10 research outputs found

    MvfR-regulon inhibitors rescue PA14-macrophage cytotoxicity.

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    <p>PA14-induced killing of Raw264.7 macrophage cells was determined minus and plus 100 µM inhibitor. Error bars represent mean +/− SEM of at least 3 replicates. Differences between PA14 + vehicle and the samples PA14 + M64, PA14 + M62, PA14 + M59, PA14 + M51, PA14 + M50, or PA14 + M27 are statistically significant (<i>p</i><0.01, one way ANOVA, Dunnett's test). Differences between MvfR and MvfR + M64 (<i>p</i>>0.05) or vehicle and M64 (<i>p</i>>0.05) are not statistically significant (unpaired t test). Notably, M64 does not alter cytotoxicity of <i>mvfR</i> cells, and is itself non-cytotoxic.</p

    The most potent inhibitors reduce 2-AA production and the formation of antibiotic tolerant persisters.

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    <p><b>a.</b> 2-AA levels in presence of 10 µM inhibitor. Error bars show mean +/− SD of at least 2 replicates. <b>b.</b> Observed fold change in persister cell concentrations of PA14 cultures with 10 µM inhibitor or with 0.75 mM 2-AA. Untreated PA14 cells and <i>mvfR-</i> cells were the positive and negative controls, respectively. Error bars show mean +/− SEM of at least 3 replicates. Differences between PA14 and the samples M34, M50, M62, M59, M51, M64 or mvfR- (<i>p</i><0.01) as well as between PA14 and the samples PA14 + 2-AA or M29 (<i>p</i><0.01) are statistically significant (one way ANOVA, Dunnett's test). <b>c.</b> Observed fold change in persister cell concentrations of PA14 plus 5 µM M64 in the presence of clinical antibiotics used to treat <i>P. aeruginosa</i> infections: amikacin (blue), levofloxacin (purple), ciprofloxacin (orange) and meropenem (red). All values were normalized to control cultures in 0.01% DMSO. Error bars show mean +/− SEM of at least 3 replicates. Differences between control and the samples amikacin, levofloxacin, ciprofloxacin or meropenem are statistically significant (<i>p</i><0.01, one way ANOVA, Dunnett's test).</p

    M64 inhibits <i>P. aeruginosa</i> persistence in the mouse burn and infection models.

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    <p>PA14 CFU quantification in muscle (<b>a</b>) underlying or (<b>b</b>) adjacent to the abdominal infection site in mice infected with PA14 and treated with ciprofloxacin (10 mg/kg), and minus (green) or plus (red) M64 (4 mg/kg). Ciprofloxacin and M64 were administered by intravenous injection 6 hours post-infection and then twice a day. Ciprofloxacin was administered for 4 days as described until no CFUs were detected in the muscle samples. Ciprofloxacin administration was stopped at day 4 to allow for the potential emergence and detection of antibiotic-tolerant cells. M64 was administered for 6 days, up until antibiotic-tolerant cells were detected in the PA14 + ciprofloxacin only group. Error bars represent mean +/− SEM of at least 3 replicates. d.l., detection limit.</p

    Chemical structures of 17 MvfR-regulon inhibitors identified by whole cell HTS, and their corresponding inhibition of HAQ and pyocyanin production.

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    <p>HHQ (dark grey bars), PQS (white bars), HQNO (light grey bars), and pyocyanin (black bars) levels were quantified plus or minus 50 µg/mL of each compound. Structures labelled in red share the common benzamide-benzimidazole core.</p

    M64 reduces pyocyanin production in <i>P. aeruginosa</i> clinical multi-drug resistant strains.

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    <p><b>a.</b> Quantitative pyocyanin production in multi-drug resistant clinical <i>P. aeruginosa</i> isolates plus (red) and minus (black) 5 µM M64. A representative image of qualitative pyocyanin production, visible as green media, in PA14 culture +/− M64, is shown above the histogram. <b>b.</b> Antibiotic resistance profile of <i>P. aeruginosa</i> clinical strains and their respective isolation sites from infected patients. Amik. = amikacin, Gent. = gentamycin, Mero. = meropenem, Pip. = piperacin, Tobra. = tobramycin, Cefe. = cefepime, Aze. = azetromycin, Cip. = ciprofloxacin. R = resistant; I = intermediate; S = sensitive.</p

    Structure and biological activity of benzamide-benzimidazole derivatives for inhibition of HAQ and pyocyanin production.

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    <p>The HTS compounds are shaded purple, the 2<sup>nd</sup> generation commercially available derivatives are shaded white, and the 2<sup>nd</sup> generation synthetic derivatives are shaded green. Alterations to the M56 benzamide-benzamidazole core structure are marked in red. HHQ, PQS, and pyocyanin (pyo.) levels were quantified in response to 10 µM compounds, and 1 µM of the most potent compounds: M34, M51, M62, M50, M59, and M64.</p

    Magnetic resonance imaging of M64 inhibition of macrophage recruitment at a burn and infection site.

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    <p><b>a–e. </b><i>In vivo</i> positive contrast imaging of mice infected with PA14, plus and minus M64. The off resonance imaging transverse relaxation in the rotating frame (ORI-T2ρ) images were transformed to signal to noise ratio (SNR) images and thresholded in units of image standard deviation. <b>a., b.</b> The positive-contrast images are presented in pseudocolor, thresholded to signal greater than three in dimensionless SNR units, and superimposed on a FLASH image. For image processing, regions of interest (ROI) were drawn around the burn region and the total thresholded signal intensity was integrated within each ROI. Similar slices were chosen at the same anatomical location in all mice. <b>c., d.</b> 3-dimensional graphs of pixel intensities show an intense peak in the burn area for the PA14 control mouse, with this peak reduced by M64. <b>e.</b> Signal was measured in units of SNR, thresholded at three standard deviations, and measured within ROIs at the level of the burn and infection. The noise threshold was estimated by fitting the image background to a Rician distribution. Error bars depict standard error of the mean image intensity in the ROI. Error bars depict mean +/− SD of at least 3 replicates. Difference between PA14 and PA14 + M64 is statistically significant (<i>p</i><0.05, unpaired t test).</p

    κ-Opioid Receptor Agonists Activate Virulence of P. aeruginosa against Probiotic Bacteria and C. elegans

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    <div><p>Error bars, mean ± SD.</p><p>(A and B) The exposure of P. aeruginosa PAO1 to U-50,488, 200 μM, increases the inhibiting effect of its extracellular milieu (conditioned media) on the growth of probiotic microorganisms (A) L. plantarum and (B) L. rhamnosus GG.</p><p>(C and D) The exposure of P. aeruginosa PAO1 to dynorphin, 100 μM, increases the inhibiting effect of its extracellular milieu (conditioned media) on the growth of probiotic microorganisms (C) L. plantarum and (D) L. rhamnosus GG.</p><p>(E) The extracellular milieu of P. aeruginosa PAO1 mutant ΔMvfR exposed to dynorphin, 100 μM, did not inhibit the growth of probiotic microorganism L. rhamnosus GG.</p><p>(F and G) P. aeruginosa PAO1 but not mutant ΔMvfR exposed to (F) U-50,488, 200 μM, or (G) dynorphin, 100 μM, suppressed the production of new progeny in C. elegans.</p></div

    Dynorphin Binds to P. aeruginosa In Vitro, and Enters the Bacterial Cell Cytoplasm

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    <div><p>(A–C) Binding of dynorphin to <i>P. aeruginosa;</i> (A) negative control demonstrating no dynorphin staining when cells were not incubated with dynorphin; (B) negative control demonstrating no dynorphin staining when cell were incubated with dynorphin but primary anti-dynorphin antibodies were omitted from staining procedure; and (C) positive staining (brown color) of P. aeruginosa incubated with dynorphin followed by whole procedure of immunostaining.</p><p>(D) Immunoelectron microscopy of P. aeruginosa PAO1 cells incubated with dynorphin, 100 μM. Black arrows show 10-nm gold spots indicating the presence of dynorphin.</p></div

    Proposed Activation and Effectors Pathways of P. aeruginosa in Response to Host Stress (Intestinal I/R Injury)

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    <div><p>(1) Dynorphin is released by intestinal tissues and accumulates in the lumen during ischemia/reperfusion and penetrates the plasma membrane of P. aeruginosa (dark green arrows).</p><p>(2) Dynorphin synergizes with PQS via MvfR to increase the transcription of <i>pqsABCDE</i> leading to the production of HAQs, including HQNO and HHQ.</p><p>(3) Increased HQNO production suppresses the growth of Lactobacillius spp., rendering the intestinal epithelium more vulnerable to invasion and the action of cytotoxins of P. aeruginosa (red arrows).</p><p>(4) HHQ is the immediate precursor of PQS [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030035#ppat-0030035-b023" target="_blank">23</a>], and both compounds play an important role in bacterial cell-to-cell communication [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030035#ppat-0030035-b023" target="_blank">23</a>] (yellow and blue arrows).</p><p>(5) PQS induces the expression of <i>pqsABCDE</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030035#ppat-0030035-b063" target="_blank">63</a>], and is required for <i>phzA1-G1</i>expression<i>,</i> the gene responsible for PCN production (blue arrows). 6.) The release of PCN can induce neutrophils apoptosis and damage epithelial cells [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030035#ppat-0030035-b064" target="_blank">64</a>] (green arrows) allowing for immuno-evasion and deeper penetration of bacteria.</p></div
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