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Excision of an unstable pathogenicity island in Salmonella enterica serovar Enteritidis is induced during infection of phagocytic cells.

Quiroz TS, Nieto PA, Tobar HE, Salazar-Echegarai FJ, Lizana RJ, Quezada CP, Santiviago CA, Araya DV, Riedel CA, Kalergis AM, Bueno SM - PLoS ONE (2011)

Bottom Line: Importantly, we also found that one type of excision occurred at higher rates when S.Enteritidis was residing inside murine phagocytic cells.These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Católica de Chile, Santiago, Chile.

ABSTRACT
The availability of the complete genome sequence of several Salmonella enterica serovars has revealed the presence of unstable genetic elements in these bacteria, such as pathogenicity islands and prophages. This is the case of Salmonella enterica serovar Enteritidis (S. Enteritidis), a bacterium that causes gastroenteritis in humans and systemic infection in mice. The whole genome sequence analysis for S. Enteritidis unveiled the presence of several genetic regions that are absent in other Salmonella serovars. These regions have been denominated "regions of difference" (ROD). In this study we show that ROD21, one of such regions, behaves as an unstable pathogenicity island. We observed that ROD21 undergoes spontaneous excision by two independent recombination events, either under laboratory growth conditions or during infection of murine cells. Importantly, we also found that one type of excision occurred at higher rates when S. Enteritidis was residing inside murine phagocytic cells. These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells.

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Exposure to peroxide induces the excision of ROD21 from the S. Enteritidis chromosome.S. Enteritidis strain PT1 was grown in LB medium and then approximately 3√ó109 CFUs were transferred to fresh LB medium (LB) or to N-minimal medium (N), and incubated for 2 or 18 additional hours. Hydrogen peroxide was added at a final concentration equal to 0.25 mM during the last 30 min of incubation (N+H) and either genomic DNA or RNA was isolated. (A) The frequency of attB-1 excision was quantified by qPCR using genomic DNA and was expressed as a relative value equal to the ratio between the copy number of attB-1 over the copy number of rpoD gene. (B) SEN1970 expression was determined by qPCR using cDNA and expressed as a relative value (the ratio between the copy number of SEN1970 and the copy number of rpoD). The results are the average of three independent experiments. **; <0.01, one-way ANOVA and Tukey post test.
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pone-0026031-g006: Exposure to peroxide induces the excision of ROD21 from the S. Enteritidis chromosome.S. Enteritidis strain PT1 was grown in LB medium and then approximately 3√ó109 CFUs were transferred to fresh LB medium (LB) or to N-minimal medium (N), and incubated for 2 or 18 additional hours. Hydrogen peroxide was added at a final concentration equal to 0.25 mM during the last 30 min of incubation (N+H) and either genomic DNA or RNA was isolated. (A) The frequency of attB-1 excision was quantified by qPCR using genomic DNA and was expressed as a relative value equal to the ratio between the copy number of attB-1 over the copy number of rpoD gene. (B) SEN1970 expression was determined by qPCR using cDNA and expressed as a relative value (the ratio between the copy number of SEN1970 and the copy number of rpoD). The results are the average of three independent experiments. **; <0.01, one-way ANOVA and Tukey post test.

Mentions: Given that phagocytic cells produce reactive oxygen species upon phagocytosis of bacteria [24], whether oxidative stress could increase the frequency of ROD21 excision was evaluated. S. Enteritidis PT1 was grown in LB medium until OD600 equal to 0.6 and then 3.6√ó109 bacteria were incubated in N medium, which mimics the intracellular conditions found inside eukaryotic cells (i.e. reduced magnesium concentration [25]). At 30 min before the end of the incubation in N medium, bacteria were treated with 0.25 mM hydrogen peroxide and the frequency of ROD21 excision was determined by qPCR. As shown in figure 6A, ROD21 excision was significantly higher in bacteria grown for 18 h in N medium and treated with peroxide, as compared to the same strain grown in N medium alone.


Excision of an unstable pathogenicity island in Salmonella enterica serovar Enteritidis is induced during infection of phagocytic cells.

Quiroz TS, Nieto PA, Tobar HE, Salazar-Echegarai FJ, Lizana RJ, Quezada CP, Santiviago CA, Araya DV, Riedel CA, Kalergis AM, Bueno SM - PLoS ONE (2011)

Exposure to peroxide induces the excision of ROD21 from the S. Enteritidis chromosome.S. Enteritidis strain PT1 was grown in LB medium and then approximately 3√ó109 CFUs were transferred to fresh LB medium (LB) or to N-minimal medium (N), and incubated for 2 or 18 additional hours. Hydrogen peroxide was added at a final concentration equal to 0.25 mM during the last 30 min of incubation (N+H) and either genomic DNA or RNA was isolated. (A) The frequency of attB-1 excision was quantified by qPCR using genomic DNA and was expressed as a relative value equal to the ratio between the copy number of attB-1 over the copy number of rpoD gene. (B) SEN1970 expression was determined by qPCR using cDNA and expressed as a relative value (the ratio between the copy number of SEN1970 and the copy number of rpoD). The results are the average of three independent experiments. **; <0.01, one-way ANOVA and Tukey post test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198454&req=5

pone-0026031-g006: Exposure to peroxide induces the excision of ROD21 from the S. Enteritidis chromosome.S. Enteritidis strain PT1 was grown in LB medium and then approximately 3√ó109 CFUs were transferred to fresh LB medium (LB) or to N-minimal medium (N), and incubated for 2 or 18 additional hours. Hydrogen peroxide was added at a final concentration equal to 0.25 mM during the last 30 min of incubation (N+H) and either genomic DNA or RNA was isolated. (A) The frequency of attB-1 excision was quantified by qPCR using genomic DNA and was expressed as a relative value equal to the ratio between the copy number of attB-1 over the copy number of rpoD gene. (B) SEN1970 expression was determined by qPCR using cDNA and expressed as a relative value (the ratio between the copy number of SEN1970 and the copy number of rpoD). The results are the average of three independent experiments. **; <0.01, one-way ANOVA and Tukey post test.
Mentions: Given that phagocytic cells produce reactive oxygen species upon phagocytosis of bacteria [24], whether oxidative stress could increase the frequency of ROD21 excision was evaluated. S. Enteritidis PT1 was grown in LB medium until OD600 equal to 0.6 and then 3.6√ó109 bacteria were incubated in N medium, which mimics the intracellular conditions found inside eukaryotic cells (i.e. reduced magnesium concentration [25]). At 30 min before the end of the incubation in N medium, bacteria were treated with 0.25 mM hydrogen peroxide and the frequency of ROD21 excision was determined by qPCR. As shown in figure 6A, ROD21 excision was significantly higher in bacteria grown for 18 h in N medium and treated with peroxide, as compared to the same strain grown in N medium alone.

Bottom Line: Importantly, we also found that one type of excision occurred at higher rates when S.Enteritidis was residing inside murine phagocytic cells.These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Católica de Chile, Santiago, Chile.

ABSTRACT
The availability of the complete genome sequence of several Salmonella enterica serovars has revealed the presence of unstable genetic elements in these bacteria, such as pathogenicity islands and prophages. This is the case of Salmonella enterica serovar Enteritidis (S. Enteritidis), a bacterium that causes gastroenteritis in humans and systemic infection in mice. The whole genome sequence analysis for S. Enteritidis unveiled the presence of several genetic regions that are absent in other Salmonella serovars. These regions have been denominated "regions of difference" (ROD). In this study we show that ROD21, one of such regions, behaves as an unstable pathogenicity island. We observed that ROD21 undergoes spontaneous excision by two independent recombination events, either under laboratory growth conditions or during infection of murine cells. Importantly, we also found that one type of excision occurred at higher rates when S. Enteritidis was residing inside murine phagocytic cells. These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells.

Show MeSH
Related in: MedlinePlus