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. 2007 Oct 3:8:24.
doi: 10.1186/1471-2172-8-24.

Mycobacterium tuberculosis 6-kDa early secreted antigenic target (ESAT-6) protein downregulates lipopolysaccharide induced c-myc expression by modulating the extracellular signal regulated kinases 1/2

Niladri Ganguly  1 , Pham H Giang, Sandip K Basu, Fayaz Ahmad Mir, Imran Siddiqui, Pawan Sharma
Affiliations

Mycobacterium tuberculosis 6-kDa early secreted antigenic target (ESAT-6) protein downregulates lipopolysaccharide induced c-myc expression by modulating the extracellular signal regulated kinases 1/2

Niladri Ganguly et al. BMC Immunol. .

Abstract

Background: Mycobacterium tuberculosis (Mtb) causes death of 2-3 million people every year. The persistence of the pathogenic mycobacteria inside the macrophage occurs through modulation of host cell signaling which allows them, unlike the other non-pathogenic species, to survive inside the host. The secretory proteins of M. tuberculosis have gained attention in recent years both as vaccine candidates and diagnostic tools; they target the immune system and trigger a putatively protective response; however, they may also be involved in the clinical symptoms of the disease.

Results: Our studies showed that RD-1-encoded secretory protein ESAT-6 is involved in modulation of the mitogen-activated protein (MAP) kinase-signaling pathway inside the macrophage. ESAT-6 induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the cytoplasm but not in the nucleus, which normally is the case for MAP kinases. ESAT-6 also antagonized LPS-induced ERK1/2 phosphorylation in the nucleus. Stimulation of cells by ESAT-6 along with sodium orthovanadate (a tyrosine phosphatase inhibitor) restored phosphorylation of ERK1/2 in the nucleus, suggesting active dephosphorylation of ERK1/2 by some putative phosphatase(s) in the nucleus. Further, ESAT-6 was found to down regulate the expression of LPS-inducible gene c-myc in an ERK1/2-dependent manner.

Conclusion: This study showed the effect of secretory proteins of M. tuberculosis in the modulation of macrophage signaling pathways particularly ERK1/2 MAP kinase pathway. This modulation appears to be achieved by limiting the ERK1/2 activation in the nucleus which ultimately affects the macrophage gene expression. This could be a mechanism by which secretory proteins of Mtb might modulate gene expression inside the macrophages.

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Figures

Figure 1
Figure 1
ESAT-6 induced phosphorylation of ERK1/2 in cytoplasm but not in nucleus. 10 × 106 RAW264.7 cells were stimulated with 5 μg/ml of recombinant ESAT-6 for 0, 15, 30, 60 and 120 minutes; cytoplasmic and nuclear extracts were run on gel and probed with anti-phospho-ERK1/2 antibody. (A) phosphorylation of ERK1/2 in cytoplasm. (C) phosphorylation of ERK1/2 in nucleus. (B) and (D) Total ERK1/2 in the cytoplasmic and nuclear extracts respectively at different time points to confirm equal loading of samples in all the lanes. (E) and (G) represents phosphorylated p38 in cytoplasm and the nucleus respectively. (F) and (H) shows the total p38 protein in cytoplasm and nucleus respectively. Data is a representative from three experiments.
Figure 2
Figure 2
LPS induced phosphorylation of ERK1/2 in both cytoplasm and nucleus. RAW264.7 cells were stimulated with 0.1 μg/ml of bacterial LPS for 0, 15, 30, 60 and 120 minutes and probed for phospho-ERK1/2 as before. (A) Phosphorylation of ERK1/2 in cytoplasm upon stimulation with LPS(0.1 μg/ml). (C) phosphorylation of ERK1/2 in nucleus. The data is a representative of three independent experiments. (B) and (D) Total ERK1/2 in cytoplasm and nucleus normalized for protein content. Costimulation of RAW264.7 cells with LPS (0.1 μg/ml) and 5 μg/ml of ESAT-6 for 0, 15, 30, 60 and 120 minutes. (E) ERK1/2 phosphorylation in cytoplasm upon stimulation with 5 μg/ml of ESAT-6 and 0.1 μg/ml of LPS. (G) Phosphorylation of ERK1/2 in the nucleus with both LPS and ESAT-6. (F) and (H) Total ERK1/2 protein in cytoplasm and nucleus respectively.
Figure 3
Figure 3
Stimulation with ESAT-6 in presence of sodium orthovanadate caused appearance of phospho-ERK1/2 in the nucleus. Stimulation of RAW264.7 cells with 5 μg/ml of ESAT-6 and 1 mM Na3VO4 for 0, 15, 30, 60 and 120 minutes. (A) Phosphorylation of ERK1/2 in cytoplasm. (C) ERK1/2 phosphorylation in the nucleus. (B) and (D) Total ERK1/2 in cytoplasm and nucleus respectively. (E) and (G) Phosphorylation of ERK1/2 in cytoplasm and the nucleus respectively upon treatment with 1 mM Na3VO4 for 0, 15, 30, 60 and 120 minutes. (F) and (H) Total ERK1/2 in cytoplasm and nucleus respectively to show equal loading of proteins in all the lanes. The data is representative of three independent experiments.
Figure 4
Figure 4
Densitomteric analysis of the western blots. The densitomteric analysis for the ERK blots for unstimulated cells, ESAT-6 and/or LPS, ESAT-6 and/or NaV are shown. The upper graph represents plot of cytoplasmic extracts and the lower graph represents nuclear extract. The data represented as fold change. The unstimulated cells were given a value of 1.00.
Figure 5
Figure 5
ESAT-6 downregulated LPS-induced ERK kinase activity. (A) and (B) represent the autoradiogram of the ERK kinase assay using Myelin basic protein (MBP) as a substrate in cytoplasm and nucleus respectively. Lane.1. Unstimulated cells, Lane.2. Cells stimulated with 5 μg/ml ESAT-6, Lane.3. Cells stimulated with 0.1 μg/ml of LPS, Lane.4. Cells stimulated with LPS and ESAT-6, Lane.5. Cells stimulated with 1 mM Na3VO4 and 5 μg/ml ESAT-6, Lane.6. Cells stimulated with 1 mM Na3VO4. (C) and (D) represents the graph showing fold change of the densitometric values obtained from the densitometric studies of the autoradiogram of (A) and (B) respectively. Unstimulated cells were given a value 1.00. The data represented as mean +/- S.D. of three independent experiments.
Figure 6
Figure 6
ESAT-6 stimulated increase in the phosphatase activity associated with ERK1/2 in the nucleus. (A) RAW264.7 cells were stimulated for different time points of 0, 15, 30, 60 and 120 minutes, the ERK-1 was immunoprecipitated from the nuclear extract and the phosphatase activity was determined, the last column where cells were stimulated with ESAT-6 for 120 minutes but no ERK-1 antibody was added (antibody control). (B) After phosphatase assay was done, the immunoprecipitate was mixed with 2× sample buffer and run on 10% SDS-PAGE and after western blotting the membrane was probed with ERK-1 antibody to confirm equal pull down of ERK1/2 in all the samples. The graph shows the mean +/- S.D. of three independent experiments.
Figure 7
Figure 7
ESAT-6 downregulated LPS induced c-myc expression. (A) RT-PCR for c-myc gene expression, Lane. 1. Unstimulated cells, Lane.2. Cells stimulated with 5 μg/ml ESAT-6, Lane.3. Cells stimulated with 0.1 μg/ml of LPS, Lane. 4. Cells stimulated with LPS and ESAT-6, Lane.5. Cells stimulated with 1 mM Na3VO4 and 5 μg/ml ESAT-6, Lane.6. Cells stimulated with 1 mM Na3VO4. Stimulation time was 120 minutes. (C) RT-PCR for c-myc gene expression, Lane.1. Unstimulated cells, Lane.2. Cells stimulated with 5 μg/ml ESAT-6, Lane.3. Cells stimulated with 1 mM Na3VO4 and 5 μg/ml ESAT-6, Lane.4. Cells stimulated with Na3VO4 and ESAT-6 and 10 μM of MEK-1 inhibitor PD98059, Lane.5. Cells simulated with Na3VO4 and ESAT-6 and 10 μM of p38 inhibitor SB203580, Lane.6. Cells stimulated with 1 mM Na3VO4, (B) and (D) RT-PCR for β-actin to confirm equal amplification in all the samples. The data is representative of two independent experiments. (E) and (F) shows the graph of densitometric analysis of Figure. 8A and 8C respectively. The data is shown as fold change; unstimulated cells were given a value of 1.00.
Figure 8
Figure 8
ESAT-6 downregulated LPS-induced expression of several genes. (A-E) RT-PCR for the genes IL-1β, Bax, Icam-1, Tnfr-1 and β-actin. The data is representative of two independent experiments. Lane.1. Unstimulated cells, Lane.2. Cells stimulated with 5 μg/ml ESAT-6, Lane.3. Cells stimulated with 0.1 μg/ml of LPS, Lane.4. Cells stimulated with LPS and ESAT-6. (F) RT-PCR for β-actin to confirm equal amplification in all the samples.
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