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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

Rv3131 induces TLR2 expression and NF-κB activation.(A) Real time expression of TLR2: RNA isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or TSA (250 ng) was reverse transcribed and real time PCR was carried out using gene specific primers. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. Data are presented as mean ± SEM of three independent experiments. (B) Surface expression of TLR2 by flow cytometry: PMA-differentiated THP-1 cells were treated with 100 ng of rRv3131 for 24 h followed by incubation with primary and secondary FITC labelled antibodies and analysed by flow cytometry. Data are expressed as mean ± SEM of percentage of cell population/MFI values from three independent experiments. (C) TLR2 protein expression: total protein isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or LPS (100 ng) was electrophoresed on polyacrylamide gel, transferred onto PVDF membrane and probed with antibodies against TLR2 and β-actin (internal control). Signal corresponding to the intensity of the band was measured using chemiluminiscence. The graph represents relative TLR2 expression levels quantified densitometrically (D) NF-κB phosphorylation: the total protein isolated was analysed using western blot with antibodies specific to phosphorylated p65.
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f4: Rv3131 induces TLR2 expression and NF-κB activation.(A) Real time expression of TLR2: RNA isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or TSA (250 ng) was reverse transcribed and real time PCR was carried out using gene specific primers. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. Data are presented as mean ± SEM of three independent experiments. (B) Surface expression of TLR2 by flow cytometry: PMA-differentiated THP-1 cells were treated with 100 ng of rRv3131 for 24 h followed by incubation with primary and secondary FITC labelled antibodies and analysed by flow cytometry. Data are expressed as mean ± SEM of percentage of cell population/MFI values from three independent experiments. (C) TLR2 protein expression: total protein isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or LPS (100 ng) was electrophoresed on polyacrylamide gel, transferred onto PVDF membrane and probed with antibodies against TLR2 and β-actin (internal control). Signal corresponding to the intensity of the band was measured using chemiluminiscence. The graph represents relative TLR2 expression levels quantified densitometrically (D) NF-κB phosphorylation: the total protein isolated was analysed using western blot with antibodies specific to phosphorylated p65.

Mentions: The possible role of Rv3131 in modulating TLR2 expression, besides its interaction on the cell surface was analysed by real time PCR. In THP-1 cells treated with rRv3131 protein, TLR2 mRNA levels were significantly upregulated (Fig. 4A) similar to that of the positive control TSA. Besides the gene expression, TLR2 expression levels on THP-1 cell surface following rRv3131 treatment were also analysed using flow cytometry analysis. A significant increase in the expression of TLR2 (Fig. 4B) but not TLR4 (data not shown) was observed in THP-1 cells when treated with rRv3131. Western blot analyses also indicated an increased expression of TLR2 in rRv3131 treated THP-1 cells (Fig. 4C). To determine whether the interaction of Rv3131 with TLR2 initiates NF-κB signalling pathway, western blot was performed. Analysis of whole cell protein isolated from THP-1 cells revealed activated form of NF- κB (p65) in rRv3131 and LPS treated preparations, but not in untreated cells (Fig. 4D).


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)

Rv3131 induces TLR2 expression and NF-κB activation.(A) Real time expression of TLR2: RNA isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or TSA (250 ng) was reverse transcribed and real time PCR was carried out using gene specific primers. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. Data are presented as mean ± SEM of three independent experiments. (B) Surface expression of TLR2 by flow cytometry: PMA-differentiated THP-1 cells were treated with 100 ng of rRv3131 for 24 h followed by incubation with primary and secondary FITC labelled antibodies and analysed by flow cytometry. Data are expressed as mean ± SEM of percentage of cell population/MFI values from three independent experiments. (C) TLR2 protein expression: total protein isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or LPS (100 ng) was electrophoresed on polyacrylamide gel, transferred onto PVDF membrane and probed with antibodies against TLR2 and β-actin (internal control). Signal corresponding to the intensity of the band was measured using chemiluminiscence. The graph represents relative TLR2 expression levels quantified densitometrically (D) NF-κB phosphorylation: the total protein isolated was analysed using western blot with antibodies specific to phosphorylated p65.
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Related In: Results  -  Collection

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f4: Rv3131 induces TLR2 expression and NF-κB activation.(A) Real time expression of TLR2: RNA isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or TSA (250 ng) was reverse transcribed and real time PCR was carried out using gene specific primers. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. Data are presented as mean ± SEM of three independent experiments. (B) Surface expression of TLR2 by flow cytometry: PMA-differentiated THP-1 cells were treated with 100 ng of rRv3131 for 24 h followed by incubation with primary and secondary FITC labelled antibodies and analysed by flow cytometry. Data are expressed as mean ± SEM of percentage of cell population/MFI values from three independent experiments. (C) TLR2 protein expression: total protein isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or LPS (100 ng) was electrophoresed on polyacrylamide gel, transferred onto PVDF membrane and probed with antibodies against TLR2 and β-actin (internal control). Signal corresponding to the intensity of the band was measured using chemiluminiscence. The graph represents relative TLR2 expression levels quantified densitometrically (D) NF-κB phosphorylation: the total protein isolated was analysed using western blot with antibodies specific to phosphorylated p65.
Mentions: The possible role of Rv3131 in modulating TLR2 expression, besides its interaction on the cell surface was analysed by real time PCR. In THP-1 cells treated with rRv3131 protein, TLR2 mRNA levels were significantly upregulated (Fig. 4A) similar to that of the positive control TSA. Besides the gene expression, TLR2 expression levels on THP-1 cell surface following rRv3131 treatment were also analysed using flow cytometry analysis. A significant increase in the expression of TLR2 (Fig. 4B) but not TLR4 (data not shown) was observed in THP-1 cells when treated with rRv3131. Western blot analyses also indicated an increased expression of TLR2 in rRv3131 treated THP-1 cells (Fig. 4C). To determine whether the interaction of Rv3131 with TLR2 initiates NF-κB signalling pathway, western blot was performed. Analysis of whole cell protein isolated from THP-1 cells revealed activated form of NF- κB (p65) in rRv3131 and LPS treated preparations, but not in untreated cells (Fig. 4D).

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