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 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

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

<i>PesA</i> deletion enhances sensitivity to ciprofloxacin.

<p>Antibiotic disk diffusion was performed on LB-agar plates spread with 10⁶ CFU bacterial cells of wild-type PA14 and <i>ΔpesA</i> mutant strains. The diameters of the inhibitory zones were measured after overnight incubation at 37°C. Data derive from three independent experiments. Values represent the mean ± SD. Statistical significance by Student’s t-Test is indicated: **<i>p</i>< 0.01.</p

Interaction of PesA with <i>pyoS3A-I</i> mRNA.

<p>A) Prediction by <i>TargetRNA</i> software of the base-pairing interactions between PesA and <i>pyoS3A-I</i> mRNA. B) <i>In vitro</i> interaction between PesA RNA and <i>pyoS3A-I</i> mRNA by an electrophoretic mobility shift assay. Increasing amounts of PesA RNA (0, 0.08, 0.15, and 0.25 pmol; lanes 1–4) or, as a negative control, <i>E</i>. <i>coli</i> RseX RNA (0.25 and 2.5 pmol; lanes 5 and 6) were incubated at 37°C for 20 min with 0.15 pmol radiolabeled <i>pyoS3A-I</i> mRNA and loaded onto a native 6% polyacrylamide gel.</p

PesA gene dissemination and expression levels among environmental, CF and COPD clinical isolates.

<p>Assays on environmental, CF and COPD isolates are shown in three panels, A, B and C, respectively. The strain-collection was plated on BHI-agar plates. After overnight growth at 37°C, culture samples were taken and processed for total RNA extraction and analysis by Northern blot, and for genomic DNA extraction. Positive or negative PCR-amplification outcomes are indicated as “+” or “-” in the “<i>pesA</i> gene amplification” row, below each Northern Blot. PAO1 and PA14 were used as controls of Northern Blot analysis and for negative or positive gene amplification, respectively.</p

PesA deletion enhances UV sensitivity similarly to <i>pyoS3</i> operon deletion.

<p>3 μl of cultures of PA14 <i>wt</i>, Δ<i>pesA and</i> Δ<i>pyoS3</i>, serially diluted 10-fold, were spotted onto LB-agar plates, and treated with UV light at the indicated doses. Surviving CFUs were observed after overnight incubation at 37°C.</p

Time course of cell viability of IB3-1 cells following bacterial infection with <i>P</i>. <i>aeruginosa</i> PA14 wild-type and Δ<i>pesA</i>.

<p>Cell viability, assessed as a reduction of MTT salt, was quantified by the optical density (OD) at 490 nm. IB3-1 cells were seeded at a density of 5 × 10⁴ cells/well into 96-well microplates, and infected with 5 × 10⁶ bacterial cells (MOI 1:100). At every time point, data are shown as the difference in OD₄₉₀ between the PA14 wild-type strain and the sRNA-deleted mutant Δ<i>pesA</i>. Uninfected cells were used as positive control of cell viability. Data derive from three independent experiments. Results are shown as the difference in the OD₄₉₀ reached at the different time points by IB3-1 cells infected by the mutant strain or non-infected, subtracted of the OD₄₉₀ reached by IB3-cells infected with the wild-type strain. Values represent the mean ± standard error of the mean (SEM). Statistical significance between wild-type and Δ<i>pesA</i> strains by Student’s t-Test is indicated: *<i>p</i>< 0.05.</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

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