Physiological effects caused by microcystin-producing and non-microcystin producing Microcystis aeruginosa on medaka fish: A proteomic and metabolomic study on liver

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Metabolic changes in Medaka fish induced by cyanobacterial exposures in mesocosms: an integrative approach combining proteomic and metabolomic analyses

Author Correction: The original version of this Article contained an error in the spelling of the author Giovanni Chiappetta, which was incorrectly given as Giovanni Chiapetta. This error has now been corrected in the HTML and PDF versions of the Article, and in the accompanying Supplementary Information document. https://doi.org/10.1038/s41598-018-20638-0International audienceCyanobacterial blooms pose serious threats to aquatic organisms and strongly impact the functioning of aquatic ecosystems. Due to their ability to produce a wide range of potentially bioactive secondary metabolites, so called cyanotoxins, cyanobacteria have been extensively studied in the past decades. Proteomic and metabolomic analyses provide a unique opportunity to evaluate the global response of hundreds of proteins and metabolites at a glance. In this study, we provide the first combined utilization of these methods targeted to identify the response of fish to bloom-forming cyanobacteria. Medaka fish (Oryzias latipes) were exposed for 96 hours either to a MC-producing or to a non-MC-producing strain of Microcystis aeruginosa and cellular, proteome and metabolome changes following exposure to cyanobacteria were characterized in the fish livers. The results suggest that a short-term exposure to cyanobacteria, producing or not MCs, induces sex-dependent molecular changes in medaka fish, without causing any cellular alterations. Globally, molecular entities involved in stress response, lipid metabolism and developmental processes exhibit the most contrasted changes following a cyanobacterial exposure. Moreover, it appears that proteomic and metabolomic analyses are useful tools to verify previous information and to additionally bring new horizons concerning molecular effects of cyanobacteria on fish

Author Correction: Metabolic changes in Medaka fish induced by cyanobacterial exposures in mesocosms: an integrative approach combining proteomic and metabolomic analyses

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PAO1 biofilm phenotypes as affected by DMSO, naringin, naringenin or OALC.

<p>(A) Fluorescence microscopy images of PAO1 cells incubated statically at 37°C for 24 hours. Cells were visualized after staining with SYTO-9 (green fluorescence for living bacteria) and propidium iodide (red fluorescence for dead bacteria) furnished in the LIVE/DEAD <i>Bac</i>Light kit. (B) Fluorescence microscopic images of biofilm formation by PAO1 cells incubated for 24 hours and then treated for 24 hours with DMSO, naringin, naringenin or OALC. Fluorescence microscopy was achieved by using a Leica DM IRE2 inverted fluorescence microscope using a 40x objective lens and images were false-colored and assembled using Adobe Photoshop.</p

Deep sexual dimorphism in adult medaka fish liver highlighted by multi-omic approach

International audienceSexual dimorphism describes the features that discriminate between the two sexes at various biological levels. Especially, during the reproductive phase, the liver is one of the most sexually dimorphic organs, because of different metabolic demands between the two sexes. The liver is a key organ that plays fundamental roles in various physiological processes, including digestion, energetic metabolism, xenobiotic detoxification, biosynthesis of serum proteins, and also in endocrine or immune response. The sex-dimorphism of the liver is particularly obvious in oviparous animals, as the female liver is the main organ for the synthesis of oocyte constituents. In this work, we are interested in identifying molecular sexual dimorphism in the liver of adult medaka fish and their sex-variation in response to hepatotoxic exposures. By developing an integrative approach combining histology and different high-throughput omic investigations (metabolomics, proteomics and transcriptomics), we were able to globally depict the strong sexual dimorphism that concerns various cellular and molecular processes of hepatocytes comprising protein synthesis, amino acid, lipid and polysaccharide metabolism, along with steroidogenesis and detoxification. The results of this work imply noticeable repercussions on the biology of oviparous organisms environmentally exposed to chemical or toxin issues

Synergistic activity of OALC with tobramycin against biofilm-encapsulated <i>P</i>. <i>aeruginosa</i> PAO1.

<p>PAO1 cells were incubated statically for 24 hours and then treated for 24 hours with tobramycin (100 μg mL^-1) and DMSO, naringin (4 mM), naringenin (4 mM) or OALC (200 μM final concentration). (A) OALC + tobramycin, (B) tobramycin, (C) naringenin + tobramycin, (D) DMSO + tobramycin, (E) naringin + tobramycin. Assessment of bacterial viability and microscopy were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132791#pone.0132791.g006" target="_blank">Fig 6</a>. (F) Quantification of bacterial viability. Error bars represent the standard errors of the means; all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO (Student’s <i>t</i>-tests; <i>P</i> ≤ 0.01).</p

PAO1 biofilm phenotypes as affected by DMSO, naringin, naringenin or OALC.

<p>(A) Fluorescence microscopy images of PAO1 cells incubated statically at 37°C for 24 hours. Cells were visualized after staining with SYTO-9 (green fluorescence for living bacteria) and propidium iodide (red fluorescence for dead bacteria) furnished in the LIVE/DEAD <i>Bac</i>Light kit. (B) Fluorescence microscopic images of biofilm formation by PAO1 cells incubated for 24 hours and then treated for 24 hours with DMSO, naringin, naringenin or OALC. Fluorescence microscopy was achieved by using a Leica DM IRE2 inverted fluorescence microscope using a 40x objective lens and images were false-colored and assembled using Adobe Photoshop.</p

Effect of OALC on virulence factors and acylhomoserine lactones production in <i>P</i>. <i>aeruginosa</i> PAO1.

<p>(A) rhamnolipids production, (B) pyocyanin production, (C) Elastase production. The cell density of the bacteria was assessed at 600 nm and elastase production was assessed via an elastolysis assay and calculated as the ratio between <i>A</i>₄₉₅ and <i>A</i>₆₀₀. The rhamnolipids production was measured using methylene-blue-based method and expressed in μg mL^-1. Pyocyanin was extracted, quantified by absorbance measurements at 380 nm and calculated as the ratio between <i>A</i>₃₈₀ and <i>A</i>₆₀₀. (D) Quantification of <i>N</i>-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL; grey bar) and <i>N</i>-butanoyl-L-homoserine lactone (C4-HSL; clear bar) produced by PAO1 cells. Acylhomoserine lactones were extracted and quantified by mass spectrometry. Error bars represent the standard errors of the means and all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO controls (Student’s <i>t</i>-tests; <i>P</i> ≤ 0.01). Naringenin (Nar) and naringin (Nin) were used as a quorum sensing inhibitor control and negative control, respectively.</p

Chemical structure of the bioactive compound assigned as 3β-hydroxyolean-12-en-28-al 3-<i>p</i>-coumarate (oleanolic aldehyde coumarate, OALC).

<p>Chemical structure of the bioactive compound assigned as 3β-hydroxyolean-12-en-28-al 3-<i>p</i>-coumarate (oleanolic aldehyde coumarate, OALC).</p

Effect of OALC on QS genes (<i>lasI/R and rhlI/R</i>) and global activator genes (<i>gacA</i> and <i>vfr</i>) expression in <i>P</i>. <i>aeruginosa</i> PAO1.

<p>(A) Effect of OALC on <i>lasR</i> (grey bar) and <i>lasI</i> (clear bar) expression following 18 hours of growth. (B) Effect of OALC on <i>rhlR</i> (grey bar) and <i>rhlI</i> (clear bar) expression following 18 hours of growth. (C) Effect of OALC on <i>gacA</i> (grey bar) and <i>vfr</i> (clear bar) expression following 18 hours of growth. Gene expression was measured as the β-galactosidase activity of the <i>lacZ</i> gene fusions and expressed in Miller units. Error bars represent the standard errors of the means; all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO (Student’s <i>t</i>-tests; <i>P</i> ≤ 0.01).</p