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Differential Expression and Alternative Roles of Cytoskeletal Proteins in Macrophage-Mycobacterium tuberculosis Interaction

S. Divya Sharon Reg.no 01110921008

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Dr. Satish Mundayoor Scientist G, Rajiv Gandhi Centre for Biotechnology Trivandrum

Dr. B. Anandaraj Assistant Professor Anna University of Technology Tiruchirappalli

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

Cell Culturing and Infection


THP-1: Human acute monocytic leukemia cell line Shows consistency with the behaviour of alveolar macrophages during M.tb infection THP-1 cells cultured in RPMI 1640 media and induced to differentiate using PMA M.tb(H37Rv) cultured in 7H9 Middlebrooks broth with OADC supplement McFarlands standard (no.1) was used to determine the cell number of M.tb/ml of culture Differentiated THP-1 cells were infected with M.tb(live and heat killed) with the required MOI (1:10 and 1:15)

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

Protein Selection and Identification


The spots were identified and analysed visually and using PDQuest
MASCOT was used to identify the protein based on the m/z ratio obtained after MS Cofilin and Vimentin chosen

2D Electrophoresis
Sample preparation
pH 3-10

Rehydration and loading of IPG strips(16h) Isoelectric focusing

Loading of IPG strips in 12% SDS gel


Mol. Wt. SDS PAGE

Gel staining and visualization


Molecular marker 200kDa to 6kDa

Macrophage Control

Macrophage infected with live M.tb

Vimentin : Mol.wt. 54Kda pI - 5.1 Cofilin : Mol. Wt. 18.5 kDa pI- 8.2

Macrophage infected with heat killed Mt.b

qRT- PCR
mRNA isolated and quantified Isolated mRNA subjected to single step qRT-PCR GAPDH used as the reference gene

Gene Expression Analysis


GAPDH was used as reference gene as minimum variation was observed in its expression pattern The Gene expression levels were analyzed using QbasePLUS from Biogazelle Results showed significant up regulation of Cofilin and down regulation of Vimentin in cells infected with live M.tb

Western Blot and Densitometry


Protein isolated at 12h and 24h post-infection Quantitation was done using BCA Assay The samples were subject to immunoblotting on PVDF membrane and densitometry analysis was performed using QuanitityOne -actin was used as the reference protein Variations consistent with qRT-PCR results were observed in the case of both vimentin and cofilin

Protein Isolation and Estimation

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)

Western Blot of Vimentin with Multiple Bands

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

THP-1+ M.tb Live

THP-1+ M.tb Heat Killed

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

The specificity of their role can be established by studying their phosphorylation


pattern and the expression of associated proteins

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