INSERM U447, Institut Pasteur de Lille, 1 rue du Professeur Calmette, F-59019 Lille Cedex, France
Correspondence
Philip Supply
philip.supply{at}pasteur-lille.fr
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ABSTRACT |
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INTRODUCTION |
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Mycobacterium tuberculosis possesses a complex infectious cycle, which includes intra- and extra-cellular phases, both within and outside the lungs, as well as a latency phase, suggesting that the expression of many of its genes must be subjected to regulation. However, the regulatory mechanisms governing the adaptive responses of M. tuberculosis, especially during phagocytosis, are still poorly understood. The M. tuberculosis genome contains 11 pairs of genes encoding two-component systems, in addition to a few isolated genes encoding orphan histidine kinases or response regulators (Cole et al., 1998). Several of these genes have been characterized at least partially (Dasgupta et al., 2000
; Ewann et al., 2002
; Graham & Clark-Curtiss, 1999
; Haydel et al., 1999
; Himpens et al., 2000
; Perez et al., 2001
; Sherman et al., 2001
; Supply et al., 1997
; Via et al., 1996
; Zahrt & Deretic, 2000
, 2001
). For example, an M. tuberculosis strain with a mutation in the phoP gene was found to be impaired in intra-cellular growth within macrophages, one of the major target cells of M. tuberculosis, and its virulence was found to be attenuated in mice (Perez et al., 2001
). MprAMprB is required for persistence in murine infection (Zahrt & Deretic, 2001
) and DevRDevS is induced in response to hypoxia and required to survive it (Boon & Dick, 2002
; Park et al., 2003
; Sherman et al., 2001
). MtrAMtrB was found to be essential for survival, as so far it has not been possible to obtain mtrA knockout strains of M. tuberculosis (Zahrt & Deretic, 2000
). In addition, mtrA has been shown to be upregulated upon phagocytosis in Mycobacterium bovis BCG but not in M. tuberculosis (Via et al., 1996
). The upregulating mechanism in M. bovis BCG has not yet been identified. The PrrAPrrB system has been found to be induced after macrophage phagocytosis and to be transiently required during the early stages of the macrophage infection (Ewann et al., 2002
; Graham & Clark-Curtiss, 1999
). The PrrAPrrB system belongs to a wide subfamily of two-component systems, of which OmpREnvZ is the prototype. Many members of this subfamily have been demonstrated to be autoregulated. However, autoregulation is not a general rule, as illustrated by the hilA gene in Salmonella typhimurium (Bajaj et al., 1996
; Lucas et al., 2000
). Here, we investigated the possible autoregulation of prrA and its role in the induction of the expression of this gene upon phagocytosis of M. tuberculosis.
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METHODS |
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PrrA and the cytoplasmic domain of PrrB were produced as His6-tagged recombinant proteins using pQE-30 (Qiagen). The prrA-coding sequence was amplified by PCR using the oligonucleotide pair 5'-AAAAGATCTATGGGCGGCATGGACACTGGTGTGA-3' and 5'-AAAAAGCTTATTCTGCATACGCAGCACGAATCCGACT-3'; these primers include a BglII and a HindIII restriction site, respectively (underlined). The cytoplasmic domain of PrrB was identified by alignment against the EnvZ and SenX3 sequences (Forst et al., 1989; Himpens et al., 2000
) as the C-terminal part of the protein starting from amino acid 204. The corresponding DNA sequence was amplified by PCR using oligonucleotides 5'-GCGGATCCATCGAGATCGCCGAGGC-3' and 5'-TTTAAGCTTCTAACTGGGTCCGGGAAGGC-3', containing a BamHI and a HindIII restriction site, respectively (underlined). The amplified fragments were digested with BamHI and HindIII and inserted into pQE30 restricted by the same enzymes to yield pQE30-PrrA and pQE30-PrrB, respectively.
Purification of the His6-tagged recombinant proteins under native conditions.
The recombinant E. coli strains containing pQE-PrrA and pQE-PrrB were grown in 1 l LB medium containing 100 µg ampicillin ml-1 and 25 µg kanamycin ml-1. When the OD600 value reached 1·21·4, expression of the genes encoding the recombinant proteins was induced with 1mM IPTG for 3 h. The cells were then harvested by centrifugation, resuspended in 5 ml lysis buffer (300 mM NaCl, 50 mM Na2HPO4, 10 mM imidazole, pH 8) per gram of fresh weight. The cells were then lysed using a French Press under a pressure of 1000 p.s.i. (6·9 MPa). The lysates were clarified by centrifugation at 10 000 g for 20 min. The supernatants were filtered using a 0·45 µm filter before loading onto a 1·5 ml Ni-NTA column (Qiagen) equilibrated in lysis buffer. The column was washed first with lysis buffer until the OD280 value reached less than 0·01 and then with 5 ml lysis buffer containing 50 mM imidazole. The proteins were eluted with 7 ml lysis buffer containing 250 mM imidazole, and fractions of 1·5 ml were collected. The fractions were analysed by SDS-PAGE, using a 12·5 % polyacrylamide gel, and Coomassie blue staining. After dialysis overnight against PBS (137 mM NaCl, 2·7 mM KCl, 10 mM Na2HPO4, 2 mM KHPO4) containing 10 % (v/v) glycerol, the protein concentrations were measured using BCA kit (Pierce); the proteins were dispensed into aliquots and then stored at -20 °C.
Phosphorylation assays.
Phosphorylation assays were performed using 2 µg of His6PrrB incubated for 20 min at 37 °C in the presence of 10 µCi of [-32P]ATP (3000 Ci mmol-1, 111 TBq mmol-1; Amersham Biosciences) in 20 µl of phosphorylation buffer containing 100 mM Tris/HCl (pH 8·0), 50 mM KCl and 5 mM MnCl2. For phosphotransfer assays, 20 µl of phosphorylation buffer containing 10 µg of His6PrrA were subsequently added. The reactions were stopped by the addition of 5 µl of 0·5 M EDTA (pH 8·0), and the incubation mixtures were subjected to SDS-PAGE using a 12·5 % polyacrylamide gel. After electrophoresis and Coomassie blue staining, the gel was dried and exposed for autoradiography to an X-ray film (Biomax; Kodak).
Electrophoretic mobility shift assays.
The promoter region of the prrAprrB operon, PprrA/B, was amplified by PCR using primers prrA/PF (5'-tcggggattgtcgacaccatc-3') and prrA/PR (5'-ccatttgcctgattaccgtc-3'). The amplified fragment containing the entire intergenic region separating prrA from its flanking gene (Rv0904) was sequenced and then labelled by T4 kinase (Roche diagnostics) using 10 µCi of [-32P]ATP (3000 Ci mmol-1; Amersham Biosciences). One nanogram of the labelled PCR fragment was incubated for 20 min at room temperature with 02 µg of His6PrrA, in 10 µl binding buffer containing 2 mM Tris/HCl (pH 8·0), 0·4 mM MgCl2, 10 mM KCl, 200 µM DTT, 10 % (v/v) glycerol and 0·01 % Nonidet P40. The reaction mixtures were loaded onto a 12 % (w/v) polyacrylamide/45 mM Tris-borate/1 mM EDTA (pH 8·0) native gel and subjected to electrophoresis. After drying, the gel was exposed to an X-ray film. When the effect of phosphorylation on binding was tested, His6PrrA was phosphorylated prior to the assay in the same phosphorylation buffer as above containing 0·5 mM ATP instead of radiolabelled ATP.
Bone-marrow macrophage infection and flow cytometry analysis.
Murine bone-marrow-derived macrophages were prepared and grown as described previously (Ewann et al., 2002). The infection assays were performed with 2x105 cells per well in 24-well cell culture clusters (Techno Plastic products). After removing the culture medium, 1 ml of a suspension of the M. tuberculosis Mt103 wild-type or of the Mt21D3 prrA/B mutant strain (Ewann et al., 2002
), each containing pFluo1Sm, or of BCG containing pFluo1Sm was added to obtain an m.o.i. of 10 : 1. Control wells containing non-infected macrophages were filled with 1 ml fresh culture medium. After 4 h incubation at 37 °C, the cells were washed three times in PBS to remove extracellular bacteria. The cells were then scraped and resuspended in 300 µl PBS; the fluorescence was analysed using a FACSVantage apparatus (BD Bioscience). The non-infected macrophage suspension was used as a reference to define the macrophage cell population, to exclude free bacilli and to eliminate the effects of macrophage autofluorescence.
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RESULTS |
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Autoregulation of the prrAprrB operon
To confirm autoregulation of the prrAprrB operon, we introduced pFluo1Sm, a plasmid containing the gfp reporter gene under control of the prrAprrB promoter region, into the M. tuberculosis Mt103 wild-type strain or the Mt21D3 prrA/B mutant derivative (Ewann et al., 2002). Since prrAprrB is not expressed in axenic culture conditions, and since the expression of this operon is induced early after macrophage phagocytosis (Graham & Clark-Curtiss, 1999
; Ewann et al., 2002
), murine bone-marrow-derived macrophages were infected with the recombinant strains. A BCG strain containing the same construct and a BCG strain containing pJFX4 and constitutively expressing gfp were used as controls. The fluorescence of the infected macrophages was measured by flow cytometry. Induction of fluorescence was readily observed with macrophages infected by BCG or M. tuberculosis Mt103 containing the prrA : : gfp construct (Fig. 4
b, c). In contrast, no fluorescence was detected when the macrophages were infected with the M. tuberculosis prrA/B mutant containing the prrA : : gfp construct (Fig. 4d
). Although effects of different copy numbers of the prrA : : gfp plasmid in the wild-type and in the prrA/B mutant can not be totally ruled out, these results, taken together with the results of the mobility shift assays, indicate that the prrAprrB operon is autoregulated in M. tuberculosis.
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DISCUSSION |
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Increased binding to the prrAprrB promoter region was observed upon phosphorylation of PrrA, which is consistent with the fact that the intracellular activation of the prrAprrB operon actually depends on both the presence and activity of the PrrAPrrB two-component system itself. The in vitro transphosphorylation was relatively inefficient under the conditions used in this study, as only a minor fraction of PrrA could be phosphorylated via PrrB. Therefore, the observed effect of PrrA phosphorylation on DNA binding was probably not optimal. Modifications of the Mn2+ concentrations or the replacement of Mn2+ by other bi-valent ions did not significantly improve the enzymic activities (data not shown). It may be possible that optimal phosphotransfer requires additional factors yet to be identified or requires PrrB sequences that are absent from His6PrrB.
Several other M. tuberculosis genes have been shown to be induced concomitantly to prrA during the first days of the macrophage infection (Graham & Clark-Curtiss, 1999). These genes encode proteins with various functions, such as sigma factors and cation transporters, as well as proteins involved in lipid and cell-wall metabolism or in intracellular invasion or persistence in mice (Chitale et al., 2001
; Cole et al., 1998
; Kolattukudy et al., 1997
; Manganelli et al., 1999
; McKinney et al., 2000
). The potential control of intracellular induction of these genes by the PrrAPrrB system can now be investigated using a strategy similar to that described here and used to demonstrate prrA autoregulation. This approach may perhaps be complemented by a non-targeted, albeit more delicate, proteomic and transcriptomic analysis of the available prrA/B mutant grown intracellularly.
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ACKNOWLEDGEMENTS |
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Received 27 May 2003;
revised 17 September 2003;
accepted 19 September 2003.