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

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

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

Additional file 3: of Mapping genetic determinants of host susceptibility to Pseudomonas aeruginosa lung infection in mice

Effect of the Pairl1 locus on survival in A/J, C3H/HeOuJ and heterozygotes. (DOCX 106 kb

Additional file 5: of Mapping genetic determinants of host susceptibility to Pseudomonas aeruginosa lung infection in mice

List of the most promising candidates genes for susceptibility to acute P. aeruginosa pulmonary infection. (DOCX 27 kb

Genotypic and phenotypic characteristic of <i>P. aeruginosa</i> sequential isolates from CF patients.

<p>Three clonal lineages (AA, KK and MF) of <i>P. aeruginosa</i> strains were isolated at the onset of chronic colonization (early: AA2, KK1, KK2, MF1) or several years after acquisition and before patient's death (late: AA43, AA44, KK71, KK72, MF51). Clonality of strains was assessed by Pulsed Field Gel Electrophoresis and was reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648-Bragonzi1" target="_blank">[4]</a>. Multiple phenotypic traits changed during genetic adaptation to the CF lung and included <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648-Bragonzi2" target="_blank">[14]</a>: (a) motility defect, (b) mucoid phenotype, (c) protease reduction, (d) siderophore reduction, (e) hemolysis reduction, (f) LasR phenotype, (g) growth rate reduction. In addition, lipopolysaccharide (LPS) lipid A (h) and peptidoglycan (PGN) muropeptides (i) were analysed exclusively in the lineage AA showing specific structural modifications temporally associated with CF lung infection as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648-Cigana1" target="_blank">[5]</a>. Additional data were reported in the online data supplement (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648.s002" target="_blank">Table S1</a></b>).</p

Lung inflammatory response in susceptible A/J and resistant C3H/HeOuJ <i>P</i>. <i>aeruginosa</i>-infected mice.

<p>The number of neutrophils (<b>A</b>), macrophages (<b>B</b>), lymphocytes (<b>C</b>) and epithelial cells (<b>D</b>) recruited in the airways were determined in the BALF from A/J (n = 12) (blue bar) and C3H/HeOuJ (n = 12) (green bar) mice by cytospin after 6, 12 and 18 hours of <i>P. aeruginosa</i> infection with 5×10⁶ CFU of AA2 clinical isolate. Bars represent mean values and the error bars the standard error of the mean (SEM). The data are pooled from two independent experiments. Statistical significance by Mann-Whitney U test is indicated: *p<0.05, **p<0.01, ***p<0.001.</p

Correlation between survival percent and initial infection dose of clonal pair of early/late <i>P. aeruginosa</i> isolates in C57Bl/6NCrl.

<p>C57Bl/6NCrl mice were infected with different doses of <i>P. aeruginosa</i> strains from AA (<b>A</b>) and KK (<b>B</b>) clonal lineages. Survival of infected mice was followed over a period of 4 days and is indicated as a cumulative percent. Higher doses of late <i>P. aeruginosa</i> strains (AA43, AA44, KK71, KK72) are required for mortality when compared to early strains (AA2, KK1 and KK2). Two to three independent experiments were pooled (nr of mice: 5–18 as detailed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648.s003" target="_blank">table S2</a></b>). Statistical analysis of pair wise comparisons for early and late strains are indicated *** p<0.001 (Mantel-Cox test).</p

C57Bl/6NCrl and BALB/cAnNCrl inbred mouse strains exhibit a similar susceptibility after infection with clonal pair of early/late <i>P. aeruginosa</i> isolates.

<p>C57Bl/6NCrl (<b>A, B</b>) and BALB/cAnNCrl (<b>C, D</b>) mice were infected with 5×10⁶ cfu/lung of <i>P. aeruginosa</i> strains from AA (<b>A, C</b>) and 1×10⁷ cfu/lung KK (<b>B, D</b>) clonal lineages. Survival of infected mice was followed over a period of 4 days. Early strains (AA2, KK1 and KK2) were lethal while late strains (AA43, AA44, KK71, KK72) were attenuated in acute virulence. Two to three independent experiments were pooled (nr of mice: 5–18 as detailed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648.s004" target="_blank">table S3</a></b>). Statistical analysis was calculated for pair wise comparisons between early and late strains (* p<0.05; ** p<0.01; *** p<0.001, Mantel-Cox test).</p