Additional file 2: Table S2. of Host genetic diversity influences the severity of Pseudomonas aeruginosa pneumonia in the Collaborative Cross mice

Change in body weight of each CC line after P. aeruginosa airway. (DOCX 51 kb

Additional file 3: Table S3. of Host genetic diversity influences the severity of Pseudomonas aeruginosa pneumonia in the Collaborative Cross mice

Bonferroni’s Multiple Comparison Tests of recorded traits (MST and CBW1) among CC lines after P. aeruginosa airway infection. (DOCX 74 kb

Additional file 1: Table S1. of Host genetic diversity influences the severity of Pseudomonas aeruginosa pneumonia in the Collaborative Cross mice

Survival Time of each CC lines after P. aeruginosa airway infection. (DOCX 51 kb

Effect of QS dysregulation caused by <i>qteE</i> mutation on pyocyanin production in <i>P. aeruginosa</i>.

<p>Levels of pyocyanin measured in cell-free supernatants from cultures of the indicated strains grown till A₆₀₀≈3.5. C, no plasmid; EV, pBBR1MCS-5 empty vector; pQteE, pBBR1MCS-5 derivative plasmid for <i>qteE</i> expression.</p

Effect of QS dysregulation caused by <i>qteE</i> mutation on <i>P. aeruginosa</i> virulence-related phenotypes.

<p>Levels of (A) 3OC₁₂-HSL, (B) elastase, (C) proteases produced along growth by <i>P. aeruginosa</i> wild type (black lines), <i>qteE</i> (red lines) and <i>rsaL</i> strains (green line) carrying the pBBR1MCS-5 empty vector, or by the <i>qteE</i> strain carrying the pQteE plasmid (pBBR1MCS-5-derived) for the expression of <i>qteE</i> (dashed red line). Values are the means (± standard deviations) of at least three independent experiments.</p

Schematic representation of QteE- and RsaL-dependent regulation of the <i>P. aeruginosa las</i> QS system.

<p>In the pre-quorum period, QteE binds to the LasR receptor and prevents the binding of the LasR-3OC₁₂-HSL complex to the <i>rsaL</i>-<i>lasI</i> bidirectional promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112105#pone.0112105-Siehnel1" target="_blank">[20]</a>, hence delaying the onset of the QS response. Once the quorum has been reached, the LasR/3OC₁₂-HSL complex triggers the transcription of both <i>rsaL</i> and <i>lasI</i> genes. The consequent increase of 3OC₁₂-HSL levels, and thus of activated LasR, generates a positive feedback loop also responsible for the increase of RsaL levels. RsaL binding to the <i>rsaL</i>-<i>lasI</i> bidirectional promoter represses the expression of both <i>rsaL</i> and <i>lasI</i> genes, thus counteracting the positive feedback loop. This circuit provides 3OC₁₂-HSL homeostasis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112105#pone.0112105-Rampioni3" target="_blank">[24]</a>. Solid arrows represent positive control; T-shaped lines represent negative control; dashed arrows indicate information flow; curved arrows represent the transcription start points of the indicated genes.</p

Effect of QS dysregulation caused by <i>qteE</i> and <i>rsaL</i> mutations on <i>P. aeruginosa</i> pathogenesis in mice.

<p>C57Bl/6 mice were infected with the indicated strains embedded in agar beads. (A) Mice mortality induced by bacteremia (light grey) and survival (white) were evaluated on challenged mice. (B) Clearance (white) and capacity to establish chronic airways infection (dark grey) were determined on surviving mice after 14 days from challenge. The results are averages of three independent experiments. Statistical significance is indicated by an asterisk comparing <i>P. aeruginosa</i> wild type versus <i>qteE</i> or <i>rsaL</i> strains (<i>p</i><0.05).</p

The PAPI-1 pathogenicity island-encoded small RNA PesA influences <i>Pseudomonas aeruginosa</i> virulence and modulates pyocin S3 production

<div><p>Small non-coding RNAs (sRNAs) are post-transcriptional regulators of gene expression that have been recognized as key contributors to bacterial virulence and pathogenic mechanisms. In this study, we characterized the sRNA PesA of the opportunistic human pathogen <i>Pseudomonas aeruginosa</i>. We show that PesA, which is transcribed within the pathogenicity island PAPI-1 of <i>P</i>. <i>aeruginosa</i> strain PA14, contributes to <i>P</i>. <i>aeruginosa</i> PA14 virulence. In fact, <i>pesA</i> gene deletion resulted in a less pathogenic strain, showing higher survival of cystic fibrosis human bronchial epithelial cells after infection. Moreover, we show that PesA influences positively the expression of pyocin S3 whose genetic <i>locus</i> comprises two structural genes, <i>pyoS3A</i> and <i>pyoS3I</i>, encoding the killing S3A and the immunity S3I proteins, respectively. Interestingly, the deletion of <i>pesA</i> gene results in increased sensitivity to UV irradiation and to the fluoroquinolone antibiotic ciprofloxacin. The degree of UV sensitivity displayed by the PA14 strain lacking PesA is comparable to that of a strain deleted for <i>pyoS3A</i>-<i>I</i>. These results suggest an involvement of pyocin S3 in DNA damage repair and a regulatory role of PesA on this function.</p></div

PesA positively regulates the expression of both the <i>pyoS3A</i> and <i>pyoS3I</i> translational fusions in PA14.

<p>Comparison of the sfGFP and mCherry activities expressed in arbitrary units (AU) resulting from the translational fusion of (A) <i>lacZ</i>::<i>pyos3A-I</i>::<i>sfGFP</i>, (B) <i>pyoS3I</i>::<i>sfGFP</i> and (C) <i>Cherry</i>::<i>pyoS3A-I</i>::<i>sfGFP</i> combined with the control vector (pGM931) or the plasmid overexpressing PesA (pGM-<i>pesA</i>), in PA14 wild-type and PA14 Δ<i>pesA</i>. The strains were grown to an OD₆₀₀ of 1.8 in LB medium supplemented with gentamicin and carbenicillin, to maintain pBBR1- and pGM- plasmids, respectively, and arabinose, to induce PesA overexpression. Cells were harvested and treated for sfGFP and mCherry activity determination by measuring fluorescence polarization FP_485/535 and fluorescence intensity FI_590/635, respectively. sfGFP and mCherry activities are expressed as ratio FP_485/535/Abs₅₉₅ and FI_590/635/Abs₅₉₅, respectively. Data derive from three independent experiments. Values represent the mean ± SD. Statistical significance by Student’s t-Test is indicated: *p<0.05; **<i>p</i>< 0.01.</p

PesA expression is induced by temperature and low availability of oxygen.

<p>Levels of PesA RNA in: A) Wild-type PA14 grown in BHI at 20°C (lanes 1 and 4), 37°C (lanes 3 and 6) or following 20 min of acclimation (AC) from 20 to 37°C (lanes 2 and 5). Culture samples were taken at middle (OD₆₀₀ of 0.8) and late (OD₆₀₀ of 1.8) exponential growth phase. B) Wild-type PA14 grown in BHI anaerobically (NO₃⁻; lanes 1 and 2), aerobically (O₂, lane 6) and aerobically until an OD₆₀₀ of 0.8 and then shifted to anaerobic conditions (O₂ → NO₃⁻; lanes 3, 4 and 5). Samples were taken 20 and 150 min after the shift to anaerobic conditions (t₂₀ and t₁₅₀). After sampling, cell cultures were processed for total RNA extraction and analysis by Northern blot. Intensities of the bands of PesA were quantified and normalized to those of 5S RNA in the same lane. Values are expressed as arbitrary units (AU) in the histograms below each Northern blot and represent the mean ± Standard Deviation (SD) of three independent experiments.</p