Académique Documents
Professionnel Documents
Culture Documents
External Guide
Internal Guide
Dr. Satish Mundayoor Scientist G, Rajiv Gandhi Centre for Biotechnology Trivandrum
Introduction
Mycobacterium tuberculosis
Etiologic agent of TB in humans Non-motile, rod-shaped, acid-fast bacterium Identified and described on 24th March, 1882 by Robert Koch
Tuberculosis
Disease affects 1.8 billion people/year
M.tb (SEM)
Extrapulmonary Tuberculosis (infection may spread from lungs to other parts of the body)
Commonly used drugs: rifampin (RIF) isoniazid (INH), pyrazinamide (PZA ) and ethambutol (EMB)
Poorly treated patients can develop MDR or XDR TB
Objectives
To understand the role of cytoskeletal proteins during M.tb infection by analyzing their differential expression To identify proteins of interest using 2D electrophoresis and MS To analyze the expression of the proteins of interest at the mRNA level using qRT-PCR To comparatively quantitate the protein using western blot
2D electrophoresis
1. Protein Isolation Protein isolation was done 24 h post-infection Isolated protein quantified using 2Dquant Kit 2. IEF
IPG strips were loaded with the isolated protein samples IEF done using the Bio Rad PROTEAN IEF unit This caused the proteins to separate based on their pI
3. Equilibration The strips were equilibrated in urea, SDS, DTT and IAA to prepare them for SDS PAGE
Continued
4.SDS PAGE The strips were loaded on top of 12% SDS gels and run at 100V Separation of proteins based on their molecular weight occurs during this phase The gels were then stained with coomassie G250
Gels were destained with ddH2O and visualized using a gel doc
The proteins can be observed as spots on the gel
2D Electrophoresis
Sample preparation
pH 3-10
Macrophage Control
Vimentin : Mol.wt. 54Kda pI - 5.1 Cofilin : Mol. Wt. 18.5 kDa pI- 8.2
qRT- PCR
mRNA isolated and quantified Isolated mRNA subjected to single step qRT-PCR GAPDH used as the reference gene
Protein Isolated from infected cells after the required time period of infection 12h,24h Isolated protein estimated using BCA Assay
OD at 570 nm
2 OD at 570 nm 1.5 1 0.5 0 0 1000 2000 3000 Conc g/ml OD at 570 nm Linear (OD at 570 nm)
Multiple bands were observed for vimentin. Band patterns indicated the lysis of vimentin by caspases after proapoptotic stimuli
Phosphoprotein Staining
Phosphoprotein staining of 2D gels were done Pro-Q Diamond stain was used for staining
The gels were visually examined and no significant variations were found
Protein spots that were suspected to be phosphorylated forms of vimentin and cofilin were cut and send for MS analysis
Phosphoprotein Staining
THP-1
Conclusion
Significant variation in the expression of vimentin and cofilin was observed Biphasic regulation of vimentin observed during infection with M.tb live Significant upregulation of cofilin observed These variations suggest active role of these proteins in the post-infection response of the host cell The roles played by these proteins may be proapoptotic or antiapoptotic They may aid the host or the pathogen