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

Interleukin 10 modulation of neutrophil subsets infiltrating lungs during Streptococcus pneumoniae infection

Interleukin-10 production and lung neutrophil infiltration are two essential components of the balanced immune response to pneumonia caused by Streptococcus pneumoniae. Here we describe the existence of two neutrophil subsets in lungs during experimental S. pneumoniae infection in mice, which have different size, granularity and expression of activation markers. During infection, both neutrophils subsets were increased in the lungs of IL-10 producing mice, however this increment was significantly higher in the absence of this cytokine. These results suggest that IL-10 is a key cytokine that regulates lung inflammation during bacterial infection caused by specific neutrophil subsets infiltrating the lungs. Keywords: Streptococcus pneumoniae, N1 neutrophils, N2 neutrophils, Interleukin-1

Conjugal transfer of the pathogenicity island ROD21 in Salmonella enterica serovar Enteritidis depends on environmental conditions.

Unstable pathogenicity islands are chromosomal elements that can be transferred from one bacterium to another. Salmonella enterica serovar Enteritidis (S. Enteritidis) is a pathogenic bacterium containing such unstable pathogenicity islands. One of them, denominated ROD21, is 26.5 kb in size and capable of excising from the chromosome in certain culture conditions, as well as during bacterial infection of phagocytic cells. In this study we have evaluated whether ROD21 can be effectively transferred from one bacterium to another. We generated a donor and several recipient strains of S. Enteritidis to carry out transfer assays in liquid LB medium. These assays showed that ROD21 is effectively transferred from donor to recipient strains of S. Enteritidis and S. Typhimurium. When Escherichia coli was used as the recipient strain, ROD21 transfer failed to be observed. Subsequently, we showed that a conjugative process was required for the transfer of the island and that changes in temperature and pH increased the transfer frequency between Salmonella strains. Our data indicate that ROD21 is an unstable pathogenicity island that can be transferred by conjugation in a species-specific manner between Salmonellae. Further, ROD21 transfer frequency increases in response to environmental changes, such as pH and temperature

Schematic representation of the modified genetic regions of the strains used in the <i>S.</i> Enteritidis-to-<i>S.</i> Enteritidis mating assays.

<p>(<b>A</b>) Donor strain, showing excision type 1 and 2; (<b>B</b>) recipient strain and (<b>C</b>) transconjugant strain. Numbers above of each scheme represent the coordinates in the chromosome of <i>S.</i> Enteritidis. Asparagine tRNA genes are designated as <i>asnT-1</i>, <i>-2</i> and <i>-3</i>. DRS stands for <u>D</u>irect <u>R</u>epeated <u>S</u>equence. Black arrows represent the inserted antibiotic gene (<i>aph</i> or <i>cat</i>). Thin arrows represent primers used for PCR verification of the strains: (1) left-end of ROD21 (1,011 bp), (2) DRS (914 bp), (3) right-end (903 bp), (4) insertion of <i>cat</i> gene (1,030 bp without <i>cat</i> insert, 1,943 bp with <i>cat</i> insert), and (5) insertion of <i>aph</i> gene (2,469 bp). Below each scheme, agarose gels show PCR products obtained for the recipient and transconjugant <i>S.</i> Enteritidis strain.</p

Excision of ROD21 in different conditions.

<p>Donor <i>S.</i> Enteritidis strain was grown in LB medium and (<b>A</b>) LB medium supplemented with hydrogen peroxide, (<b>B</b>) lower pH (pH 5), and (<b>C</b>) lower and higher temperatures (23°C and 43°C respectively) in order to evaluate excision of ROD21. The frequency of <i>attB1</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>attB1</i> over the copy number of <i>rpoD</i> gene. Error bars represent standard deviation from the mean (N = 3). ***, P<0.001, **, P = 0.016 (one-way ANOVA and Tukey's post-test).</p

Transfer of ROD21 to <i>S.</i> Typhimurium.

<p>(<b>A</b>) Scheme of the genome of the <i>S.</i> Typhimurium recipient strain with <i>cat</i> gene inserted in the putAP region (black arrow) and double-resistant strain with <i>aph</i> gene inserted in ROD21 and <i>cat</i> gene inserted in the putAP region (black arrows). Numbers above each scheme represent the coordinates in the chromosome of <i>S.</i> Typhimurium. Asparagine tRNA genes are designated as <i>asnT-1</i>, <i>-2</i> and <i>-3</i>. DRS stands for <u>D</u>irect <u>R</u>epeated <u>S</u>equence. Thin arrows represent primers used for PCR verification of the strains: (1) left-end of ROD21 (1,011 bp), (2) DRS (914 bp), (3) right-end of ROD21 (903 bp), (4) insertion of <i>cat</i> gene (1,838 bp), (5) insertion of <i>aph</i> gene (2,469 bp), and (6) left-end of Gifsy-1 (1,110 bp). Below the scheme, agarose gels show PCR products obtained for the recipient and transconjugant <i>S.</i> Typhimurium strains. (<b>B</b>) Schematic representation showing the location of a 50 bp region present in <i>S.</i> Enteritidis, downstream DRS, but absent in <i>S.</i> Typhimurium. The figure below shows the alignments of the theoretical sequence of the above mentioned region for <i>S.</i> Enteritidis PT4 and <i>S.</i> Enteritidis 14028 and the experimental sequences obtained for <i>S.</i> Enteritidis PT1, <i>S.</i> Typhimurium 14028 and 7 transconjugants of <i>S.</i> Typhimurium-ROD21 obtained in a representative transfer assay. (<b>C</b>) Survival of C57BL/6 mice intragastrically infected with 1×10⁵ CFU of <i>S.</i> Typhimurium wild type and <i>S.</i> Typhimurium-ROD21 strain. Data shown are the averages of three independent experiments, which included 4 mice per group. ***, P<0.05 Kaplan-Meier and Mantel-Cox post test.</p

ROD21 transfer requires excision.

<p>Transfer frequency of ROD21 from donor <i>S.</i> Enteritidis, from <i>S.</i> Enteritidis ΔDRSΔSEN1970, from <i>S.</i> Enteritidis ΔSEN1980 and from <i>S.</i> Enteritidis/pBAD-SEN1998 supplemented with arabinose to (<b>A</b>) recipient <i>S.</i> Enteritidis and (<b>B</b>) recipient <i>S.</i> Typhimurium. Graph shows the frequency of ROD21 transfer, expressed as the ratio between transconjugant bacteria and the total amount of donors calculated in each assay. Results are the average of four independent experiments (Y axis are expressed as reverse of log values). ***, P<0.001, Student's <i>t</i> test. ND = Non-detected.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Transfer of ROD21 to <i>S.</i> Enteritidis and <i>S.</i> Typhimurium in different environmental conditions.

<p>Strains were subjected to transfer assays in LB medium and (<b>A</b>) LB medium supplemented with hydrogen peroxide, (<b>B</b>) pH 7 and pH 5, and (<b>C</b>) 23°C and 43°C. Error bars represent standard deviation from the mean (N = 3). NS = non-significant, Student t-Test (Two tailed). ***, p<0.001 one-way ANOVA and Tukey post-test.</p

Primers and probes used in this study.

<p>The location of primers pairs from 1 to 6 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090626#pone-0090626-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090626#pone-0090626-g002" target="_blank">2</a>. Lowercase indicate the region that hybridizes to the 5′ or 3′ end of the pKD3 or pKD4 plasmids.</p