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

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

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

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

Image_2_Persistent Salmonella enterica serovar Typhimurium Infection Increases the Susceptibility of Mice to Develop Intestinal Inflammation.tif

<p>Chronic intestinal inflammations are triggered by genetic and environmental components. However, it remains unclear how specific changes in the microbiota, host immunity, or pathogen exposure could promote the onset and exacerbation of these diseases. Here, we evaluated whether Salmonella enterica serovar Typhimurium (S. Typhimurium) infection increases the susceptibility to develop intestinal inflammation in mice. Two mouse models were used to evaluate the impact of S. Typhimurium infection: the chemical induction of colitis by dextran sulfate sodium (DSS) and interleukin (IL)-10^−/− mice, which develop spontaneous intestinal inflammation. We observed that S. Typhimurium infection makes DSS-treated and IL-10^−/− mice more susceptible to develop intestinal inflammation. Importantly, this increased susceptibility is associated to the ability of S. Typhimurium to persist in liver and spleen of infected mice, which depends on the virulence proteins secreted by Salmonella Pathogenicity Island 2-encoded type three secretion system (TTSS-2). Although immunization with a live attenuated vaccine resulted in a moderate reduction of the IL-10^−/− mice susceptibility to develop intestinal inflammation due to previous S. Typhimurium infection, it did not prevent bacterial persistence. Our results suggest that persistent S. Typhimurium infection may increase the susceptibility of mice to develop inflammation in the intestine, which could be associated with virulence proteins secreted by TTSS-2.</p

Table_1_Persistent Salmonella enterica serovar Typhimurium Infection Increases the Susceptibility of Mice to Develop Intestinal Inflammation.docx

<p>Chronic intestinal inflammations are triggered by genetic and environmental components. However, it remains unclear how specific changes in the microbiota, host immunity, or pathogen exposure could promote the onset and exacerbation of these diseases. Here, we evaluated whether Salmonella enterica serovar Typhimurium (S. Typhimurium) infection increases the susceptibility to develop intestinal inflammation in mice. Two mouse models were used to evaluate the impact of S. Typhimurium infection: the chemical induction of colitis by dextran sulfate sodium (DSS) and interleukin (IL)-10^−/− mice, which develop spontaneous intestinal inflammation. We observed that S. Typhimurium infection makes DSS-treated and IL-10^−/− mice more susceptible to develop intestinal inflammation. Importantly, this increased susceptibility is associated to the ability of S. Typhimurium to persist in liver and spleen of infected mice, which depends on the virulence proteins secreted by Salmonella Pathogenicity Island 2-encoded type three secretion system (TTSS-2). Although immunization with a live attenuated vaccine resulted in a moderate reduction of the IL-10^−/− mice susceptibility to develop intestinal inflammation due to previous S. Typhimurium infection, it did not prevent bacterial persistence. Our results suggest that persistent S. Typhimurium infection may increase the susceptibility of mice to develop inflammation in the intestine, which could be associated with virulence proteins secreted by TTSS-2.</p

Data_Sheet_1_Persistent Salmonella enterica serovar Typhimurium Infection Increases the Susceptibility of Mice to Develop Intestinal Inflammation.docx

<p>Chronic intestinal inflammations are triggered by genetic and environmental components. However, it remains unclear how specific changes in the microbiota, host immunity, or pathogen exposure could promote the onset and exacerbation of these diseases. Here, we evaluated whether Salmonella enterica serovar Typhimurium (S. Typhimurium) infection increases the susceptibility to develop intestinal inflammation in mice. Two mouse models were used to evaluate the impact of S. Typhimurium infection: the chemical induction of colitis by dextran sulfate sodium (DSS) and interleukin (IL)-10^−/− mice, which develop spontaneous intestinal inflammation. We observed that S. Typhimurium infection makes DSS-treated and IL-10^−/− mice more susceptible to develop intestinal inflammation. Importantly, this increased susceptibility is associated to the ability of S. Typhimurium to persist in liver and spleen of infected mice, which depends on the virulence proteins secreted by Salmonella Pathogenicity Island 2-encoded type three secretion system (TTSS-2). Although immunization with a live attenuated vaccine resulted in a moderate reduction of the IL-10^−/− mice susceptibility to develop intestinal inflammation due to previous S. Typhimurium infection, it did not prevent bacterial persistence. Our results suggest that persistent S. Typhimurium infection may increase the susceptibility of mice to develop inflammation in the intestine, which could be associated with virulence proteins secreted by TTSS-2.</p