Screening for Biologically Annotated Drugs That Trigger Triacylglycerol Accumulation in the Diatom Phaeodactylum

Abstract

Microalgae are a promising feedstock for the production of triacylglycerol (TAG) for a variety of potential applications, ranging from food and human health to biofuels and green chemistry. However, obtaining high TAG yields is challenging. A phenotypic assay for the accumulation of oil droplets was developed to screen a library of 1,200 drugs, annotated with pharmacology information, to select compounds that trigger TAG accumulation in the diatom Phaeodactylum tricornutum Using this screen, we identified 34 molecules acting in a dose-dependent manner. Previously characterized targets of these compounds include cell division and cell signaling effectors, membrane receptors and transporters, and sterol metabolism. Among the five compounds possibly acting on sterol metabolism, we focused our study on ethynylestradiol, a synthetic form of estrogen that is used in contraceptive pills and known for its ecological impact as an endocrine disruptor. Ethynylestradiol impaired the production of very-long-chain polyunsaturated fatty acids, destabilized the galactolipid versus phospholipid balance, and triggered the recycling of fatty acids from membrane lipids to TAG. The P. tricornutum transcriptomic response to treatment with ethynylestradiol was consistent with the reallocation of carbon from sterols to acetyl-coenzyme A and TAG. The mode of action and catabolism of ethynylestradiol are unknown but might involve several up-regulated cytochrome P450 proteins. A fatty acid elongase, Δ6-ELO-B1, might be involved in the impairment of very-long-chain polyunsaturated fatty acids and fatty acid turnover. This phenotypic screen opens new perspectives for the exploration of novel bioactive molecules, potential target genes, and pathways controlling TAG biosynthesis. It also unraveled the sensitivity of diatoms to endocrine disruptors, highlighting an impact of anthropogenic pollution on phytoplankton.

Figure 1.

Calibration of the multiparametric assay for the phenotypic screen. The assay was developed using a strain expressing a Histone H4 protein fused to EYFP at the N terminus. Graphs show the correlations between measured fluorescence and cell concentrations after a 48-h incubation in ESAW 1N1P and 0N1P media, respectively. A, Cell abundance. The YFP fluorescence was used to evaluate cell abundance in a calibrated population grown in 1N1P medium by measuring the excitation/emission fluorescence at 515/530 nm. B, Chlorophyll fluorescence. The fluorescence of chlorophyll was used as a basic evaluation of cell physiological status by measuring the excitation/emission fluorescence at 440/680 nm. C, Nile Red fluorescence. The content in TAG was evaluated by staining with Nile Red and measuring the fluorescence at 530/580 nm. Data correspond to biological triplicates ± sd. Correlation coefficients were 0.94 or less. Solid squares represent cells grown in 1N1P medium (+ Nitrogen), and circles represent cells grown in 0N1P medium (− Nitrogen). RFU, Relative fluorescence units.

Figure 2.

Multiparametric screen of the Prestwick Chemical Library. Using the developed assay, P. tricornutum cells were incubated for 48 h with compounds of the Prestwick Chemical Library at a final concentration of 10 µm in 0.5% (v/v) DMSO. Multiple parameters were then measured as described in “Materials and Methods.” A, Cell abundance. The concentration of cells was estimated using a strain expressing a Histone H4 protein fused to EYFP based on the fluorescence at 515/530 nm. B, TAG levels. The content of TAG was evaluated by staining with Nile Red and measuring the fluorescence at 530/580 nm. C, Chlorophyll levels. The fluorescence of chlorophyll was measured at 440/680 nm.

Figure 3.

Selection of compounds based on primary and secondary screens. Based on the primary screen, two criteria were used to select compounds for the secondary screen: (1) low impact on cell abundance and/or chlorophyll level and (2) increase of Nile Red staining level. A, Primary screen, correlation between chlorophyll level and cell abundance detection. Values obtained from chlorophyll fluorescence levels and cell abundance based on YFP fluorescence were plotted. Linear correlation is given by the formula y = 0.9409x – 2.2011 (R² = 0.8189). B, Primary screen, comparison of Nile Red staining and cell abundance. The dashed line shows spots corresponding to compounds increasing Nile Red staining above 120% of the average per plate while maintaining a cell abundance above 50% of that of the control. C, Secondary screen. Parallel assays were repeated at 10 µm in technical duplicates, using 160 compounds selected following the primary screen. The dashed line shows spots corresponding to compounds increasing Nile Red staining above 120% of the average per plate while maintaining a cell abundance above 50% of that of the control. Error bars indicate sd.

Figure 4.

Dose-response analyses of secondary screen-selected compounds. Dose-response analyses were performed as described in “Materials and Methods,” in triplicate, at compound concentrations of 1.5, 3, 6, 12, 25, 50, and 100 µm. Cell abundance was evaluated based on the fluorescence of a Histone H4 protein fused to EYFP and expressed as a percentage of the untreated control (100% = YFP level at 0 µm compound). TAG accumulation was evaluated based on Nile Red staining, normalized by cell abundance, and expressed as a percentage of the untreated control (100% = Nile Red level per cell at 0 µm compound).

Figure 5.

Compounds interfering with sterol metabolism. The pathway is adapted from Fabris et al. (2014). The biosynthesis of isopentenyl pyrophosphate (Isopentenyl-PP) and farnesyl pyrophosphate (Farnesyl-PP), which is common to all isoprenoids, is initiated by the reduction of HMG-CoA into mevalonate. Epoxysqualene is the last noncyclic intermediate in the pathway. The molecular diversity of sterols is then generated downstream of cycloartenol and involves cytochrome P450 cyclooxygenases. The connection with FA and TAG biosynthesis occurs at the level of acetyl-CoA. Sterol and TAG appear as competing sinks for carbon. FA synthesis is an iterative process adding two carbons (2C) per iteration, producing FAs of 16 or 18 carbons. FAs can be elongated further in the cytosol and are used for the biosynthesis of polar and storage glycerolipids, including TAG. Inhibitors are shown in red at the level of their primary enzymatic targets. ?, Putative inhibitors in the downstream part of sterol metabolism acting on cytochrome P450 enzymes; *, the Ro48-8071 effect was described previously by Fabris et al. (2014). Fluorescence imaging of treated wild-type cells highlights chlorophyll fluorescence (440/680 nm), allowing the detection of chloroplasts (Chl) and Nile Red staining (530/580 nm) of oil droplets enriched in TAG. Bars = 5 µm.

Figure 6.

Glycerolipid profile following treatment with ethynylestradiol. P. tricornutum cells were cultivated in 1N1P medium until a cell density of 1 × 10⁶ cells mL⁻¹ was reached. Cells were then incubated for 48 h in the presence or absence of compounds, as indicated. Lipids were extracted, separated by thin-layer chromatography (TLC), and analyzed as described in “Materials and Methods.” A, Glycerolipid profiles following treatments with 30 µm ethynylestradiol and simvastatin. Glycerolipids are expressed in nmol per million cells. B, Fatty acid profiles of membrane glycerolipids following treatment with 30 µm ethynylestradiol. C, Fatty acid profile of TAG following treatment with 30 µm ethynylestradiol. Data correspond to biological triplicates ± sd. ASQ, Acylsulfoquinovosyldiacylglycerol; DAG, diacylglycerol; Ethy, ethynylestradiol; FFA, free fatty acid; PE, phosphatidylethanolamine; PI, phosphatidylinositol; Simva, simvastatin. *, P < 0.05 (Student’s t test). Error bars indicate sd.

Figure 7.

K-means clustering of P. tricornutum differentially expressed genes in response to increasing doses of ethynylestradiol. Only differentially expressed genes with a Log₂FC > 1 in at least one of the contrasts (i.e. comparing ethynylestradiol supplies at 10 versus 0 µm, 20 versus 0 µm, or 20 versus 10 µm) and with P < 0.05 (Wald test) were considered for further analyses. A partition of differentially expressed genes was performed using the k-means method, with the number of partitions set to six and clustering based on a Euclidian distance (Liu et al., 2014). Two clusters comprise genes down-regulated following treatments: group 1 (DR₁), containing genes with the strongest magnitude in expression decline (in the −2 to −4 Log₂FC range); and group 5 (DR₂), with moderate but significant expression decline (Log₂FC ∼ −1). Four clusters comprise genes up-regulated following ethynylestradiol treatments: groups 2, 3, 4, and 6 (UR₁, UR₂, UR₃, and UR₄, respectively).