Excision of an Unstable Pathogenicity Island in Salmonella enterica Serovar Enteritidis Is Induced during Infection of Phagocytic Cells

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

Deletion of a prophage-like element causes attenuation of Salmonella enterica serovar Enteritidis and promotes protective immunity

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a wide host range serovar belonging to the S. enterica genus. Worldwide, it is one of the most frequent causes of food borne disease. Similar to S. Typhimurium, some virulence genes of S. Enteritidis are located in pathogenicity islands and prophages. In this study we have generated a mutant strain of S. Enteritidis lacking a prophage-like element, denominated φSE12. The resulting mutant strain was attenuated and promoted protective immunity in infected mice. Although S. Enteritidis strains lacking the complete prophage φSE12 remained capable of surviving inside phagocytic cells, they showed a significantly reduced capacity to colonize internal organs and failed to cause lethal disease in mice. Consistent with these data, infection with S. Enteritidis strains lacking prophage φSE12 promoted the production of anti-. Salmonella IgG antibodies and led to protection against a challenge with virulent strains of S. Enteritidis. The

Exposure to peroxide induces the excision of ROD21 from the <i>S.</i> Enteritidis chromosome.

<p><i>S.</i> Enteritidis strain PT1 was grown in LB medium and then approximately 3×10⁹ 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. (<b>A</b>) The frequency of <i>attB-1</i> excision was quantified by qPCR using genomic DNA and was expressed as a relative value equal to the ratio between the copy number of <i>attB-1</i> over the copy number of <i>rpoD</i> gene. (<b>B</b>) <i>SEN1970</i> expression was determined by qPCR using cDNA and expressed as a relative value (the ratio between the copy number of <i>SEN1970</i> and the copy number of <i>rpoD</i>). The results are the average of three independent experiments. **; <0.01, one-way ANOVA and Tukey post test.</p

ROD21 excision can be generated by means of two different recombination events.

<p>Amplification of <i>attB</i> and <i>attP</i> sequences generated after type 1 and type 2 excisions were detected by nested PCR in LK5, PT4, PT1 and PT21 strains of <i>S.</i> Enteritidis, using primer pairs described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026031#pone-0026031-t002" target="_blank">Table 2</a> and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026031#pone-0026031-g002" target="_blank">Figure 2</a>. PCR products for <i>attB</i> (<b>A</b>) and <i>attP</i> (<b>B</b>) sequences for each type of excision were resolved in 1% agarose gels. The sequence of each PCR product was obtained (chromatograms in each Figure) and compared with the <i>attB</i> and <i>attP</i> sequences deduced for type 1 and type 2 excisions (labeled as theoretical). <i>attB</i> and <i>attP</i> sequences are highlighted in red in both alignments and chromatograms. Expected size for each PCR product: 591 bp for type 1 excision <i>attB</i>, 657 bp for type 2 excision <i>attB</i>, 995 bp for type 1 excision <i>attP</i> and 1050 bp for type 2 excision <i>attP</i>.</p

Primers used in this study.

<p>*Coordinates are those of the <i>S. enterica</i> serovar Enteritidis PT4 NTCT NCTC13349 sequence.</p><p>**Italics indicate the region that anneals to the 5′ or 3′ end of a mini <i>Tn</i>10 transposon.</p