903, the DNA segment was amplified as for the construction of the suicide vector, by using the 5'KanR/EcoRV and 3'KanR/EcoRV primers.
Generation of recombinant NAT proteins.
Recombinant M. smegmatis NAT and M. tuberculosis NAT and its fragments were produced by heterologous expression in E. coli using the pET28b-BL21(DE3)plysS expression system. Overexpressed NATs were either purified using the hexa-histidine tag to aid purification (if soluble) or were concentrated in inclusion bodies (if insoluble) as described earlier (Payton et al., 2001 ).
Analysis of NAT activities.
Cell pellets of 10 ml exponential phase M. smegmatis cultures were resuspended in 1 ml 50 mM Tris/HCl, 2 mM EDTA, pH 8·0 containing 1 mM Pefabloc (protease inhibitor; Pentapharm) and disrupted by sonication at 4 °C (3 times for 5 min at 7 µm amplitude with 2 min intervals between them). Cell debris was removed by centrifugation (12000 g, 20 min, 4 °C) and NAT activity was determined in the supernatant by the detection of N-acetylation of INH as previously described (Payton et al., 1999 ; Olson et al., 1977
).
SDS-PAGE, Western blot analysis and protein preparation for immunization.
SDS-PAGE and Western blotting were as described previously (Payton et al., 2001 ). In each case where purified recombinant proteins were used, equal amounts were loaded onto the gels.
To raise antibodies against M. smegmatis and M. tuberculosis NATs, the recombinant proteins were generated in E. coli. Protein concentration was greater than 5 mg (l culture)-1 including both the soluble and insoluble forms. The proteins were concentrated in inclusion bodies (insoluble fraction) and were subjected to SDS-PAGE. The bands of the proteins were excised from gels after staining with Coomassie brilliant blue and were used for immunization of rabbits as previously described (Stanley et al., 1996 ), except that only Incomplete Freunds Adjuvant was used.
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RESULTS |
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The knockout KO9 appeared to grow more slowly on agar than the parent strain. In liquid cultures, there was an extension of approximately 15 h in the lag phase, in comparison to KO9 to the wild-type M. smegmatis. However, after the lag phase, exponential phase growth rates were very similar. The growth rate was also determined for M. smegmatis transformed with pAGAN 40 alone and this matched the wild-type M. smegmatis with the same length of lag phase (Fig. 4).
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DISCUSSION |
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Targeted disruption of nat resulted in a delayed entry into exponential phase growth, extending the length of the lag phase (Fig. 4). In view of this observation, it may be that the presence of NAT is required during early growth, resulting in the extended lag phase for the knockout. The other possibility is that the other genes in the cluster are also affected by the removal of nat, caused either by a change of message stability or by a lack of transcription depending on the location of the nat gene within the cluster. It would be very important to identify the structure of the cluster in which nat is found for comparison with the putative clusters of M. bovis (Bacille CalmetteGuérin BCG) and M. tuberculosis, where nat appears to be the last gene (Payton et al., 2001
). It is also important to determine the nature of the other genes within the cluster. Nevertheless, the results presented here do suggest that NAT might have an endogenous function associated with growth.
Previous studies have shown that the heterologous expression of M. tuberculosis NAT in M. smegmatis results in a three-fold increase in resistance to INH (Payton et al., 1999 ). It was therefore not surprising to observe an increased sensitivity to INH in the M. smegmatis nat knockout mutant (Fig. 5
), albeit a small difference. INH must be activated by oxidation to be effective against M. tuberculosis (Zhang & Young, 1993
). It is very unlikely that N-acetyl-INH could be activated, as is the pro-drug INH, through the catalase peroxidase (KatG) pathway (Zhang & Young, 1993
; Zhang et al., 1996
). The combination of the level of NAT activity, coupled with different rates of KatG activity, for example, may contribute to the effective concentration of active, oxidized INH inside mycobacterial cells. Therefore cells with reduced NAT activity due to lower expression or mutation in the gene may have altered INH sensitivity. To determine whether the nat gene has a similar endogenous role in the pathogenic M. tuberculosis, these studies must now be carried out with M. tuberculosis.
The lack of a specific antibody recognizing only the M. tuberculosis NAT has been a major limitation in this research. The antiserum that we describe here against M. tuberculosis NAT is highly specific. We have mapped the predominant epitope to the C-terminal part of the molecule. Since the sequences of M. tuberculosis and M. bovis BCG NATs are identical, the antibody will also be useful for comparing nat expression in M. bovis BCG, another slow growing strain of mycobacteria. In preliminary experiments it has been demonstrated, using this antibody for Western blot analysis, that NAT is found in growing cultures of M. bovis BCG and M. tuberculosis H37Rv (Upton et al., 2001 ). This antiserum will also be extremely useful in determining the levels of NAT in clinical isolates of M. tuberculosis that differ in their sensitivity to INH.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 8 May 2001;
revised 8 August 2001;
accepted 13 August 2001.