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Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni.

Hwang S, Kim M, Ryu S, Jeon B - PLoS ONE (2011)

Bottom Line: Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide.Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type.Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea.

ABSTRACT
CosR (Campylobacter oxidative stress regulator; Cj0355c) is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a leading foodborne pathogen causing human gastroenteritis worldwide. Despite importance, the function of CosR remains completely unknown mainly because of cell death caused by its knockout mutation. To overcome this technical limitation, in this study, antisense technology was used to investigate the regulatory function of CosR by modulating the level of CosR expression. Two-dimensional gel electrophoresis (2DGE) was performed to identify the CosR regulon either by suppressing CosR expression with antisense peptide nucleic acid (PNA) or by overexpressing CosR in C. jejuni. According to the results of 2DGE, CosR regulated 32 proteins involved in various cellular processes. Notably, CosR negatively regulated a few key proteins of the oxidative stress response of C. jejuni, such as SodB, Dps, Rrc and LuxS, whereas CosR positively controlled AhpC. Electrophoretic mobility shift assay showed that CosR directly bound to the promoter region of the oxidative stress genes. DNase I footprinting assays identified 21-bp CosR binding sequences in the sodB and ahpC promoters, suggesting CosR specifically recognizes and binds to the regulated genes. Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide. Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type. Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.

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Regulation of oxidative stress defense proteins by CosR.(A) The spots of oxidative stress proteins regulated by CosR were compared at different CosR levels. The spots of CosR protein were included to demonstrate the different level of CosR in each condition. The protein spots are from 2DGE (Figure 4) and specific protein spots are indicated by arrows. “CosR−” and “CosR+” indicate CosR knockdown and CosR overexpression, respectively. (B) Binding of rCosR to the promoter region of oxidative stress genes. Radiolabeled gene probes of sodB, dps, luxS and ahpC were incubated with rCosR at different concentrations, and unlabeled DNA probes (U.P) and internal coding regions (I.C) of each target gene were used as a competitor.
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pone-0022300-g005: Regulation of oxidative stress defense proteins by CosR.(A) The spots of oxidative stress proteins regulated by CosR were compared at different CosR levels. The spots of CosR protein were included to demonstrate the different level of CosR in each condition. The protein spots are from 2DGE (Figure 4) and specific protein spots are indicated by arrows. “CosR−” and “CosR+” indicate CosR knockdown and CosR overexpression, respectively. (B) Binding of rCosR to the promoter region of oxidative stress genes. Radiolabeled gene probes of sodB, dps, luxS and ahpC were incubated with rCosR at different concentrations, and unlabeled DNA probes (U.P) and internal coding regions (I.C) of each target gene were used as a competitor.

Mentions: To identify the CosR regulon, protein expression profiles were analyzed after CosR knockdown and CosR overexpression. The effective concentration of CosR-PNA was determined by altering the treatment time and the CosR-PNA concentration to suppress CosR expression without significant viability changes. Based on the results of western blot analysis and viability tests (Figure 3), 1.5 µM CosR-PNA was determined to be the most appropriate concentration to knockdown CosR. Alternatively, the effect of CosR overexpression was evaluated with a strain that harbors an extra copy of cosR in the chromosome. Quantitative real-time PCR (qRT-PCR) and western blot analysis confirmed cosR overexpression at the transcriptional and translational levels in the strain (Figure S1). Analysis of the protein expression profiles in the CosR-overexpression strain provided complementary evidence for the protein expression changes observed in the CosR-knockdown condition. Altered CosR levels by knockdown or overexpression influenced the expression of 32 proteins involved in various cell functions, including macromolecule biosynthesis, energy metabolism, respiration, heat shock response, regulation and oxidative stress defense (Figure 4 and Table 1). CosR appeared in the two-dimensional gel as three spots located parallel with different pI values (Figures 4 and 5A), presumably due to protein modifications such as phosphorylation. Based on the results of two-dimensional gel electrophoresis (2DGE), we found that the level of CosR was reduced by PNA knockdown and elevated in the CosR-overexpression strain (Figures 4 and 5A), confirming successful control of the CosR level in both CosR knockdown and overexpression conditions. CosR knockdown resulted in a 2.1-fold decrease in the protein level of PckA (phosphoenolpyruvate carboxykinase), an essential protein in C. jejuni [32], whereas CosR overexpression increased PckA protein levels by 3-fold (Figure 4 and Table 1). CosR affected the expression level of proteins associated with copper tolerance and gene regulation (Figure 4 and Table 1); CosR negatively regulated Cj1516, a homolog of the multicopper oxidase CueO in E. coli, and CprR, which is another essential response regulator in C. jejuni [18], [33]. Notably, CosR regulated the expression of several important proteins of oxidative stress defense in C. jejuni; CosR negatively regulated SodB, Dps, LuxS and Rrc, whereas AhpC was positively regulated by CosR (Figures 4, 5A and Table 1). Electrophoretic mobility shift assay (EMSA) was performed with rCosR to determine if CosR directly binds to the promoter regions of sodB, dps, luxS and ahpC. The results showed that rCosR bound to the promoter regions, and binding of rCosR to the DNA resulted in concentration-dependent mobility shift (Figure 5B). Unlabeled target DNA fragments effectively competed with the labeled DNA probes of sodB, dps, luxS and ahpC, but the internal coding regions did not (Figure 5B), indicating specific binding of CosR to the target promoter regions. These results demonstrate that CosR is a pleiotropic regulator, particularly modulating the expression of oxidative stress genes in C. jejuni.


Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni.

Hwang S, Kim M, Ryu S, Jeon B - PLoS ONE (2011)

Regulation of oxidative stress defense proteins by CosR.(A) The spots of oxidative stress proteins regulated by CosR were compared at different CosR levels. The spots of CosR protein were included to demonstrate the different level of CosR in each condition. The protein spots are from 2DGE (Figure 4) and specific protein spots are indicated by arrows. “CosR−” and “CosR+” indicate CosR knockdown and CosR overexpression, respectively. (B) Binding of rCosR to the promoter region of oxidative stress genes. Radiolabeled gene probes of sodB, dps, luxS and ahpC were incubated with rCosR at different concentrations, and unlabeled DNA probes (U.P) and internal coding regions (I.C) of each target gene were used as a competitor.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139631&req=5

pone-0022300-g005: Regulation of oxidative stress defense proteins by CosR.(A) The spots of oxidative stress proteins regulated by CosR were compared at different CosR levels. The spots of CosR protein were included to demonstrate the different level of CosR in each condition. The protein spots are from 2DGE (Figure 4) and specific protein spots are indicated by arrows. “CosR−” and “CosR+” indicate CosR knockdown and CosR overexpression, respectively. (B) Binding of rCosR to the promoter region of oxidative stress genes. Radiolabeled gene probes of sodB, dps, luxS and ahpC were incubated with rCosR at different concentrations, and unlabeled DNA probes (U.P) and internal coding regions (I.C) of each target gene were used as a competitor.
Mentions: To identify the CosR regulon, protein expression profiles were analyzed after CosR knockdown and CosR overexpression. The effective concentration of CosR-PNA was determined by altering the treatment time and the CosR-PNA concentration to suppress CosR expression without significant viability changes. Based on the results of western blot analysis and viability tests (Figure 3), 1.5 µM CosR-PNA was determined to be the most appropriate concentration to knockdown CosR. Alternatively, the effect of CosR overexpression was evaluated with a strain that harbors an extra copy of cosR in the chromosome. Quantitative real-time PCR (qRT-PCR) and western blot analysis confirmed cosR overexpression at the transcriptional and translational levels in the strain (Figure S1). Analysis of the protein expression profiles in the CosR-overexpression strain provided complementary evidence for the protein expression changes observed in the CosR-knockdown condition. Altered CosR levels by knockdown or overexpression influenced the expression of 32 proteins involved in various cell functions, including macromolecule biosynthesis, energy metabolism, respiration, heat shock response, regulation and oxidative stress defense (Figure 4 and Table 1). CosR appeared in the two-dimensional gel as three spots located parallel with different pI values (Figures 4 and 5A), presumably due to protein modifications such as phosphorylation. Based on the results of two-dimensional gel electrophoresis (2DGE), we found that the level of CosR was reduced by PNA knockdown and elevated in the CosR-overexpression strain (Figures 4 and 5A), confirming successful control of the CosR level in both CosR knockdown and overexpression conditions. CosR knockdown resulted in a 2.1-fold decrease in the protein level of PckA (phosphoenolpyruvate carboxykinase), an essential protein in C. jejuni [32], whereas CosR overexpression increased PckA protein levels by 3-fold (Figure 4 and Table 1). CosR affected the expression level of proteins associated with copper tolerance and gene regulation (Figure 4 and Table 1); CosR negatively regulated Cj1516, a homolog of the multicopper oxidase CueO in E. coli, and CprR, which is another essential response regulator in C. jejuni [18], [33]. Notably, CosR regulated the expression of several important proteins of oxidative stress defense in C. jejuni; CosR negatively regulated SodB, Dps, LuxS and Rrc, whereas AhpC was positively regulated by CosR (Figures 4, 5A and Table 1). Electrophoretic mobility shift assay (EMSA) was performed with rCosR to determine if CosR directly binds to the promoter regions of sodB, dps, luxS and ahpC. The results showed that rCosR bound to the promoter regions, and binding of rCosR to the DNA resulted in concentration-dependent mobility shift (Figure 5B). Unlabeled target DNA fragments effectively competed with the labeled DNA probes of sodB, dps, luxS and ahpC, but the internal coding regions did not (Figure 5B), indicating specific binding of CosR to the target promoter regions. These results demonstrate that CosR is a pleiotropic regulator, particularly modulating the expression of oxidative stress genes in C. jejuni.

Bottom Line: Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide.Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type.Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea.

ABSTRACT
CosR (Campylobacter oxidative stress regulator; Cj0355c) is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a leading foodborne pathogen causing human gastroenteritis worldwide. Despite importance, the function of CosR remains completely unknown mainly because of cell death caused by its knockout mutation. To overcome this technical limitation, in this study, antisense technology was used to investigate the regulatory function of CosR by modulating the level of CosR expression. Two-dimensional gel electrophoresis (2DGE) was performed to identify the CosR regulon either by suppressing CosR expression with antisense peptide nucleic acid (PNA) or by overexpressing CosR in C. jejuni. According to the results of 2DGE, CosR regulated 32 proteins involved in various cellular processes. Notably, CosR negatively regulated a few key proteins of the oxidative stress response of C. jejuni, such as SodB, Dps, Rrc and LuxS, whereas CosR positively controlled AhpC. Electrophoretic mobility shift assay showed that CosR directly bound to the promoter region of the oxidative stress genes. DNase I footprinting assays identified 21-bp CosR binding sequences in the sodB and ahpC promoters, suggesting CosR specifically recognizes and binds to the regulated genes. Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide. Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type. Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.

Show MeSH
Related in: MedlinePlus