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

ROD21 excision frequency increases when <i>S.</i> Enteritidis infects phagocytic cells.

<p>Bone marrow-derived DCs (<b>A</b>) and J774.3 macrophages (<b>B</b>) were infected with <i>S.</i> Enteritidis (MOI equal to 25). After 2, 18 and 24 h post infection (hpi) intracellular bacteria were recovered and the copy number of the <i>attB</i> 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 <i>attB-1</i> sequence determined for intracellular and extracellular bacteria. The DNA amount was normalized by calculating the copy number of the <i>rpoD</i> 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 (<b>C</b>) and J774.3 (<b>D</b>) 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.</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

Schematic representation of ROD21 in the chromosome of <i>S.</i> Enteritidis.

<p>(<b>A</b>) Representation of the genetic location of the genes coding for the asparagine tRNA (<i>asnT-1, -2 and -3</i>) in the chromosome of <i>S.</i> Typhimurium and <i>S.</i> Enteritidis and the exact location of ROD21 in the chromosome of <i>S.</i> Enteritidis. Black and dark gray arrows represent those genes shared between both serovars and light gray arrows represent genes found only in ROD21 of <i>S.</i> Enteritidis. Numbers next to each scheme are coordinates in the chromosome of <i>S.</i> Typhimurium and <i>S.</i> Enteritidis. DRS stand for <u>D</u>irect <u>R</u>epeated <u>S</u>equence (<i>attR</i>). (<b>B</b>) The alignments of DRS and <i>asnT-1</i>, <i>asnT-2</i> (<i>attL</i>) and <i>asnT-3</i> show that the DRS is identical to the last 22 bp of the <i>asnT</i> genes.</p

Schematic representation of excisions type 1 and type 2 of ROD21 and the respective episomal elements generated.

<p>(<b>A</b>) Schematic representation of ROD21 and its surrounding region in the chromosome of the <i>S.</i> Enteritidis NCTC13349 strain. Light gray arrows indicate genes that are part of ROD21, black arrows indicate neighboring genes located outside ROD21 and dark gray arrows show neighboring genes specifically contained between the DRS limiting ROD21 and the <i>asnT-3</i> gene. Portions of the chromosome involved in type 1 and 2 excisions are shown by connecting the respective recombining DRS/tRNAs (dotted lines). Numbered arrows indicate the regions where the primers used in this study hybridize. (<b>B</b>) Schematic representation of the <i>attB-1</i> and <i>attP-1</i> sites formed after type 1 excision and the genes remaining in both the chromosome of <i>S.</i> Enteritidis and the episomal element. (<b>C</b>) Schematic representation of the <i>attB-2</i> and <i>attP-2</i> sites formed after type 2 excision, and the genes remaining in both the chromosome of <i>S.</i> Enteritidis and in the episomal element. Primer pairs used to detect the chromosomal excisions and episomal elements are indicated as black arrows.</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

Open reading frames (ORFs) in ROD21 Pathogenicity island and comparison with <i>serU</i> island of the UPEC CFT073.

<p>Open reading frames (ORFs) in ROD21 Pathogenicity island and comparison with <i>serU</i> island of the UPEC CFT073.</p