Extracellular DNA degradation by pneumococcus EndA is independent of the competence regulon and cell autolysis.

<p>(A) The <i>rpsL</i> PCR products (30 µg/ml) were exposed to the wild-type D39 and isogenic mutants for the indicated time intervals, or in the presence or absence of ATA or 2% choline chloride. The integrity of DNA was visualized by agarose gel electrophoresis. (B) Competence-deficient pneumococcal mutants degrade salmon sperm DNA efficiently. Pneumococcal cells were streaked onto the THB agar supplemented with salmon sperm DNA in the presence or absence of ATA or choline chloride. After 24 hr, DNA degradation was visualized by flooding the plates with HCl. Three independent experiments were performed for both A and B with similar results. The data from one typical experiment are shown.</p

Competence-independent DNA degradation is caused by EndA secreted by pneumococcus into the culture medium.

<p>(A) The <i>rpsL</i> PCR products (30 µg/ml) were exposed to cell-free supernatants from D39 and its isogenic Δ<i>comD</i> and Δ<i>lytA</i> mutants collected during growth at the indicated time intervals. The integrity of DNA was visualized by agarose gel electrophoresis. (B) Bacterial cell lysates and cell-free supernatants from D39, Δ<i>comD</i> and Δ<i>lytA</i> cultured in THB were subjected to SDS-PAGE in a gel incorporated with 15 µg/ml of salmon sperm DNA. After renaturation, the gel was stained with ethidium bromide to visualize bands of DNA clearance by EndA. The experiments were performed independently three times. The results from one typical experiment are shown.</p

Pneumococcal strains cultured in CTM have impaired ability to degrade DNA.

<p>Pneumococcal strains D39, R6, 0100993, Tigr4 and their isogenic derivatives were grown in CTM to 10⁸/ml concentration, washed and resuspended in fresh THB or CTM. The <i>rpsL</i> PCR products (30 µg/ml) were exposed to the pneumococcal cells for the indicated time intervals. The integrity of DNA was visualized by agarose gel electrophoresis. The experiments were repeated independently three times. The results from one typical experiment are shown.</p

Pneumococcal strains grown in the CTM secrete lower amounts of EndA when compared to the same bacterial strains grown in the THB.

<p>EndA activity in the culture supernatant of D39 was determined and compared between pneumococcus growing in THB and CTM by in-gel digestion assay (A), and by quantitative DNA degradation assay in D39 cultured supernatant serially diluted with fresh THB (B). The experiments were performed independently in triplicates and repeated three times. The means ± SD of one representative experiment are shown.</p

EndA-mediated competence-independent DNA degradation is conserved in multiple pneumococcal strains and is growth medium-dependent.

<p>(A) Pneumococcal strains D39, R6, 0100993, Tigr4 and their isogenic derivatives were cultured in THB to 10⁸/ml concentration, washed and resuspended in fresh THB or CTM. The <i>rpsL</i> PCR products (30 µg/ml) were exposed to the pneumococcal strains for the indicated time intervals. The integrity of DNA was visualized by agarose gel electrophoresis. The experiments were repeated independently three times. The results from one typical experiment are shown. (B) Growth kinetics of D39 in THB versus CTM. The experiments were performed independently in triplicates and repeated three times. The means ± SD of one representative experiment are shown.</p

Nucleolytic activity of EndA during competence induction by CSP1.

<p>PCR-amplified P-32 labeled donor DNA (5 μ-3) was exposed to Δ<i>comA</i> cells in the presence or absence of 400 ng/ml CSP1. The Δ<i>endA</i> cells and THB were used as controls. Experiments were performed in triplicates. (A) Scanned image of small hot DNA fragments (<100 bp) and nucleotides spotted on filter paper and exposed to a phosphoimager cassette. (B) Quantification of dots (n = 3) in A. *<i>p</i><0.05 when comparing the densitometry number of <i>ΔcomA</i> supplemented with CSP1 against <i>ΔcomA</i> alone at 30 min and 90 min post CSP1 exposure.</p

Induction of competence with CSP1 does not appreciably increase EndA-mediated DNA degradation.

<p>(A–B) Induction of both <i>comX</i> expression (A) and genetic transformation (B) in the JC0923 cells grown in THB or CTM with or without exposure to 400 ng/ml CSP1. Genetic transformation was performed with the addition of 30 ug/ml <i>rpsL</i> PCR products. Experiments were performed in triplicates and repeated three times. The means ± SD of one typical experiment are shown. (C) Integrity of the <i>rpsL</i> DNA exposed to THB-grown JC0923 cells in the presence or absence of CSP1. (D) Measurement of dsDNA integrity (from C) with ethidium bromide (excitation: 300 nm; emission: 600 nm). Experiments were performed in triplicates and repeated three times. The means ± SD of one typical experiment are shown.</p

EGFR inhibitor and antioxidant GSH prevent the inhibition of PCN-mediated activation of AKT and ERK1/2 signaling.

<p>A549 cells were exposed to the EGFR inhibitor AG1478 (<b>A–B</b>) or GSH (<b>C–D</b>) for 90 min before the addition of PCN (12.5 µg/ml) for 12 hr. Control cells were exposed to same volume of sterile H₂O. Cytoplasmic proteins were probed with anti-phospho–specific antibodies. (<b>A–B</b>) The phosphorylation of AKT and ERK1/2 in A549 cells treated with AG1478 and PCN. (<b>C–D</b>) The phosphorylation of AKT and ERK1/2 in A549 cells treated with GSH and PCN. Right panels represent densitometry analysis of western blots. The experiments were repeated three times with similar results. Densitometry data represent the mean ± SD from all three experiments. <i>*p</i><0.05 when cells that were exposed to PCN were compared to control cells exposed to the same volume of sterile water. **<i>p</i><0.05 when cells that were exposed to AG1478 or GSH were compared to cells exposed to PCN alone.</p

<i>Pseudomonas aeruginosa</i> Pyocyanin Activates NRF2-ARE-Mediated Transcriptional Response via the ROS-EGFR-PI3K-AKT/MEK-ERK MAP Kinase Signaling in Pulmonary Epithelial Cells

<div><p>The redox-active pyocyanin (PCN) secreted by the respiratory pathogen <i>Pseudomonas aeruginosa</i> generates reactive oxygen species (ROS) and causes oxidative stress to pulmonary epithelial cells. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) confers protection against ROS-mediated cell death by inducing the expression of detoxifying enzymes and proteins via its binding to the cis-acting antioxidant response element (ARE). However, a clear relationship between NRF2 and PCN-mediated oxidative stress has not been established experimentally. In this study, we investigated the induction of NRF2-ARE response by PCN in the pulmonary epithelial cells. We analyzed the effect of PCN on NRF2 expression and nuclear translocation in cultured human airway epithelial cells, and in a mouse model of chronic PCN exposure. NRF2-dependent transcription of antioxidative enzymes was also assessed. Furthermore, we used inhibitors to examine the involvement of EGFR and its downstream signaling components that mediate NRF2-ARE-activation in response to PCN. PCN enhances the nuclear NRF2 accumulation and activates the transcription of ARE-mediated antioxidant genes. Furthermore, PCN activates NRF2 by inducing the EGFR-phosphoinositide-3-kinase (PI3K) signaling pathway and its main downstream effectors, AKT and MEK1/2-ERK1/2 MAP kinases. Inhibition of the EGFR-PI3K signaling markedly attenuates PCN-stimulated NRF2 accumulation in the nucleus. We demonstrate for the first time that PCN-mediated oxidative stress activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway, leading to nuclear NRF2 translocation and ARE responsiveness in pulmonary epithelial cells.</p></div

Nuclear translocation of NRF2 is increased in PCN-mediated goblet cell hyperplasia in mouse airways.

<p>Mouse lungs (groups of 12) were exposed to sterile water control (<b>A</b> and <b>C</b>) or PCN (<b>B</b> and <b>D</b>). (<b>A–B</b>) Lung sections were PAS stained for mucins (red color) and photographed with a light microscope. (<b>C–D</b>) Lung sections were stained with an anti-NRF2 primary antibody and visualized with a goat anti-Rabbit DyLight® 488-conjugated secondary antibody. Nuclei were stained with DAPI. Images were photographed using confocal fluorescence microscope. (<b>E</b>) Quantification of PAS-positive cells in control versus PCN-exposed airways. (<b>F</b>) Quantification of goblet cells with nuclear NRF2 in control versus PCN-exposed airways.</p