Background
Tuberculosis, the disease caused by
Mycobacterium tuberculosis (Mtb), is the leading cause of human mortality, claiming nearly 3 million lives every year [
1]. The naïve or resting macrophages are extremely prone to invasion by Mtb bacilli and are unable to mount any anti-bacterial response associated with activated macrophages [
2‐
7]. Thus, the resting macrophage seems to provide an ideal niche where intracellular tubercle bacilli may reside, replicate and persist [
8,
9]. The proteins that are secreted by mycobacteria have gained particular attention in the recent years both as vaccine candidates and virulence factors [
10‐
18]. Some of these proteins like CFP-10 and ESAT-6 are encoded by the RD-1 region of Mtb genome, a region consistently deleted in all BCG vaccine strains of
M. bovis [
19‐
22].
Mitogen-activated protein kinases (MAPK) are evolutionarily conserved enzymes that are important in signal transduction. They play a diverse role in cell proliferation, cell death, cytokine production and cell differentiation. Three main families of MAPKs are found in mammalian cells: c-Jun-N-terminal kinases (JNK 1, 2 and 3); the extracellular signal-regulated kinases 1/2 (ERK1/2); and the p38 MAPK (p38 α, β, γ and δ) [
23]. They play diverse roles in the cell, ranging from apoptosis, cell differentiation, cell proliferation, stress response, to production of proinflammatory cytokines etc. [
24‐
31]. Targeting the MAP kinase pathway is one of the favorable strategies adopted by the pathogens to survive inside the macrophages [
32]. Mycobacteria modulate MAPK signaling to promote their survival in the host cells. Studies on MAPKs have been done using virulent and attenuated strains of mycobacteria.
M. avium has two strains; smooth transparent (SmT) and smooth opaque (SmO) which represent a more virulent and a less virulent phenotype, respectively. Both SmT and SmO induced early phosphorylation of p38 upon infection; however, only the attenuated strain elicited sustained activation of p38 MAPK. The virulent strains of mycobacteria caused greater inhibition of MAP kinases, particularly ERK1/2 pathway, as compared to the avirulent strains [
33,
34]. However, the molecular mechanisms involved in this phenomenon have not been investigated. Here, we show for the first time that ESAT-6 protein can modulate the ERK1/2 group of MAP kinases by limiting its activation in the nucleus. The MAP kinase-inducible transcription factor c-Myc is known to enhance cell proliferation as well as apoptosis [
35,
36]. Here we show that by modulating the MAP kinase ERK1/2, ESAT-6 down regulates the LPS-induced
c-myc gene expression in the macrophages.
Discussion
The present study demonstrates that ESAT-6 modulated the ERK1/2 group of MAP kinase. We also found that this modulation was achieved by inhibition of phosphorylation of ERK1/2 in the nucleus. However, the phosphorylation of another MAP kinase p38 was not affected by ESAT-6. Nevertheless, LPS, a general macrophage activator [
39‐
47] triggered phosphorylation of ERK1/2 in both cytoplasm and the nucleus. This showed that the limited activation of ERK1/2 in the nucleus was specific for ESAT-6 stimulation. Costimulation of cells with LPS and ESAT-6 dampened the ERK1/2 phosphorylation in the nucleus compared to that obtained with LPS alone; clearly ESAT-6 was exerting a strong inhibitory effect on the phosphorylation of ERK1/2 in the nucleus. Our finding that the treatment of cells with Na
3VO
4, a tyrosine phosphatase inhibitor [
48] along with ESAT-6 caused pERK1/2 to appear in the nucleus indicated that there was some phosphatase(s) activity in the nucleus that was triggered upon stimulation with ESAT-6. Moreover, when this phosphatase activity was suppressed by Na
3VO
4, the pERK1/2 reappeared in the nucleus. The results of kinase assay further corroborated our observations from western blotting that phosphorylation of ERK1/2 was concomitant with its activation. Measurement of phosphatase activity associated with ERK1/2 in the nucleus showed that there was an increase in this activity over the given time period; this finding was consistent with our observation that following treatment with both ESAT-6 and a phosphatase inhibitor (Na
3VO
4) there was an increase in phosphorylation of ERK1/2. It was already established that ERK1/2 after getting phosphorylated in cytoplasm translocates to the nucleus [
37]; therefore at zero minute, we observed little pERK1/2 in the nucleus (Fig.
1C). Our findings tend to suggest that although with increase in the ERK1/2 phosphorylation in the cytoplasm of the ESAT-6-stimulated cells, pERK1/2 must have migrated to the nucleus, but increasing phosphatase activity in the nucleus, again associated with ESAT-6 stimulation, dephosphorylated the pERK1/2 coming from the cytoplasm; therefore no pERK1/2 was detectable in the nuclear extract. Since MAP kinases undergo rapid turnover in the nucleus, the levels of total ERK1/2 in the nucleus remained constant over the experimental time period.
The
c-myc is one of the early response genes that encode a transcription factor c-Myc, which is a key regulator of cell proliferation and apoptosis. Since
c-myc expression was reported to occur through Ras/Raf/MEK/ERK pathway [
24,
49‐
52], we studied the effect of ESAT-6 on
c-myc expression in RAW264.7 cells. ESAT-6 itself did not have any effect on
c-myc expression over the basal level. However the LPS induced
c-myc expression was found to be downregulated by ESAT-6 compared to LPS stimulation alone. Again treatment with ESAT-6 along with 1 mM Na
3VO
4 increased the level of
c-myc compared to that observed with ESAT-6 alone while Na
3VO
4 alone did not have any effect on
c-myc levels. These results can be explained by the dampening of LPS-induced ERK1/2 phosphorylation in the nucleus by ESAT-6. As noted above, treatment with Na
3VO
4 along with ESAT-6 resulted in an increased level of ERK1/2 activation in the nucleus compared to ESAT-6 alone. This differential activation of ERK1/2 pathway resulted in differential
c-myc expression. To further confirm the role of ERK1/2 pathway in
c-myc expression, we determined
c-myc expression in the presence of MEK-1 inhibitor PD98059 [
53,
54] and p38 MAP kinase inhibitor SB203580 [
55,
56] along with Na
3VO
4 and ESAT-6. PD98059 downregulated
c-myc levels while SB203580 did not have any effect on
c-myc levels. The activation of ERK1/2 pathway in nucleus upon treatment with Na
3VO
4 and ESAT-6 was abrogated by PD98059 and hence
c-myc levels were downregulated. Since SB203580 did not have any effect on
c-myc expression, p38 MAP kinase was not involved in the gene expression. It confirmed the earlier observations of p38 phosphorylation from western blotting where there was no inhibition in p38 activation in cytoplasm or nucleus by ESAT-6.
Although there are reports that CFP-10 forms a 1:1 complex with ESAT-6 [
57]; however other studies [
58] have shown that there is discordance between secretion of CFP-10 and ESAT-6. Okkels and colleagues [
59] have shown that there are as many as 8 different forms of ESAT-6 and that the acetylation of ESAT-6 was required for complexation with CFP-10. Another study has shown that ESAT-6 as well as the CFP-10:ESAT-6 complex inhibited the PI-3 kinase-Akt signaling, indicating that the active component involved in downregulating the macrophage signaling was the ESAT-6 [
60]. Our studies with CFP-10 and CFP-10:ESAT-6 complex did not show any inhibition of the ERK1/2 phosphorylation in cytoplasm or nucleus of the RAW264.7 cells (see Additional file
1). It has also been shown that ESAT-6 binds to the Toll-like receptor-2 (TLR-2) and not TLR-4 on the surface of RAW264.7 macrophages, and causes inhibition of activation of transcription factors NF-κB and Interferon regulatory factors (IRFs) through the Akt kinase pathway [
60]. Our studies suggest yet another mechanism,
viz., modulation of the ERK arm of the MAP kinase pathway, by which ESAT-6 could bring about deactivation of the host cell.
Methods
Reagents and Antibodies
Bacterial lipopolysaccharide (LPS) and p-nitro phenylphosphate (p-NPP) and other fine chemicals were obtained from Sigma, St. Louis, MO, USA. Antibodies against ERK-1 and phospho-ERK1/2 were obtained from Santa Cruz Biotech, CA, USA. Tissue culture medium RPMI-1640 and the antibiotics penicillin and streptomycin and fetal bovine serum were from Life Technologies, USA.
Maintenance of cell line
Murine macrophage cell line RAW264.7 transformed with Abelson murine leukemia virus, originally obtained from ATCC, was routinely maintained in RPMI-1640 medium containing 2 mM glutamine, 100 μg/ml of penicillin and streptomycin and 10% fetal bovine serum at 5% CO2 in a humidified atmosphere at 37°C.
Cloning, expression and purification of recombinant Mycobacterial (Mtb) ESAT-6 protein
The open reading frame Rv3875, encoding ESAT-6 (GenBank Accession no. AF420491) of M. tuberculosis, was amplified by PCR from the genomic DNA of a local clinical isolate, by using the following primers: forward, 5'-GGAATTCCATATG ACAGAGCAGCAGTGGAATTTCG-3', reverse, 5'-CCGCTCGAG TGCGAACATCCCAGTGACGTTGC-3' (Nde I and Xho I sites, respectively, are underlined). The PCR product obtained here was cloned in the pGEM-T-Easy® vector and the nucleotide sequence of the gene revalidated. Full-length authentic gene was then sub-cloned into bacterial expression vector pET23b+; this vector yielded satisfactory levels of polyhistdine-tagged recombinant ESAT-6 protein expressed as an insoluble protein in E. coli. From the inclusion bodies, the protein was extracted using 8 M Urea pH 8.0. Recombinant ESAT-6 was purified by nickel-nitrilotriacetic acid (Ni2+-NTA) metal affinity chromatography according to the manufacturer's recommendations for purification of proteins under denaturing conditions. After purification, the pure fractions of protein were pooled together and the urea was removed by dialysing against 10 mM Na2HPO4, pH 8.0. The dialysed protein was aliquoted and kept at -20°C. The endotoxin level in the protein did not exceed 0.03 endotoxin units as done by E-toxate kit (Sigma).
Western blot analysis
For western blotting, 10 × 106 RAW264.7 cells were seeded per well of 12-well tissue culture plate in 1 ml of RPMI-1640 medium containing 10% FBS; cells were stimulated with 5 μg/ml of recombinant ESAT-6 for 0, 15, 30, 60 and 120 minutes. After stimulation, cells were harvested and lysed in 300 μl of lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM PMSF, 1 mM sodium orthovanadate (Na3VO4), 1 mM sodium fluoride, 1 μg/ml each of Leupeptin, Pepstatin A and Aprotinin, and 1% NP-40) for 20 minutes on ice. The cell lysates so obtained were cleared by centrifugation at 13,000 rpm, the supernatant represented the cytoplasmic extract; the nuclear pellet was washed and resuspended in the nuclear extraction buffer (20 mM HEPES pH 7.9, 400 mM KCl, 10 mM EDTA, 10 mM EGTA), kept on ice for 40 minutes with intermittent vortexing. Finally, the suspension was centrifuged at 13,000 rpm at 4°C, the supernatant was the nuclear extract. The protein contents of the cytoplasmic as well as nuclear extracts were estimated by the Bradford method and was then run on gel.
Phosphatase assay
For determination of phosphatase activity, 40 × 106 RAW264.7 cells were plated per well in a 6-well tissue culture plate (Nunc, Roskilde, Denmark) in 2 ml of complete medium. Cells were stimulated with 5 μg/ml of ESAT-6 for 0, 15, 30, 60 and 120 minutes. After stimulation, cells were harvested and lysed in 2 ml of lysis buffer for 20 minutes at 4°C, then the suspension was centrifuged at 13,000 rpm and the supernatant was discarded; the nuclear pellet was washed and suspended in 300 μl of nuclear extraction buffer and kept on ice for 40 minutes with intermittent vortexing. Then the suspension was centrifuged at 13,000 rpm and the supernatant was 'nuclear extract'. To the nuclear extract so prepared was added 20 μl of 30% ProteinA-agarose, and kept on nutator for 1 hour at 4°C (pre-clearing); to the cleared supernatant was added 4 μl of anti-ERK-1 antibody and kept on nutator for 1.5 hours at 4°C, followed by addition of 40 μl of 30% ProteinA-agarose; this mixture was kept on nutator for another 1 hour, then the Protein A-agarose beads carrying immunoprecipitated ERK were pelleted at 2,000 rpm; the pellet (immunoprecipitate) was washed thrice with wash buffer (50 mM HEPES pH 7.5, 2.5 mM MgCl2, 5% glycerol and 0.05% TritonX-100), and suspended in 100 μl of substrate solution (1 mg of p-nitrophenyl phosphate in 1 ml of buffer containing 50 mM MES pH 6.0, 1 mM EDTA and 0.1% Triton X-100) and kept at 37°C for 30 minutes. Then the agarose beads were pelleted at 2,000 rpm and the supernatant from each reaction tube was dispensed, 100 μl/well, into a 96-well micro-ELISA plate; to each such well 5 μl of 10 N NaOH to stop the reaction and the absorbance of resultant yellow color read at 405 nm using a microplate reader.
Kinase Assay for ERK1/2
For ERK1/2 kinase assay, ERK1/2 was immunoprecipitated from untreated and LPS and/or ESAT-6 treated RAW264.7 cells (2 × 107/treatment) for 60 minutes. Then cells were lysed and cytoplasmic and nuclear extracts were prepared. From the extracts, ERK was immunoprecipitated using anti-ERK-1 antibody. The immunoprecipitates were washed with wash buffer (20 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 2 mM DTT, 1 mM pNPP and 10 μM sodium orthovanadate) and then incubated with 20 μl of kinase reaction buffer (20 mM Tris-HCl pH7.5, 20 mM MgCl2, 2 mM DTT, 10 μM ATP, 10 μCi γ-32P-ATP and 5 μg MBP). The reaction was carried out at 30°C for 10 minutes. The reaction was terminated by addition of equal volume of 2× SDS loading buffer followed by boiling for 5 min. The reaction mixtures were subjected to SDS polyacrylamide gel electrophoresis. Dried gels were then exposed to X-ray films and the amount of [32P]-ATP incorporation in the substrate were ascertained by autoradiography followed by densitometric analysis.
Reverse transcription-PCR
Total RNA was isolated from 10 × 106 RAW264.7 cells, using 1 ml of TriZOL Reagent (Invitrogen Inc., Carlsbad, CA, USA); the total RNA was then quantified and converted to cDNA using Superscript II reverse transcriptase (Invitrogen Inc., USA). The cDNA was then used for amplification by PCR. The PCR was done using the Taq DNA polynerase (Biotools, B&M Lab, S.A., Spain). The PCR conditions were as follows: 94°C – 5 minutes (hot start), 94°C – 1 minute (denaturation), 55°C – 1 minute (annealing), 72°C – 1 minute (extension), 72°C – 10 minutes (final extension). The primers for amplification of c-myc: Forward: 5'-TCC TGT ACC TCG TCC GAT TC-3', Reverse: 5'-AAT TCA GGG ATC TGG TCA CG-3', IL-1β: Forward: 5'-TGG CAA CTG TTC CTG AAC TCA A-3', Reverse: 5'-TCC ACG GGA AAG ACA CAG GTA-3', Icam-1: Forward: 5'-TCT CGG AAG GGA GCC AAG TAA-3', Reverse: 5'-CTC TTG CCA GGT CCA GTT CC-3', Tnfr-1: Forward: 5'-CCC CAC CTC TGT TCA GAA ATG G-3', Reverse: 5'-TAC TTC CAG CGT GTC CTC GT-3', Bax: Forward: 5'-CTG AGC TGA CCT TGG AGC AG-3', Reverse: 5'-CCA GCC CAT GAT GGT TCT GAT-3', β-actin: Forward: 5'-CTA TGC TCT CCC TCA CGC CA-3', Reverse: 5'-CCG CTC GTT GCC AAT AGT GAT-3'.
Authors' contributions
NG performed the western blot analysis, kinase assay and phosphatase assay. SKB and IS did the RT-PCR experiments. PHG and FAM helped in the expression and purification of recombinant ESAT-6 and in the western blot analysis. PS and NG were responsible for conceptualizing and designing the study as well as for writing the manuscript. All authors have read and approved the final manuscript.