Chemical defence by mono-prenyl hydroquinone in a freshwater ciliate, Spirostomum ambiguum
Several species of ciliates produce and accumulate low molecular weight toxic compounds in specialised membrane-bound ejectable organelles: extrusomes. These compounds can be used in predator–prey interaction for killing prey as well as for chemical defence. Here, we describe the isolation and characterisation of 2-(3-methylbut-2-enyl)benzene-1,4-diol(mono-prenyl hydroquinone), the extrusomal defensive toxin of the freshwater heterotrich ciliate Spirostomum ambiguum. The toxin was purified at homogeneity by RP-HPLC, and its structural characterisation was carried out through NMR and MS measurements. In vivo experiments involving S. ambiguum and Climacostomum virens in predator–prey interaction, and the analysis of cytotoxic activity of mono-prenyl hydroquinone on a panel of free-living freshwater ciliates, indicated that the toxin is very effective in S. ambiguum’s chemical defence.
KeywordsPrenyl hydroquinone Extrusomes Chemical defence Secondary metabolites Natural compounds
Spirostomum ambiguum is a large, elongated, colourless freshwater ciliate (800–2,000 × 48–60 μm) belonging to the order Heterotrichida. The species is very common in the sludge-water contact zone of wells, ponds, sewage ponds, lakes, oxbows, ditches, and in the sediments of alpha- to beta-mesosaprobien rivers (Foissner et al., 1992). In the past 30 years a number of studies have been devoted to analysing the morphological and molecular basis of predator–prey interaction in heterotrich ciliates, and particular attention was focused on the important role of specialised ejectable membrane-bound organelles, generally called extrusomes, in the immobilization and capture of prey, and in defence from predators (Hausmann, 2002; Rosati & Modeo, 2003; Dettner, 2010). Offensive extrusomes exclusively involved in catching and killing the prey (toxicysts or related organelles) have been extensively studied in carnivorous ciliates, amongst which Didinium, Dileptus, Lacrymaria, Homalozoon and Litonotus can be cited as typical representatives. On the other hand, ciliates such as Blepharisma, Stentor, Climacostomum and Spirostomum display different kinds of extrusomes (called cortical granules, or pigment granules when they contain pigments), which have been reported to be primarily, but not exclusively, involved in chemical defence against predators (Harumoto et al., 1998; Miyake et al., 2001; Sera et al., 2006).
Pigment granules of B. japonicum contain five different red pigments, called blepharismins, recently chemically synthesised and structurally related to two well known photodynamic and toxic pigments: hypericin, extracted from the plant Hypericum, and stentorin, extracted from the ciliate S. coeruleus (Cameron & Riches, 1995; Dai et al., 1995; Yoshioka et al., 2008). To date, the three main functions that have been ascribed to blepharismins are photoreception, chemical defence against predators and protection against UV radiation (Giese, 1981; Miyake et al., 1990; Harumoto et al., 1998). In addition to physiological functions, blepharismins were also demonstrated to exert an antibiotic effect against the Gram-positive bacterium Staphylococcus aureus (Pant et al., 1997). Similar to blepharismins, the blue pigment stentorin was also structurally characterised and synthesised, and shown to be able to mediate light-induced response and chemical defence against raptorial ciliates (Iio et al., 1995; Miyake et al., 2001). Instead, differently from coloured and photodynamic pigments, the colourless toxins climacostol and spirostomin, extracted from cortical granules of Climacostomumvirens and Spirostomum teres, respectively, appeared to be exclusively related to predator–prey interaction (Miyake et al., 2003; Masaki et al., 2004; Sera et al., 2006). With particular regard to climacostol, it should be noted that this toxin was reported to act both as a defensive molecule against predators, as well as an offensive molecule that can paralyse prey before ingestion (Sugibayashi & Harumoto, 2000; Miyake et al., 2003; Terazima & Harumoto, 2004). To date, the structures of climacostol and spirostomin have been described, and both molecules have also been chemically synthesised (Abe & Mori, 2001; Masaki et al., 2004; Sera et al., 2006; Fiorini et al., 2010).
The aims of this study were (a) to isolate, purify, and characterise the toxic compound used by the freshwater ciliate S. ambiguum for chemical defence, and (b) to evaluate the role of the purified toxic compound in the defence strategy of S. ambiguum against predators, by performing an array of cytotoxicity tests on a panel of free-living ciliated protozoa.
Materials and methods
Cell cultures and preparation of extrusome-deficient cells
Spirostomum ambiguum stock Pol-5 (collected in Policoro (MT), Italy) was cultured in bacterised medium as described in Buonanno & Ortenzi (2010). Climacostomum virens stock W-24, kindly provided by Dr. Tavrovskaya (Institute of Cytology, Russian Academy of Sciences, St. Petersburg), Blepharisma japonicum stock R1072 (Harumoto et al., 1998), Coleps hirtus (collected in Genga (AN), Italy), and Euplotes aediculatus (Colorado strain), kindly provided by Dr. J. Kloetzel (University of Baltimore, Maryland, USA) were cultured in a balanced salt solution called SMB (Miyake, 1981) and fed with the flagellate Chlorogonium elongatum grown as described by Buonanno et al. (2005). Paramecium bursaria (collected in Macerata, Italy), Paramecium tetraurelia stock 51 (Miyake et al., 2003) and Spirostomum teres stock Pol-1 (Buonanno, 2005) were cultured in bacterised culture medium. All ciliates were cultured and handled at room temperature (23°C ± 1) and used in experiments 1 day after they had consumed the food.
Extrusome-deficient cells of S. ambiguum were obtained according to a protocol described in Buonanno (2011) and based on a cold-shock induction of extrusome discharge. Briefly, a dense suspension of ciliates (~30,000/ml) were quickly mixed in a 1:5 ratio with ice-cooled SMB, at 0°C for 1 min, and then centrifuged at about 50×g to separate the cells from the supernatant. Precipitated cells were washed twice, resuspended in SMB at room temperature for 2 h, and then used in experiments. The extrusome-deficient cells obtained by this procedure were healthy as control cells (Miyake et al., 2003; Buonanno, 2011). The supernatant containing the discharge from the extrusomes (here termed as ‘toxin-enriched supernatant’) was used immediately or dried by vacuum centrifuge and stored at −20°C until use.
Toxin extraction and purification
Cells of S. ambiguum were dipped in aqueous 75% ethanol for 24 h at 4°C. The extract, including cell debris, was centrifuged at 4,000×g for 10 min, and the clear supernatant was recovered and dried by vacuum centrifuge. The dry ethanol extract, or the toxin-enriched supernatant recovered by cold-shock extraction, was resuspended in 60% methanol, loaded by multiple injections on an Ascentis RP-C18 semi-preparative column (Supelco, Bellefonte, PA, USA, 10 × 250 mm, 5 μm), operated on a YL9100 high-pressure liquid chromatography system from Young Lin Instrument (Hogye-dong, Anyang, Korea). The column was equilibrated in water, and eluted using a linear gradient of methanol (0–100% in 100 min) at a flow rate of 2 ml/min with UV detection at 254 and 280 nm. Fractions were collected every 1 min, dried by vacuum centrifuge, and stored at −20°C until use. The identification of fractions corresponding to peaks of interest was performed on the basis of toxicity assay results (see below). Approximately, 100 μg of pure toxin were obtained from 3.8 to 4 mg of dry ethanol extract of 1:1 of cell suspension (6,000 cells/ml), and 7–10 μg of toxin from 100 ml of cell suspension (6,000 cells/ml) subjected to cold-shock.
All solvents used for toxin purification were of analytical grade (Merck, Darmstadt, Germany) or HPLC grade (Riedel deHaen-Sigma Chemical Co, St. Louis, MO, USA). The Chromabond C18 preparative column used for flash chromatography was obtained from Merck (Darmstadt, Germany). HPLC/DAD measurements were performed using a high-pressure liquid chromatography system (Hewlett-Packard model HP1100 series, Walderbron, Germany). The photodiode array (PDA) detector (Agilent 1100 series) was set at the wavelengths of 215 and 280 nm. LC/ESI-MS analysis was carried out on a reverse phase column (Agilent Zorbax Eclipse XDB-C18 4.6 × 150 mm, 3.5 μm) with CH3CN/H2O 7:3, 0.9 ml/min, split UV/MS 7/3, λ 280 nm, 5 μl injected, mounted on a Hewlett-Packard HP1100 HPLC-UV Diode Array system mated with an Esquire® LC Bruker-Daltonics ion trap mass spectrometer. Mass spectra were obtained with an ESI source in negative-ion mode. MS conditions: source temperature 300°C, nebulising gas N2 4 l/min, positive-ion mode, ISV 4 kV, OV 38.3 V, scan range 100–300 Da. To analyse the mass and UV, the following software was used: DataAnalysis (Version 3.0, Bruker Daltonik GmbH) and LC/MSD ChemStation (Agilent Technologies), respectively. NMR spectra were recorded on a Bruker-Avance 400 MHz NMR spectrometer using a 5 mm BBI probe with 90° proton pulse length of 9.4 μs at a transmission power of 0 db. Chemical shifts (δ) are recorded in ppm with CD3OD (δH 3.31 ppm and δC 49.0 ppm) as internal standards with multiplicity (s singlet, d doublet, t triplet and m multiplet).
Predator–prey experimental design and cytotoxicity tests
Untreated or extrusome-deficient cells of S. ambiguum (50 specimens) were mixed with 10 cells of C. virens in 250 μl of SMB in a depression slide, and the outcomes of encounters between the two species were observed under a stereomicroscope equipped with a DV3000 camera (BEL Engineering, Italy). The effects of the interaction were analysed in 40 independent encounters for both treated and untreated S. ambiguum.
To quantitatively analyse the predator–prey interactions between C. virens and extrusome-deficient cells of S. ambiguum, 10 cells of C. virens were mixed with 5, 10, 20 and 40 cells of either untreated or extrusome-deficient cells of S. ambiguum in 250 μl of SMB. These mixtures (here termed 10C-5S, 10C-10S, 10C-20S and 10C-40S, respectively) were made in nine replicates, for both extrusome-deficient and untreated cells of S. ambiguum, and were observed under a stereomicroscope after 24 h. Untreated cells were handled in the same way as the extrusome-deficient cells, except that they were mixed with SMB at room temperature instead of SMB at 0°C. The data represent the mean ± standard error (SE) of nine independent determinations, and the significance of the differences between the mean values was examined by Student’s t test with the minimum level of significance set at P < 0.05.
To evaluate cytotoxicity of the purified compound, fractions collected by HPLC were resuspended in 200 μl of SMB containing 70% ethanol and then diluted in 300 μl of SMB. Aliquots (10 μl) of each fraction, or SMB containing 70% ethanol as control, were added to samples of C. virens cells (10 specimens in 240 μl of SMB) and then assayed for cytotoxicity. The cytotoxicity was evaluated as the percentage of surviving cells after 24 h of incubation in a dark moist chamber at room temperature.
For dose–response experiments, triplicate samples of 10 ciliate cells were placed in depression slides containing 250 μl SMB and increasing concentrations of toxin (from 0.1 to 2.0 μg/ml). The number of surviving cells was counted, after 24 h, and the median lethal concentrations (LC50) were estimated on the basis of a concentration–survival curve, essentially according to the procedure described by Buonanno (2009). The LC50 was evaluated by nonlinear regression analysis using GraphPad Prism 4 software (GraphPad Software, San Diego, CA). Data are presented as the mean ± SE of three independent determinations with the minimum level of significance set at P < 0.001.
Isolation, purification and structural characterisation of the toxin of S. ambiguum
In a previous study, it was observed that when the predatory microturbellarian Stenostomum sphagnetorum tries to attack a specimen of S. ambiguum it is forced to regurgitate the captured prey, suggesting that a chemical defence is adopted by the ciliate temporarily trapped in the predator’s pharynx (Buonanno, 2011).
To investigate if the defence mechanism in S. ambiguum is mediated by toxins possibly secreted by extrusomes, similar to other ciliates that actively avoid predation, we primarily focused our attention on the isolation of the toxic compound produced by the ciliate.
Two different experimental approaches were used to isolate the toxic compound from cells of S. ambiguum. In one of these, we induced the discharge of ciliate extrusomes by cold-shock, and subjected the toxin-enriched supernatant to RP-HPLC fractionation. The identification of the peaks of interest was performed by assaying the cytotoxicity of every fraction against the predatory ciliate C. virens.
For LC–MS and NMR analyses, the dry ethanol extract was partitioned between ethyl acetate and water. The organic extract (~4 mg) was initially fractionated by flash chromatography by using an acetonitrile/water gradient elution. The fraction containing UV absorbing compounds (by thin layer chromatography analysis), or the toxic compound obtained by semi-preparative HPLC, was then subjected to analytical HPLC by using an acetonitrile/water isocratic elution to afford a pure compound which was later analysed by a combination of spectroscopic methods (1D and 2D NMR, MS) and comparison with the literature data (Howard et al., 1979; Yang et al., 2007).
The mass spectrum of this compound as obtained by negative-ion mode electrospray ionization [ESI(−)] showed a pseudo molecular ion [M−H]− at m/z 177 and UV absorption at 290 nm. A characteristic 1H-NMR spectrum of three aromatic protons, two doublets at δH 6.57 (J = 8.5 Hz) and 6.52 (J = 3.0 Hz), and a double doublet at δH 6.43 (J = 8.5 and 3.0 Hz) indicated the presence of a mono-substituted 2-hydroquinone structure. The presence of one prenyl chain and its position on the aromatic ring was established by 2D NMR and MS spectra. All spectra data were in agreement with previously published data for both natural (Howard et al., 1979) and synthetic 2-(3-methylbut-2-enyl)benzene-1,4-diol (mono-prenyl hydroquinone) (Connon & Blechert, 2003; Yang et al., 2007).
Defence strategy of Spirostomum ambiguum against predators
The defensive behaviour of S. ambiguum was investigated by comparing the predator–prey interaction involving extrusome-deficient or untreated ciliate cells with the predator C. virens.
Cytotoxicity of mono-prenyl hydroquinone
Dose–response experiments were performed to evaluate the effects of mono-prenyl hydroquinone on the viability (normal morphology and locomotion) of S. ambiguum and seven other species of freshwater free-living ciliates.
Prenylated-hydroquinone derivatives are metabolites of ubiquitous occurrence and have been isolated from fungi, algae, plants, animals and bacteria. The biosynthesis of these compounds is mixed, with the prenylated portion of 1–9 isoprene units that derives from the mevalonate pathway, and the hydroquinone moiety that derives from shikimic acid (Scheepers et al., 2006). It is known that some prenylated hydroquinones play an important role in many biological processes including cellular respiration, photosynthesis and electron transport, but anticancer, antimutagenic, antimicrobial and anti-inflammatory properties have been also described for a number of these compounds (De Rosa et al., 1994; Terencio et al., 1998; Yang et al., 2007; Baby & Sujatha, 2011). Furthermore, an ecological role was suggested for some prenylated hydroquinones, which have been reported to exert a defensive action against predators (Park et al., 1992; Sladic & Gašic, 2006).
In this study, we report the isolation, purification, structural characterization and biological activity of mono-prenyl hydroquinone produced by the ciliated protozoan S. ambiguum.
The data obtained by LC–MS and NMR analyses suggest that ciliates of the genus Spirostomum are able to synthesise species-specific metabolites deriving from different biogenetic routes; in fact, in our hands S. ambiguum has led to the isolation of mono-prenyl hydroquinone, whilst Sera et al. (2006) isolated spirostomin ((2,5-dimethyl-5,6,7,8-tetrahydronaphtalene-1,4-dione)-8,6′-(pyrane-2′,5′-dione)) from S. teres (Fig. 2). Whereas prenyl hydroquinones (and quinones), either linear or cyclic, are metabolites of mixed biogenesis, and aromatic prenyltransferases are expected to catalyse the transfer of the dimethylallyl diphosphate moiety (DMAPP, C5) to the aromatic acceptor molecule (Heide, 2009), spirostomin is thought to derive from intra-molecular cyclizations of a C14 linear polyketide backbone.
The evidence that phylogenetically close organisms can produce biogenetically distant specific secondary metabolites is not a novelty (Pietra, 2002), and should come as no surprise when the species share the same micro-environment. In fact, even in the absence of predator–prey interaction, as in the case of S. teres and S. ambiguum, competition for nutrients and space may justify the production and use of chemical deterrents by the two organisms.
Mono-prenyl hydroquinone, together with prenylquinone, was first isolated from the terrestrial plant Phagnalon saxitale (Bohlmann & Kleine, 1966). Later, the same compounds and 6-hydroxy-2,2-dimethylchromene extracted from the ascidian Aplidium californicum, were structurally characterised and synthesised (Howard et al., 1979). Interestingly, in an extensive investigation of chemical defences in ascidians, it was demonstrated that mono-prenyl hydroquinone produced by A. californicum had no effect on feeding by carnivorous coral reef fishes or invertebrates, and to date the ecological role of this compound remains unclear (Lindquist et al., 1992). In contrast, our data indicate that in S. ambiguum the same compound is effectively involved in the defence against predators. In fact, untreated cells of S. ambiguum, which are physiologically equipped with mono-prenyl hydroquinone stored in their extrusomes, appear significantly less vulnerable to the predatory ciliate C. virens than extrusome-deficient cells.
Immediately after the interaction between specimens of S. ambiguum and C. virens, predator cells start to swim backwards, suggesting that the cell–cell contact triggers the discharge of extrusomes in S. ambiguum to deter the attack of C. virens. The rapid reaction of C. virens to the chemical defence adopted by S. ambiguum can be explained considering that in most cases the discharge of extrusomes after mechanical and/or chemical stimuli takes place in milliseconds (Hausmann, 1978). Interestingly, S. ambiguum cells also show rapid contraction after an attack by C. virens. This is not surprising if we remember that, similar to S. ambiguum, C. virens carries a well-known toxin, climacostol, stored in its extrusomes which can also be used as an offensive molecule to paralyse prey before ingestion. Consequently, it is likely that the contraction observed S. ambiguum cells during an attack by C. virens is induced by the discharge of extrusomes by the predator.
The use of toxic compounds to mediate prey–predator interaction is well documented in single-celled eukaryotes. In particular, dinoflagellates and ciliates among free-living alveolates can be considered as a rich mine of low-molecular weight bioactive molecules, which in the last two decades have attracted the attention of ecologists, biologists, physiologists and chemists (Tillmann, 2004; Pucciarelli et al., 2008; Guella et al., 2010a, b).
In line with the data presented in this work, studies carried out on other ciliate toxins, such as climacostol, euplotins, blepharismins, stentorin, spirostomin, revealed that the compounds are utilised to inhibit competitors, to attack prey, or as means of defence from predators via cell–cell contact. With regard to the mechanism of action, euplotin C and climacostol have been shown to activate programmed cell death by impairing mitochondrial membrane potential and inducing ROS generation (Cervia et al., 2007; Buonanno et al., 2008; Muto et al., 2011). In the case of blepharismins, it was proposed that the induction of ion-permeable channel formation in cell membranes may play an important role in the defensive function of the toxins against predatory ciliates (Muto et al., 2001).
However, at present, the only data available for mono-prenyl hydroquinone of S. ambiguum regard cytotoxicity. Six of the eight species of free-living ciliates used in dose–response experiments turned out to be moderately to highly sensitive to the toxin, whereas it appears poorly active against B. japonicum and S. ambiguum. The low activity of mono-prenyl hydroquinone against S. ambiguum, which produces it as a defensive weapon, was expected, in line with what was observed for the toxins produced by other ciliates, i.e. blepharismins (Harumoto et al., 1998), stentorin (Miyake et al., 2001), euplotin C (Cervia et al., 2009) and climacostol (Buonanno & Ortenzi, 2010).
On the other hand, a possible explanation for the unexpected high resistance of B. japonicum (which produces blepharismins) to mono-prenyl hydroquinone may be found in the biochemical affinities between these toxins.
In general, the hydroquinones’ cytotoxicity can be correlated with their redox and electrophilic properties. Indeed, hydroquinones are known (a) to undergo enzymatic oxidative activation generating, after a complex cascade of biochemical processes, hydroxyl radicals which are thought to be responsible for most oxygen radical toxicity (Henry & Wallace, 1996), and (b) to promptly react with thiol-containing nucleophiles such as glutathione (Guilivi & Cadenas, 1994). Both the redox quinone/hydroquinone cycling and their electrophilic properties result in oxidative stress and, ultimately, in cell death. Since in blepharismins the quinone/hydroquinone redox system is embedded in the same polycyclic structure, it is expected that they can demonstrate the same biochemical properties of prenyl-hydroquinones and may neutralise the biochemical effects (ROS generation and electrophilic additions) of the latter.
To summarise, it must be considered that S. ambiguum can usually interact and share the same habitat with predators such as C. virens and C. hirtus, or competitors such as E. aediculatus, P. tetraurelia and S. teres. To improve fitness and evolutionary success it may therefore be useful to reduce both predation and competition for food resources, and at least in S. ambiguum, the adoption of a chemical shield seems to be an appropriate and effective choice.
In this study, we demonstrated that the freshwater heterotrich ciliate S. ambiguum protects itself from predators by adopting a chemical defence, which is mediated by a toxin stored into the cell extrusomes, and identified as mono-prenyl hydroquinone. In addition to its main function in reducing predation, the cytotoxic activity of mono-prenyl hydroquinone on a panel of free-living ciliates suggested that the toxin could also be used in limiting the presence of competitors for food resources.
In a general perspective, the data presented in this study not only represent an advance in the knowledge of the defence strategies in protists, but also open the way to further investigations in chemical ecology, especially considering that toxin-secreting microorganisms are attracting the attention of an increasing number of researchers due to their key role in regulating community structure in aquatic microhabitats.
Financial support was provided by the CARIMA Foundation (Fondazione Cassa di Risparmio della Provincia di Macerata) and Regione Marche. The authors are very grateful to Dr. Gill Philip for the linguistic revision of the text.
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