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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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Negative regulation of sodB by CosR.(A) qRT-PCR analysis of sodB transcription. Results are expressed as the mean and standard deviation from three independent experiments. (B) sodB transcription determined by primer extension assays at different CosR levels. The doublet bands show two sodB transcriptional start sites in C. jejuni, and the top band is the major. “CosR−” and “CosR+” indicate CosR knockdown and CosR overexpression, respectively. (C) Enzymatic activity of SodB at different CosR levels. In these experiments, CosR knockdown was achieved with 1.5 µM CosR-PNA. These results show the representative of three independent experiments.
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pone-0022300-g007: Negative regulation of sodB by CosR.(A) qRT-PCR analysis of sodB transcription. Results are expressed as the mean and standard deviation from three independent experiments. (B) sodB transcription determined by primer extension assays at different CosR levels. The doublet bands show two sodB transcriptional start sites in C. jejuni, and the top band is the major. “CosR−” and “CosR+” indicate CosR knockdown and CosR overexpression, respectively. (C) Enzymatic activity of SodB at different CosR levels. In these experiments, CosR knockdown was achieved with 1.5 µM CosR-PNA. These results show the representative of three independent experiments.

Mentions: Although SodB is a key enzyme contributing to the detoxification of O2−, its regulation has not been well understood in C. jejuni. Since the results of 2DGE, EMSA and DNase I footprinting assays indicated that CosR may regulate sodB transcription (Figures 4, 5, and 6A), qRT-PCR and primer extension assays were performed to further confirm the regulation of sodB transcription by CosR. In both assays, antisense inhibition of CosR upregulated sodB transcription, whereas CosR overexpression reduced the level of sodB transcription compared to the wild type (Figures 7A and 7B). To examine if the changes in SodB expression would affect its enzyme activity, the superoxide dismutase colorimetric assay was performed. Consistently, the superoxide dismutase activity was enhanced by CosR knockdown and attenuated by CosR overexpression (Figure 7C). These results demonstrated that CosR regulates sodB transcription and affects the SodB enzyme activity.


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)

Negative regulation of sodB by CosR.(A) qRT-PCR analysis of sodB transcription. Results are expressed as the mean and standard deviation from three independent experiments. (B) sodB transcription determined by primer extension assays at different CosR levels. The doublet bands show two sodB transcriptional start sites in C. jejuni, and the top band is the major. “CosR−” and “CosR+” indicate CosR knockdown and CosR overexpression, respectively. (C) Enzymatic activity of SodB at different CosR levels. In these experiments, CosR knockdown was achieved with 1.5 µM CosR-PNA. These results show the representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139631&req=5

pone-0022300-g007: Negative regulation of sodB by CosR.(A) qRT-PCR analysis of sodB transcription. Results are expressed as the mean and standard deviation from three independent experiments. (B) sodB transcription determined by primer extension assays at different CosR levels. The doublet bands show two sodB transcriptional start sites in C. jejuni, and the top band is the major. “CosR−” and “CosR+” indicate CosR knockdown and CosR overexpression, respectively. (C) Enzymatic activity of SodB at different CosR levels. In these experiments, CosR knockdown was achieved with 1.5 µM CosR-PNA. These results show the representative of three independent experiments.
Mentions: Although SodB is a key enzyme contributing to the detoxification of O2−, its regulation has not been well understood in C. jejuni. Since the results of 2DGE, EMSA and DNase I footprinting assays indicated that CosR may regulate sodB transcription (Figures 4, 5, and 6A), qRT-PCR and primer extension assays were performed to further confirm the regulation of sodB transcription by CosR. In both assays, antisense inhibition of CosR upregulated sodB transcription, whereas CosR overexpression reduced the level of sodB transcription compared to the wild type (Figures 7A and 7B). To examine if the changes in SodB expression would affect its enzyme activity, the superoxide dismutase colorimetric assay was performed. Consistently, the superoxide dismutase activity was enhanced by CosR knockdown and attenuated by CosR overexpression (Figure 7C). These results demonstrated that CosR regulates sodB transcription and affects the SodB enzyme activity.

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