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A putative nitroreductase from the DosR regulon of Mycobacterium tuberculosis induces pro-inflammatory cytokine expression via TLR2 signaling pathway.

Peddireddy V, Doddam SN, Qureshi IA, Yerra P, Ahmed N - Sci Rep (2016)

Bottom Line: In THP-1 cells treated with Rv3131 protein, a significant upsurge in the surface expression, overall induction and expression of mRNA of TLR2 was observed when analysed by flow cytometry, western blotting and real time PCR, respectively.Activation of TLR2 by Rv3131 resulted in the phosphorylation of NF- κβ.Results of this study indicate a strong immunogenic capability of Rv3131 elicited via the activation of TLR2 signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad 500046 India.

ABSTRACT
Tuberculosis caused by Mycobacterium tuberculosis is a global encumbrance and it is estimated that nearly one third population of the world acts as a reservoir for this pathogen without any symptoms. In this study, we attempted to characterise one of the genes of DosR regulon, Rv3131, a FMN binding nitroreductase domain containing protein, for its ability to alter cytokine profile, an essential feature of M. tuberculosis latency. Recombinant Rv3131 stimulated pro-inflammatory cytokines in THP-1 cells and human peripheral blood mononuclear cells in a time and dose dependent manner. In silico analyses using docking and simulations indicated that Rv3131 could strongly interact with TLR2 via a non-covalent bonding which was further confirmed using cell based colorimetric assay. In THP-1 cells treated with Rv3131 protein, a significant upsurge in the surface expression, overall induction and expression of mRNA of TLR2 was observed when analysed by flow cytometry, western blotting and real time PCR, respectively. Activation of TLR2 by Rv3131 resulted in the phosphorylation of NF- κβ. Results of this study indicate a strong immunogenic capability of Rv3131 elicited via the activation of TLR2 signalling pathway. Therefore, it can be surmised that cytokine secretion induced by Rv3131 might contribute to establishment of M. tuberculosis in the granulomas.

No MeSH data available.


Related in: MedlinePlus

Modelled 3D-structure of Rv3131 and its interaction with TLR2.(A) Three dimensional structure of Rv3131 determined using homology modelling: the elements of protein secondary structure were coloured and labelled (helices and sheets displayed in cyan and pink, respectively). (B) Docking study of Rv3131 with TLR2: the residues of TLR2 and Rv3131 are coloured in green and cyan, respectively. The residues presenting interaction among both the proteins are labelled and shown as stick model in element colours (green/cyan colour represents carbon, blue represents nitrogen, and red represents oxygen). The black dashed lines represent hydrogen bonds. (C) Rv3131 interacts with TLR2: HEK-Blue 293 hTLR-2 cells were treated with various concentrations (10 ng–250 ng) of rRv3131 and interaction of Rv3131 with TLR2 was measured spectrophotometrically by the colour change read at 620 nm due to the activity of SEAP. *p < 0.05; ***p < 0.0005.
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f3: Modelled 3D-structure of Rv3131 and its interaction with TLR2.(A) Three dimensional structure of Rv3131 determined using homology modelling: the elements of protein secondary structure were coloured and labelled (helices and sheets displayed in cyan and pink, respectively). (B) Docking study of Rv3131 with TLR2: the residues of TLR2 and Rv3131 are coloured in green and cyan, respectively. The residues presenting interaction among both the proteins are labelled and shown as stick model in element colours (green/cyan colour represents carbon, blue represents nitrogen, and red represents oxygen). The black dashed lines represent hydrogen bonds. (C) Rv3131 interacts with TLR2: HEK-Blue 293 hTLR-2 cells were treated with various concentrations (10 ng–250 ng) of rRv3131 and interaction of Rv3131 with TLR2 was measured spectrophotometrically by the colour change read at 620 nm due to the activity of SEAP. *p < 0.05; ***p < 0.0005.

Mentions: Computational analysis demonstrated that Rv3131 possessed conserved nitroreductase domain spanning 236–301 residues, while TOPCONS suggested the lack of signal peptide or transmembrane region in this protein. As crystallographic/solution structure of this protein is not available, BLASTp was used to find the structural templates and a template (PDB ID: 2YMV, nitroreductase from Mycobacterium smegmatis) with 37% identity was retrieved from PDB data bank. Modeller was used to generate structures using single template and the best structure was selected on the basis of different parameters including low DOPE score. To analyse the quality of structure, Ramachandran plot was generated and it revealed two residues (L327 and L328) in disallowed region. To improve quality of the structure, modelling was performed further with two templates i.e. PDB ID: 2YMV and 2B67. The quality of the structure was evaluated using Ramachandran plot acquired via Procheck and results of the validated model were as follows: most favoured regions contained 89.9%; additional allowed regions had 9.4%; generously allowed regions had 0.7% and disallowed regions contained 0.0% residues. Secondary structure of modelled Rv3131 protein revealed fourteen alpha helices and eight beta sheets (Fig. 3A). Identification of possible binding interface residues was performed using MetaPPISP programme. Protein-protein docking was performed with receptor as TLR2 and ligand as Rv3131 model using different online servers including GRAMM-X38 and PatchDock. Among the used programmes, Patchdock provided several solutions based on shape complementarity criteria, where Rv3131 was placed besides TLR2. Out of obtained complexes, best 10 were further subjected to FireDock to refine the solutions based on global energy. However, complex 7 was evolved as the best solution for residues interaction between the two proteins. As shown in Fig. 3B, Rv3131 emanates close to TLR2 and shows strong interaction due to the presence of several non-covalent bonds between them.


A putative nitroreductase from the DosR regulon of Mycobacterium tuberculosis induces pro-inflammatory cytokine expression via TLR2 signaling pathway.

Peddireddy V, Doddam SN, Qureshi IA, Yerra P, Ahmed N - Sci Rep (2016)

Modelled 3D-structure of Rv3131 and its interaction with TLR2.(A) Three dimensional structure of Rv3131 determined using homology modelling: the elements of protein secondary structure were coloured and labelled (helices and sheets displayed in cyan and pink, respectively). (B) Docking study of Rv3131 with TLR2: the residues of TLR2 and Rv3131 are coloured in green and cyan, respectively. The residues presenting interaction among both the proteins are labelled and shown as stick model in element colours (green/cyan colour represents carbon, blue represents nitrogen, and red represents oxygen). The black dashed lines represent hydrogen bonds. (C) Rv3131 interacts with TLR2: HEK-Blue 293 hTLR-2 cells were treated with various concentrations (10 ng–250 ng) of rRv3131 and interaction of Rv3131 with TLR2 was measured spectrophotometrically by the colour change read at 620 nm due to the activity of SEAP. *p < 0.05; ***p < 0.0005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837367&req=5

f3: Modelled 3D-structure of Rv3131 and its interaction with TLR2.(A) Three dimensional structure of Rv3131 determined using homology modelling: the elements of protein secondary structure were coloured and labelled (helices and sheets displayed in cyan and pink, respectively). (B) Docking study of Rv3131 with TLR2: the residues of TLR2 and Rv3131 are coloured in green and cyan, respectively. The residues presenting interaction among both the proteins are labelled and shown as stick model in element colours (green/cyan colour represents carbon, blue represents nitrogen, and red represents oxygen). The black dashed lines represent hydrogen bonds. (C) Rv3131 interacts with TLR2: HEK-Blue 293 hTLR-2 cells were treated with various concentrations (10 ng–250 ng) of rRv3131 and interaction of Rv3131 with TLR2 was measured spectrophotometrically by the colour change read at 620 nm due to the activity of SEAP. *p < 0.05; ***p < 0.0005.
Mentions: Computational analysis demonstrated that Rv3131 possessed conserved nitroreductase domain spanning 236–301 residues, while TOPCONS suggested the lack of signal peptide or transmembrane region in this protein. As crystallographic/solution structure of this protein is not available, BLASTp was used to find the structural templates and a template (PDB ID: 2YMV, nitroreductase from Mycobacterium smegmatis) with 37% identity was retrieved from PDB data bank. Modeller was used to generate structures using single template and the best structure was selected on the basis of different parameters including low DOPE score. To analyse the quality of structure, Ramachandran plot was generated and it revealed two residues (L327 and L328) in disallowed region. To improve quality of the structure, modelling was performed further with two templates i.e. PDB ID: 2YMV and 2B67. The quality of the structure was evaluated using Ramachandran plot acquired via Procheck and results of the validated model were as follows: most favoured regions contained 89.9%; additional allowed regions had 9.4%; generously allowed regions had 0.7% and disallowed regions contained 0.0% residues. Secondary structure of modelled Rv3131 protein revealed fourteen alpha helices and eight beta sheets (Fig. 3A). Identification of possible binding interface residues was performed using MetaPPISP programme. Protein-protein docking was performed with receptor as TLR2 and ligand as Rv3131 model using different online servers including GRAMM-X38 and PatchDock. Among the used programmes, Patchdock provided several solutions based on shape complementarity criteria, where Rv3131 was placed besides TLR2. Out of obtained complexes, best 10 were further subjected to FireDock to refine the solutions based on global energy. However, complex 7 was evolved as the best solution for residues interaction between the two proteins. As shown in Fig. 3B, Rv3131 emanates close to TLR2 and shows strong interaction due to the presence of several non-covalent bonds between them.

Bottom Line: In THP-1 cells treated with Rv3131 protein, a significant upsurge in the surface expression, overall induction and expression of mRNA of TLR2 was observed when analysed by flow cytometry, western blotting and real time PCR, respectively.Activation of TLR2 by Rv3131 resulted in the phosphorylation of NF- κβ.Results of this study indicate a strong immunogenic capability of Rv3131 elicited via the activation of TLR2 signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad 500046 India.

ABSTRACT
Tuberculosis caused by Mycobacterium tuberculosis is a global encumbrance and it is estimated that nearly one third population of the world acts as a reservoir for this pathogen without any symptoms. In this study, we attempted to characterise one of the genes of DosR regulon, Rv3131, a FMN binding nitroreductase domain containing protein, for its ability to alter cytokine profile, an essential feature of M. tuberculosis latency. Recombinant Rv3131 stimulated pro-inflammatory cytokines in THP-1 cells and human peripheral blood mononuclear cells in a time and dose dependent manner. In silico analyses using docking and simulations indicated that Rv3131 could strongly interact with TLR2 via a non-covalent bonding which was further confirmed using cell based colorimetric assay. In THP-1 cells treated with Rv3131 protein, a significant upsurge in the surface expression, overall induction and expression of mRNA of TLR2 was observed when analysed by flow cytometry, western blotting and real time PCR, respectively. Activation of TLR2 by Rv3131 resulted in the phosphorylation of NF- κβ. Results of this study indicate a strong immunogenic capability of Rv3131 elicited via the activation of TLR2 signalling pathway. Therefore, it can be surmised that cytokine secretion induced by Rv3131 might contribute to establishment of M. tuberculosis in the granulomas.

No MeSH data available.


Related in: MedlinePlus