Anti-<i>Pseudomonas</i> antibody production by mice infected with PAO1 or PAO1Δ<i>lasIRrhlIR</i>.

<p>Mice were infected intratracheally with 1×10³ CFU of PAO1 or PAO1Δ<i>lasIRrhlIR</i> in an 0.5% agar slurry, and bled on day 22. Sera were tested in ELISA against antigen prepared from whole killed PAO1, using isotype-specific detection antibodies for mouse IgM, IgG1, and IgG2a. Results are expressed as individual titers with a line representing the median values. There were no significant differences (Mann–Whitney <i>U</i> test) between mice infected with PAO1 or PAO1Δ<i>lasIRrhlIR</i> (QSneg).</p

Primers used in this study.

<p>Primers used in this study.</p

Phenotypic characteristics of QS mutants.

<p>Elastase production (a), protease production (b) pyocyanin production (c) twitching motility (d) rhamnolipid production (e) of QS mutants compared with parental PAO1. Rhamnolipid production only tested for <i>ΔrhlIR</i> mutant. All results represented as means ± SD of at least triplicate determinations. Differences between groups assessed with unpaired <i>t-</i>test; **<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Production of C4HSL and 3OC12HSL by QS mutants compared with PAO1.

<p>The capacity of all mutants to produce C4HSL (a) and 3OC12HSL (b) is presented. Because of interassay variability, all results are presented as percent of production by wild type PAO1. Results for 100 µM C4HSL (a) or 3OC12HSL (b) included as positive controls. Values represent mean ± SD of at least triplicate determinations. LB: LB broth (negative control). ND: not detected.</p

Expression of cytokine mRNA in lung tissue of mice 4 h after infection with PAO1 or PAO1Δ<i>lasIRrhlIR</i>.

<p>Lung tissue from mice infected with 1×10⁶ CFU of either PAO1 (hatched bars) or PAO1Δ<i>lasIRrhlI</i> (QSneg, open bars) was processed, total RNA extracted and cDNA prepared. Expression of cytokine genes was assessed in RT-qPCR using β-actin as the reference gene. All results are expressed as gene expression relative to β-actin, and are presented as the mean ± SD of five mice per group. A control group of lungs from mice that underwent anaesthesia and intratracheal infusion of PBS without bacteria was included (grey bars). Expression of all cytokines was significantly higher in mice infected with bacteria than in sham-infected mice (<i>P</i><0.01, all groups, ANOVA) but there were no significant differences between mice infected with PAO1 or PAO1Δ<i>lasIRrhlIR</i> (QSneg).</p

Leukocyte infiltrate in bronchoalveolar lavage (BAL) fluid from lungs of mice infected with PAO1 or PAO1Δ<i>lasIRrhlIR</i>.

<p>BAL fluid was collected from mice at 4 h, 24 h and 10 d after intratracheal infection with 1×10⁶ CFU of either PAO1 (hatched bars) or PAO1Δ<i>lasIRrhlIR</i> (QSneg, open bars). An aliquot of fluid from each mouse was centrifuged in a cytospin, and slides stained with DiffQuik for differential counting. A minimum of 200 cells per slide were counted. Results expressed as mean ± SD for individual BAL fluids from five mice per group at each time point. Differences between groups analysed with an unpaired <i>t</i> test. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p