Limits...
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.

Show MeSH

Related in: MedlinePlus

Antisense inhibition of CosR.(A) Antisense inhibition of CosR expression by CosR-PNA (upper). C. jejuni was incubated for 8 hrs with various concentrations of CosR-PNA. The concentration of CosR-PNA is indicated above the panel. Total protein expression at different CosR-PNA concentrations was visualized by SDS-PAGE (lower). (B) Reduction of C. jejuni viability by antisense inhibition of CosR. CosR-PNA (0, 2, or 4 µM) and the control PNA (Cont-PNA; 4 µM) were added at initial cultivation. After 10-fold serial dilution, aliquots were plated onto MH agar for viable cell counts. Results are expressed as the mean and standard deviation of three independent experiments. **: P<0.01; the statistical significance of the results was determined by two-way ANOVA analysis of variance with Bonferroni's post-tests at a 95% confidence interval using Prism software (version 5.01; GraphPad Software Inc., USA).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3139631&req=5

pone-0022300-g003: Antisense inhibition of CosR.(A) Antisense inhibition of CosR expression by CosR-PNA (upper). C. jejuni was incubated for 8 hrs with various concentrations of CosR-PNA. The concentration of CosR-PNA is indicated above the panel. Total protein expression at different CosR-PNA concentrations was visualized by SDS-PAGE (lower). (B) Reduction of C. jejuni viability by antisense inhibition of CosR. CosR-PNA (0, 2, or 4 µM) and the control PNA (Cont-PNA; 4 µM) were added at initial cultivation. After 10-fold serial dilution, aliquots were plated onto MH agar for viable cell counts. Results are expressed as the mean and standard deviation of three independent experiments. **: P<0.01; the statistical significance of the results was determined by two-way ANOVA analysis of variance with Bonferroni's post-tests at a 95% confidence interval using Prism software (version 5.01; GraphPad Software Inc., USA).

Mentions: Due to the essentiality for C. jejuni viability, it has not been possible to knockout the cosR gene, because its knockout mutation leads to cell death [17], [20]. As an alternative in this study, antisense-mediated gene silencing was utilized to knockdown cosR expression. Western blot analysis confirmed that the level of CosR expression was reduced in response to the increase in the concentration of antisense CosR-specific PNA (CosR-PNA) without affecting overall protein expression based on the results of SDS-PAGE (Figure 3A). The viability of C. jejuni was slightly reduced with 2 µM CosR-PNA and significantly decreased with 4 µM CosR-PNA (Figure 3B). A PNA oligomer with the same number of bases as CosR-PNA, but lacking significant sequence similarity to genes in the C. jejuni genome, was used as a negative control. This PNA control did not reduce bacterial viability even at 4 µM (Figure 3B). These results demonstrated that antisense-mediated gene knockdown specifically controlled the level of CosR.


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)

Antisense inhibition of CosR.(A) Antisense inhibition of CosR expression by CosR-PNA (upper). C. jejuni was incubated for 8 hrs with various concentrations of CosR-PNA. The concentration of CosR-PNA is indicated above the panel. Total protein expression at different CosR-PNA concentrations was visualized by SDS-PAGE (lower). (B) Reduction of C. jejuni viability by antisense inhibition of CosR. CosR-PNA (0, 2, or 4 µM) and the control PNA (Cont-PNA; 4 µM) were added at initial cultivation. After 10-fold serial dilution, aliquots were plated onto MH agar for viable cell counts. Results are expressed as the mean and standard deviation of three independent experiments. **: P<0.01; the statistical significance of the results was determined by two-way ANOVA analysis of variance with Bonferroni's post-tests at a 95% confidence interval using Prism software (version 5.01; GraphPad Software Inc., USA).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022300-g003: Antisense inhibition of CosR.(A) Antisense inhibition of CosR expression by CosR-PNA (upper). C. jejuni was incubated for 8 hrs with various concentrations of CosR-PNA. The concentration of CosR-PNA is indicated above the panel. Total protein expression at different CosR-PNA concentrations was visualized by SDS-PAGE (lower). (B) Reduction of C. jejuni viability by antisense inhibition of CosR. CosR-PNA (0, 2, or 4 µM) and the control PNA (Cont-PNA; 4 µM) were added at initial cultivation. After 10-fold serial dilution, aliquots were plated onto MH agar for viable cell counts. Results are expressed as the mean and standard deviation of three independent experiments. **: P<0.01; the statistical significance of the results was determined by two-way ANOVA analysis of variance with Bonferroni's post-tests at a 95% confidence interval using Prism software (version 5.01; GraphPad Software Inc., USA).
Mentions: Due to the essentiality for C. jejuni viability, it has not been possible to knockout the cosR gene, because its knockout mutation leads to cell death [17], [20]. As an alternative in this study, antisense-mediated gene silencing was utilized to knockdown cosR expression. Western blot analysis confirmed that the level of CosR expression was reduced in response to the increase in the concentration of antisense CosR-specific PNA (CosR-PNA) without affecting overall protein expression based on the results of SDS-PAGE (Figure 3A). The viability of C. jejuni was slightly reduced with 2 µM CosR-PNA and significantly decreased with 4 µM CosR-PNA (Figure 3B). A PNA oligomer with the same number of bases as CosR-PNA, but lacking significant sequence similarity to genes in the C. jejuni genome, was used as a negative control. This PNA control did not reduce bacterial viability even at 4 µM (Figure 3B). These results demonstrated that antisense-mediated gene knockdown specifically controlled the level of CosR.

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