Research Groups

Mammalian Biology: Immunology

Research Interests and Description

Staff Research Scientist: Pawan Sharma

Research Interests

Mycobacterium tuberculosis, macrophages, innate immunity, vaccine development.

Description of Research

The Group studies the role of Mtb proteins in immunomodulation of the macrophage. Recently we looked at the effect of ESAT-6 on macrophage signaling pathways. We looked at the status of extracellular signal-regulated kinases 1/2 (ERK1/2). ESAT-6 induced phosphorylation of ERK1/2 in cytoplasm, however no phosphorylated ERK1/2 could be detected in the nucleus. The macrophage activator Lipopolysaccharide (LPS) induced phosphorylation of ERK1/2 in both cytoplasm and the nucleus proving that the effect of ESAT-6 was not a property of the RAW264.7 cell line. ESAT-6 was found to dampen the LPS-induced phosphorylation of ERK1/2 in the nucleus thereby exerting an antagonistic effect. Treatment of cells with a tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4) along with ESAT-6 caused phospho-ERK1/2 to appear in the nucleus while Na3VO4 alone did not induce ERK1/2 phosphorylation in the nucleus. The activation of ERK1/2 was further confirmed by doing kinase assay after immunoprecipitating ERK1/2 from the cytoplasmic and nuclear extracts using Myelin Basic Protein (MBP) as the substrate. Measurement of ERK1/2 associated-phosphatase activity in the nucleus showed a time-dependent increase in the phosphatase activity.
To check the effect of ESAT-6 on LPS-induced gene expression, the expression of an early response gene c-myc was studied. ESAT-6 itself did not alter the c-myc expression over the basal level. However the LPS-induced c-myc expression was found to be downregulated by ESAT-6 compared to the c-myc expression by LPS stimulation alone. Again stimulation with ESAT-6 along with 1mM sodium orthovanadate (Na3VO4) increased the level of c-myc compared to ESAT-6 stimulation alone. To confirm the role of ERK1/2 pathway in c-myc expression, we determined c-myc expression in presence of MEK-1 inhibitor PD98059 and p38 MAP kinase inhibitor SB203580 along with Na3VO4 and ESAT-6. Treatment with PD98059 along with ESAT-6 and Na3VO4 downregulated c-myc levels while SB203580 did not have any effect on c-myc levels..
We compared the effect of CFP-10, ESAT-6 and CFP10:ESAT6 complex on macrophage signal transduction. It was observed that these downregulated ROS production in RAW264.7 macrophages. Lipopolysaccharide is known to induce the production of ROS. These antigens i.e. CFP-10, ESAT-6 and CFP10:ESAT6 complex were found to antagonize LPS-induced ROS production in these cells. The ROS affects intracellular signal transduction and is known to activate the transcription factor NF-κB. We first looked at the binding of NF-κB p65 subunit to the NF-κB consensus binding sequence in the nucleus as a measure of its activation; LPS was found to enhance NF-κB p65 DNA binding which was downregulated by CFP-10, ESAT-6 and the CFP10:ESAT6 complex as well as by ROS scavenger N-Acetyl cysteine. Next we determined whether LPS-induced ROS production resulted in NF-κB-dependent gene transcription in a reporter assay system. LPS was found to induce the expression of NF-κB-dependent reporter gene (Chloramphenicol acetyl transferase, cat) over the basal level. This increase in reporter gene expression was downregulated by CFP-10, ESAT-6 and the 1:1 complex. Addition of N-Acetyl cysteine, which is a ROS scavenger along with LPS stimulation led to significant reduction in reporter gene expression even below the basal level.

Recent Publications

Ganguly, N., Siddiqui, I., Sharma, P. 2008. Role of M tuberculosis RD-1 region encoded secretory proteins in protective response and virulence. Tuberculosis. In press

Singh, A., Kushwaha, H.R., Sharma, P. 2008. Molecular modeling and comparative structural account of aspartyl -semialdehyde dehydrogenase of Mycobacterium tuberculosis (H37Rv). J Mol Model 14, 249-263

Ganguly, N., Giang, P.H., Gupta, C., Basu, S.K., Siddiqui, I., Salunke, D.M., Sharma, P. 2008. Mycobacterium tuberculosis secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex inhibit lipopolysaccharide-induced NF-kB transactivation by downregulation of reactive oxidative species (ROS) production. Immunol Cell Biol 86, 96-106

Dey, A., Sharma, P., Redhu, N.S., Singh, S. 2008. Kinesin motor domain of Leishmania donovani as future vaccine candidate. Clin. Vacc. Immunol. 15, 836-842

Sivakumar, R., Dey, A., Sharma, P., Singh, S. 2008. Expression and characterization of recombinant kinesin antigen from an old Indian strain (DD8) of Leishmania donovani, and comparing it with a commercially available antigen from a newly isolated (KE16) strain of L. donovani. Infect. Genet. Evol. 8, 313-322

Ganguly, N., Giang, P.H., Basu, S.K., Mir, F.A., Siddiqui, I., Sharma, P. 2007. 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. BMC Immunology 8, 24 (doi:10.1186/1471-2172-8-24)