Limits...
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.

Show MeSH

Related in: MedlinePlus

ROD21 is lost in bacteria undergoing type 2 excision.(A) Schematic representation of tetRA insertion in the ROD21::tetRA strain. This strain was used to isolate bacteria that have lost ROD21. The ROD21::tetRA strain was grown in the contraselection BM medium and the arising colonies were tested for the presence of ROD21 by PCR. The lines denominated 1 and 2 represent the expected PCR products that would be generated if ROD21 was inserted in the chromosome. (B) Detection of ROD21 by PCR analysis in WT S. Enteritidis and in one of the ΔROD21 strains isolated in a contraselection assay. This figure shows a representative agarose gel (1%) resolving the PCR products 1 and 2 (which denote each boundary of the integrated form of ROD21) obtained for WT and ΔROD21 S. Enteritidis strains. In addition, the attB sequences generated after type 1 (attB-1) and type 2 (attB-2) excisions were also detected by PCR. As a positive control, the rpoD gene was amplified by PCR. Data shown derive from one representative S. Enteritidis ΔROD21 strain selected out of 6 strains recovered in two independent experiments in which the attB-2 sequence was detected. (C) C57BL/6 mice were orally infected with 1×106 CFUs of either WT or ΔROD21 S. Enteritidis strains and the survival rate was measured daily. Uninfected mice were included as controls. Data shown are averages of two independent experiments, each including at least 4 mice per group. (D and E) Competitive infection assays, consisting of C57BL/6 mice infected either orally or intravenously with a mixture of the WT (KnR) and ΔROD21 (CmR) S. Enteritidis strains (input ratio equal to 1∶1). After 72 h, bacteria were recovered from spleens and livers of infected mice and the ratio of WT (KnR) over ΔROD21 (CmR) S. Enteritidis was calculated and compared to the input. Data shown in graphs are average values from two independent experiments for bacteria recovered from spleens and livers after intragastric gavage (D) or intravenous (E) infections of 3 mice per group.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198454&req=5

pone-0026031-g004: ROD21 is lost in bacteria undergoing type 2 excision.(A) Schematic representation of tetRA insertion in the ROD21::tetRA strain. This strain was used to isolate bacteria that have lost ROD21. The ROD21::tetRA strain was grown in the contraselection BM medium and the arising colonies were tested for the presence of ROD21 by PCR. The lines denominated 1 and 2 represent the expected PCR products that would be generated if ROD21 was inserted in the chromosome. (B) Detection of ROD21 by PCR analysis in WT S. Enteritidis and in one of the ΔROD21 strains isolated in a contraselection assay. This figure shows a representative agarose gel (1%) resolving the PCR products 1 and 2 (which denote each boundary of the integrated form of ROD21) obtained for WT and ΔROD21 S. Enteritidis strains. In addition, the attB sequences generated after type 1 (attB-1) and type 2 (attB-2) excisions were also detected by PCR. As a positive control, the rpoD gene was amplified by PCR. Data shown derive from one representative S. Enteritidis ΔROD21 strain selected out of 6 strains recovered in two independent experiments in which the attB-2 sequence was detected. (C) C57BL/6 mice were orally infected with 1×106 CFUs of either WT or ΔROD21 S. Enteritidis strains and the survival rate was measured daily. Uninfected mice were included as controls. Data shown are averages of two independent experiments, each including at least 4 mice per group. (D and E) Competitive infection assays, consisting of C57BL/6 mice infected either orally or intravenously with a mixture of the WT (KnR) and ΔROD21 (CmR) S. Enteritidis strains (input ratio equal to 1∶1). After 72 h, bacteria were recovered from spleens and livers of infected mice and the ratio of WT (KnR) over ΔROD21 (CmR) S. Enteritidis was calculated and compared to the input. Data shown in graphs are average values from two independent experiments for bacteria recovered from spleens and livers after intragastric gavage (D) or intravenous (E) infections of 3 mice per group.

Mentions: Then, we evaluated whether the excision of ROD21 results in the loss of this pathogenicity island from the bacterial genome. To evaluate this possibility, we inserted the genes tetA and tetR downstream the gene SEN1975 in the strain of S. Enteritidis Phagotype 1 (PT1), to generate the ROD21::tetRA strain (Fig. 4A). The genes tetA and tetR confer resistance to tetracycline, but also prevent the growth of tetracycline-resistant bacteria in a medium containing zinc chloride and fusaric acid (BM medium), as described previously [21]. Therefore, only those Salmonella strains sensitive to tetracycline will grow in BM medium [21]. Nested PCR reactions showed that excision type 1 and type 2 (Fig. 2A) occurred in the modified ROD21::tetRA strain, as efficiently as observed for the wild type (WT) strain (data not shown). To evaluate if these excisions caused ROD21 loss, the ROD21::tetRA strain was grown in LB medium and then seeded on solid BM medium, as described in materials and methods. Bacteria were incubated 36 h at 37°C to select for tetracycline sensitive bacteria. A total of 35 tetracycline sensitive colonies out of 137 seeded plates were tested by PCR to evaluate ROD21 loss. We observed that only 6 out of the 35 colonies isolated had lost ROD21 from the chromosome, indicating that the frequency of ROD21 loss is 4.38×10−8. Further, PCR reactions showed that only the attB sequence generated by excision type 2 could be detected in the genome of all isolated S. Enteritidis strains (Fig. 4B), suggesting that excision type 2 was responsible of ROD21 loss in all isolated CFUs. With regard to the other 29 colonies that lost tetracycline resistance without excising ROD21, it is possible that they might had undergone other type of mutations resulting in tetracycline sensitivity, such as mutations in tetA or tetR genes [22], [23] or changes in cell membrane permeability. However, further research would be required to clarify this issue.


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)

ROD21 is lost in bacteria undergoing type 2 excision.(A) Schematic representation of tetRA insertion in the ROD21::tetRA strain. This strain was used to isolate bacteria that have lost ROD21. The ROD21::tetRA strain was grown in the contraselection BM medium and the arising colonies were tested for the presence of ROD21 by PCR. The lines denominated 1 and 2 represent the expected PCR products that would be generated if ROD21 was inserted in the chromosome. (B) Detection of ROD21 by PCR analysis in WT S. Enteritidis and in one of the ΔROD21 strains isolated in a contraselection assay. This figure shows a representative agarose gel (1%) resolving the PCR products 1 and 2 (which denote each boundary of the integrated form of ROD21) obtained for WT and ΔROD21 S. Enteritidis strains. In addition, the attB sequences generated after type 1 (attB-1) and type 2 (attB-2) excisions were also detected by PCR. As a positive control, the rpoD gene was amplified by PCR. Data shown derive from one representative S. Enteritidis ΔROD21 strain selected out of 6 strains recovered in two independent experiments in which the attB-2 sequence was detected. (C) C57BL/6 mice were orally infected with 1×106 CFUs of either WT or ΔROD21 S. Enteritidis strains and the survival rate was measured daily. Uninfected mice were included as controls. Data shown are averages of two independent experiments, each including at least 4 mice per group. (D and E) Competitive infection assays, consisting of C57BL/6 mice infected either orally or intravenously with a mixture of the WT (KnR) and ΔROD21 (CmR) S. Enteritidis strains (input ratio equal to 1∶1). After 72 h, bacteria were recovered from spleens and livers of infected mice and the ratio of WT (KnR) over ΔROD21 (CmR) S. Enteritidis was calculated and compared to the input. Data shown in graphs are average values from two independent experiments for bacteria recovered from spleens and livers after intragastric gavage (D) or intravenous (E) infections of 3 mice per group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198454&req=5

pone-0026031-g004: ROD21 is lost in bacteria undergoing type 2 excision.(A) Schematic representation of tetRA insertion in the ROD21::tetRA strain. This strain was used to isolate bacteria that have lost ROD21. The ROD21::tetRA strain was grown in the contraselection BM medium and the arising colonies were tested for the presence of ROD21 by PCR. The lines denominated 1 and 2 represent the expected PCR products that would be generated if ROD21 was inserted in the chromosome. (B) Detection of ROD21 by PCR analysis in WT S. Enteritidis and in one of the ΔROD21 strains isolated in a contraselection assay. This figure shows a representative agarose gel (1%) resolving the PCR products 1 and 2 (which denote each boundary of the integrated form of ROD21) obtained for WT and ΔROD21 S. Enteritidis strains. In addition, the attB sequences generated after type 1 (attB-1) and type 2 (attB-2) excisions were also detected by PCR. As a positive control, the rpoD gene was amplified by PCR. Data shown derive from one representative S. Enteritidis ΔROD21 strain selected out of 6 strains recovered in two independent experiments in which the attB-2 sequence was detected. (C) C57BL/6 mice were orally infected with 1×106 CFUs of either WT or ΔROD21 S. Enteritidis strains and the survival rate was measured daily. Uninfected mice were included as controls. Data shown are averages of two independent experiments, each including at least 4 mice per group. (D and E) Competitive infection assays, consisting of C57BL/6 mice infected either orally or intravenously with a mixture of the WT (KnR) and ΔROD21 (CmR) S. Enteritidis strains (input ratio equal to 1∶1). After 72 h, bacteria were recovered from spleens and livers of infected mice and the ratio of WT (KnR) over ΔROD21 (CmR) S. Enteritidis was calculated and compared to the input. Data shown in graphs are average values from two independent experiments for bacteria recovered from spleens and livers after intragastric gavage (D) or intravenous (E) infections of 3 mice per group.
Mentions: Then, we evaluated whether the excision of ROD21 results in the loss of this pathogenicity island from the bacterial genome. To evaluate this possibility, we inserted the genes tetA and tetR downstream the gene SEN1975 in the strain of S. Enteritidis Phagotype 1 (PT1), to generate the ROD21::tetRA strain (Fig. 4A). The genes tetA and tetR confer resistance to tetracycline, but also prevent the growth of tetracycline-resistant bacteria in a medium containing zinc chloride and fusaric acid (BM medium), as described previously [21]. Therefore, only those Salmonella strains sensitive to tetracycline will grow in BM medium [21]. Nested PCR reactions showed that excision type 1 and type 2 (Fig. 2A) occurred in the modified ROD21::tetRA strain, as efficiently as observed for the wild type (WT) strain (data not shown). To evaluate if these excisions caused ROD21 loss, the ROD21::tetRA strain was grown in LB medium and then seeded on solid BM medium, as described in materials and methods. Bacteria were incubated 36 h at 37°C to select for tetracycline sensitive bacteria. A total of 35 tetracycline sensitive colonies out of 137 seeded plates were tested by PCR to evaluate ROD21 loss. We observed that only 6 out of the 35 colonies isolated had lost ROD21 from the chromosome, indicating that the frequency of ROD21 loss is 4.38×10−8. Further, PCR reactions showed that only the attB sequence generated by excision type 2 could be detected in the genome of all isolated S. Enteritidis strains (Fig. 4B), suggesting that excision type 2 was responsible of ROD21 loss in all isolated CFUs. With regard to the other 29 colonies that lost tetracycline resistance without excising ROD21, it is possible that they might had undergone other type of mutations resulting in tetracycline sensitivity, such as mutations in tetA or tetR genes [22], [23] or changes in cell membrane permeability. However, further research would be required to clarify this issue.

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