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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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ROD21 excision frequency increases when S. Enteritidis infects phagocytic cells.Bone marrow-derived DCs (A) and J774.3 macrophages (B) were infected with S. Enteritidis (MOI equal to 25). After 2, 18 and 24 h post infection (hpi) intracellular bacteria were recovered and the copy number of the attB sequence generated by type 1 excision was detected by quantitative PCR, using as template the genomic DNA obtained from intracellular bacteria. Frequency of excision is expressed as the ratio between the copy number of the attB-1 sequence determined for intracellular and extracellular bacteria. The DNA amount was normalized by calculating the copy number of the rpoD gene. Data shown in graphs are average values of at least three independent experiments. The amount of intracellular bacteria recovered after 2, 18 and 24 hpi from DCs (C) and J774.3 (D) was determined by lysing either 1,000 DCs or 100 J774.3 cells with PBS-triton X100 (0.1%) and seeding the lysates in LB plates. Data shown are the average of at least 3 independent experiments.
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pone-0026031-g005: ROD21 excision frequency increases when S. Enteritidis infects phagocytic cells.Bone marrow-derived DCs (A) and J774.3 macrophages (B) were infected with S. Enteritidis (MOI equal to 25). After 2, 18 and 24 h post infection (hpi) intracellular bacteria were recovered and the copy number of the attB sequence generated by type 1 excision was detected by quantitative PCR, using as template the genomic DNA obtained from intracellular bacteria. Frequency of excision is expressed as the ratio between the copy number of the attB-1 sequence determined for intracellular and extracellular bacteria. The DNA amount was normalized by calculating the copy number of the rpoD gene. Data shown in graphs are average values of at least three independent experiments. The amount of intracellular bacteria recovered after 2, 18 and 24 hpi from DCs (C) and J774.3 (D) was determined by lysing either 1,000 DCs or 100 J774.3 cells with PBS-triton X100 (0.1%) and seeding the lysates in LB plates. Data shown are the average of at least 3 independent experiments.

Mentions: Excision type 1 of ROD21 was determined for extracellular bacteria, which were recovered from the supernatant of phagocytic cells 2 h after infection. Further, ROD21 excision was also determined for intracellular bacteria recovered from phagocytic cells at different times post-infection (2, 18 and 24 h). Then, the ratios of the ROD21 excision between intracellular bacteria and extracellular bacteria were determined (relative value in figure 5). As shown in Fig. 5A, excision rates in intracellular bacteria were increased at all time points, especially after 18 h of DCs infection (Fig. 5A) and after 2 h of macrophages infection (Fig. 5B). Similar results were obtained when the ratio of ROD21 excision between intracellular bacteria and bacteria grown in either LB or cell culture media was determined (data not shown). Finally, because similar intracellular bacterial loads were observed at all time points during infection of phagocytic cells (Fig. 5C and 5D), it is unlikely that our results could be due to variability in the amount of bacteria recovered after infection. These findings suggest that the excision of ROD21 might be induced by the environmental conditions found by S. Enteritidis inside phagocytic cells during infection.


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 excision frequency increases when S. Enteritidis infects phagocytic cells.Bone marrow-derived DCs (A) and J774.3 macrophages (B) were infected with S. Enteritidis (MOI equal to 25). After 2, 18 and 24 h post infection (hpi) intracellular bacteria were recovered and the copy number of the attB sequence generated by type 1 excision was detected by quantitative PCR, using as template the genomic DNA obtained from intracellular bacteria. Frequency of excision is expressed as the ratio between the copy number of the attB-1 sequence determined for intracellular and extracellular bacteria. The DNA amount was normalized by calculating the copy number of the rpoD gene. Data shown in graphs are average values of at least three independent experiments. The amount of intracellular bacteria recovered after 2, 18 and 24 hpi from DCs (C) and J774.3 (D) was determined by lysing either 1,000 DCs or 100 J774.3 cells with PBS-triton X100 (0.1%) and seeding the lysates in LB plates. Data shown are the average of at least 3 independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198454&req=5

pone-0026031-g005: ROD21 excision frequency increases when S. Enteritidis infects phagocytic cells.Bone marrow-derived DCs (A) and J774.3 macrophages (B) were infected with S. Enteritidis (MOI equal to 25). After 2, 18 and 24 h post infection (hpi) intracellular bacteria were recovered and the copy number of the attB sequence generated by type 1 excision was detected by quantitative PCR, using as template the genomic DNA obtained from intracellular bacteria. Frequency of excision is expressed as the ratio between the copy number of the attB-1 sequence determined for intracellular and extracellular bacteria. The DNA amount was normalized by calculating the copy number of the rpoD gene. Data shown in graphs are average values of at least three independent experiments. The amount of intracellular bacteria recovered after 2, 18 and 24 hpi from DCs (C) and J774.3 (D) was determined by lysing either 1,000 DCs or 100 J774.3 cells with PBS-triton X100 (0.1%) and seeding the lysates in LB plates. Data shown are the average of at least 3 independent experiments.
Mentions: Excision type 1 of ROD21 was determined for extracellular bacteria, which were recovered from the supernatant of phagocytic cells 2 h after infection. Further, ROD21 excision was also determined for intracellular bacteria recovered from phagocytic cells at different times post-infection (2, 18 and 24 h). Then, the ratios of the ROD21 excision between intracellular bacteria and extracellular bacteria were determined (relative value in figure 5). As shown in Fig. 5A, excision rates in intracellular bacteria were increased at all time points, especially after 18 h of DCs infection (Fig. 5A) and after 2 h of macrophages infection (Fig. 5B). Similar results were obtained when the ratio of ROD21 excision between intracellular bacteria and bacteria grown in either LB or cell culture media was determined (data not shown). Finally, because similar intracellular bacterial loads were observed at all time points during infection of phagocytic cells (Fig. 5C and 5D), it is unlikely that our results could be due to variability in the amount of bacteria recovered after infection. These findings suggest that the excision of ROD21 might be induced by the environmental conditions found by S. Enteritidis inside phagocytic cells during infection.

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