Anti-oxidants protect <i>P. aeruginosa</i> biofilms from CORM-2 inhibition.

<p>A) The addition of NAC (1 mM) or L-cysteine (100 µM) to CORM-2 (100 µM) treated <i>P. aeruginosa</i> biofilms (6 hours) restores CV staining to levels comparable to untreated biofilms. B) Treatment with CORM-2 (25 µM and 100 µM) induces bacterial ROS formation; NAC (1 mM) prevents ROS production while the addition of L-cysteine (100 µM) did alter only partially ROS levels.</p

The effect of CORM-2 on growth and biofilm formation of clinical, respiratory <i>P. aeruginosa</i> isolates.

<p>A) The addition of 100 µM CORM-2 to M9 liquid medium with glucose (left) prevents the growth of all clinical isolates compared with addition of DMSO alone (right). B) 6 hours exposure to 100 µM CORM-2 reduces the CV staining of 10/12 biofilms formed by respiratory isolates after overnight growth in plastic wells. C) 100 µM CORM-2 reduces the CV staining of 3 out of 4 biofilms formed by respiratory isolates from CF patients independently of the mucoid or non-mucoid after overnight growth in plastic wells. A similar effect was observed in mucoid-PAO1 laboratory strain PDO351 (mucA:aac+;alg+).</p

CORM-2 kills planktonic <i>P. aeruginosa</i>.

<p>The control molecule iCORM has no effect on PAO1 growth in liquid M9 medium with glucose (left panel), while CORM-2 doses >5 µM prevent growth of PAO1 (right panel).</p

CORM-2 attenuates <i>P. aeruginosa</i> biofilm formation.

<p>A) Crystal violet (CV) biomass staining decreases after CORM-2 exposure in a dose-dependent manner. Each condition was done in triplicate. B) CV staining of PAO1 biofilms treated with 100 µM CORM-2 demonstrates decreased staining of the biofilm after 2–6 hours of CORM-2 exposure compared with DMSO vehicle where the biofilm increases overtime. C) Colony counts of PAO1 released from the iCORM (100 µM) and CORM-2 (100 µM) treated biofilms demonstrate that there is a sustainable 2-log drop in bacterial viability.</p

Rich medium protect <i>P. aeruginosa</i> biofilms against CORM-2.

<p>A) Biofilm formation by <i>P. aeruginosa</i> treated with CORM-2 (100 µM) for 6 hours is similar to untreated controls when grown in LB. B) The addition of CORM-2 (100 µM) to <i>P. aeruginosa</i> growing in liquid LB medium does not result in cell death.</p

CORM-2 does not affect airway epithelial cell viability.

<p>The MTT viability assay shows that 50–200 µM of CORM2 is not toxic to airway epithelial cells after 6 hours (A) or 12 hours (B) treatment. As a control for cell death, 16HBE14o- cells were treated with saponin for 10 minutes before proceeding with the MTT staining. CORM-2 viability is expressed as percentage of non-treated control cells (DMSO).</p

CORM-2 attenuates PAO1 biofilm formation.

<p>YFP-<i>P. aeruginosa</i> were grown for 16 hours in a glass bottom dish and then visualized at selected locations for 12 additional hours using VivaView. A–D: control (CTR = no addition) biofilm formation; E-H: biofilm formation in presence of iCORM (50 µM); I–L: biofilm formation in presence of CORM2. The CORM-2 (50 µM) treated biofilm remains unchanged compared with the control biofilms that continue to mature; M–P: biofilm formation in presence of tobramycin. T indicates time; h indicates hours. Scale bars = 50 µm.</p

High doses of CORM-2 are required to reduce growth and biofilm formation of PA14.

<p>A) CORM-2 doses >100 µM (right panel) but not iCORM (left panel) prevents PA14 growth in liquid M9 medium with glucose. B) As measured by CV staining, PA14 biofilms are resistant to the low doses of CORM2 treatment (10–200 µM) that reduce PAO1 biofilm formation. C) Higher doses of CORM-2 (400 µM and 600 µM) decreases CV staining of both PA14 and PAO1 biofilm formation. Each condition was tested in triplicate.</p

CORM-2 attenuates <i>P. aeruginosa</i> colonization of respiratory epithelium.

<p>Time –lapse microscopy of bronchial epithelial cells co-cultured with PAO1 (1∶3 ratio) show that the addition of 50 µM of CORM-2 reduced microcolony formation. The time (hours) when the images were acquired is indicated at the bottom of each panel. Movies are available as supplementary online data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035499#pone.0035499.s003" target="_blank">Movies S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035499#pone.0035499.s004" target="_blank">S2</a>). Scale bars = 50 µm.</p

Bacteria integrate stimuli from the environment and decide whether to make biofilms or to move using the c-di-GMP network.

<p>A: Bow-tie architecture of c-di-GMP signaling network: c-di-GMP is synthesized by diguanylate cyclase (DGC) proteins with GGDEF domains such as WspR, DipA, and SadC, and degraded by phosphodiesterases (PDE) proteins with EAL or HD-GYP domains such as BifA, and SadR. The DGCs and PDEs could sense stimuli—such as chemoattractants which could be a signal for motility, or mechanical contact with surfaces which could be a signal for biofilm formation—and change intracellular c-di-GMP levels in response; c-di-GMP effectors—such as c-di-GMP binding proteins and riboswitch RNAs—then sense c-di-GMP levels and control phenotype outputs such as biofilm formation, motility, virulence and cell division. B: At low levels of c-di-GMP the bacteria express flagella genes and go into motile mode. C: At high levels of c-di-GMP the bacteria repress flagella genes, express biofilm genes and go into biofilm mode.</p