Protists in science communication.

Despite their high abundance and wide distribution in ecosystems, most protists remain unknown to the public. Although science communication approaches were developed in historical times to raise public awareness of these 'enigmatic' taxa, many aspects have not been considered in the spotlight of modern techniques. We present selected ideas and activities on how to attract the public to unicellular eukaryotes. We give examples of how protists can be included in educational work. We explain that trained non-experts can understand and teach others how to recognize protists, where they live, in which habitats they can be found, what they look like and why they are important. Consequently, members of the public can learn how environmental threats impact not only the lives of protists but also ours, e.g., by the accumulation of microplastics through an aquatic food web, up to fish used for human consumption. We suggest age-appropriate methods for application in workshops on protist recognition.

Exposure to the Natural Compound Climacostol Induces Cell Damage and Oxidative Stress in the Fruit Fly Drosophila melanogaster.

The ciliate Climacostomum virens produces the metabolite climacostol that displays antimicrobial activity and cytotoxicity on human and rodent tumor cells. Given its potential as a backbone in pharmacological studies, we used the fruit fly Drosophila melanogaster to evaluate how the xenobiotic climacostol affects biological systems in vivo at the organismal level. Food administration with climacostol demonstrated its harmful role during larvae developmental stages but not pupation. The midgut of eclosed larvae showed apoptosis and increased generation of reactive oxygen species (ROS), thus demonstrating gastrointestinal toxicity. Climacostol did not affect enteroendocrine cell proliferation, suggesting moderate damage that does not initiate the repairing program. The fact that climacostol increased brain ROS and inhibited the proliferation of neural cells revealed a systemic (neurotoxic) role of this harmful substance. In this line, we found lower expression of relevant antioxidant enzymes in the larvae and impaired mitochondrial activity. Adult offsprings presented no major alterations in survival and mobility, as well the absence of abnormal phenotypes. However, mitochondrial activity and oviposition behavior was somewhat affected, indicating the chronic toxicity of climacostol, which continues moderately until adult stages. These results revealed for the first time the detrimental role of ingested climacostol in a non-target multicellular organism.

Chemical Defence by Sterols in the Freshwater Ciliate Stentor polymorphus.

Heterotrich ciliates typically retain toxic substances in specialized ejectable organelles, called extrusomes, which are used in predator-prey interactions. In this study, we analysed the chemical defence strategy of the freshwater heterotrich ciliate Stentor polymorphus against the predatory ciliate Coleps hirtus, and the microturbellarian flatworm Stenostomum sphagnetorum. The results showed that S. polymorphus is able to defend itself against these two predators by deploying a mix of bioactive sterols contained in its extrusomes. Sterols were isolated in vivo and characterized by liquid chromatography-mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR), as ergosterol, 7-dehydroporiferasterol, and their two peroxidized analogues. The assessment of the toxicity of ergosterol and ergosterol peroxide against various organisms, indicated that these sterols are essential for the effectiveness of the chemical defence in S. polymorphus.

Bioactive molecules from ciliates: Structure, activity, and applicative potential.

Ciliates are a rich source of molecules synthesized to socialize, compete ecologically, and interact with prey and predators. Their isolation from laboratory cultures is often straightforward, permitting the study of their mechanisms of action and their assessment for applied research. This review focuses on three classes of these bioactive molecules: (i) water-borne, cysteine-rich proteins that are used as signaling pheromones in self/nonself recognition phenomena; (ii) cell membrane-associated lipophilic terpenoids that are used in interspecies competitions for habitat colonization; (iii) cortical granule-associated molecules of various chemical nature that primarily serve offence/defense functions.

Protistological science dissemination.

It is rare to meet protistologists who are not passionate about their study subject. The vast majority of people, however, never get the chance to hear about the work of these researchers. Although every researcher working on protists is likely to be aware of this situation, efforts made and tools employed for dissemination of knowledge are rarely documented. Following a proposal by the Italian Society of Protistology, a workshop at the 2019 VIII European Congress of Protistology in Rome, Italy, was dedicated to protistological knowledge dissemination. Through the many interventions, we discovered the diversity of efforts to reveal the protistan world to the general public, including museum exhibitions and activities, public understanding of science events, citizen science projects, specific book publications, the use of protists in teaching at all levels from primary school children to university undergraduate students, and to a global audience via social media. The participation of the workshop delegates in the discussions indicated that presentations on the wonderful world of protists to the public not only increase the visibility and accessibility of protistology research but are also very important for the scientific community. Here we report on some of the key aspects of the presentations given in the dissemination workshop.

Bioactive molecules from protists: Perspectives in biotechnology.

For hundreds of years, mankind has benefited from the natural metabolic processes of microorganisms to obtain basic products such as fermented foods and alcoholic beverages. More recently, microorganisms have been exploited for the production of antibiotics, vitamins and enzymes to be used in medicine and chemical industries. Additionally, several modern drugs, including those for cancer therapy, are natural products or their derivatives. Protists are a still underexplored source of natural products potentially of interest for biotechnological and biomedical applications. This paper focuses on some examples of bioactive molecules from protists and associated bacteria and their possible use in biotechnology.

Preliminary Data Related to the Effect of Climacostol Produced by the Freshwater Ciliate Climacostomum virens on Human Adenovirus.

The new epidemiological scenario has so far focused on the environmental circulation of human viral pathogens. Owing to the side effects of chemical disinfectants, there is an increasing need for knowledge on the use of virucidal compounds, especially those of a natural origin. Climacostol is a molecule produced by a freshwater ciliate and it exhibits activity against bacterial and fungal pathogens. We thus also speculated that there might be an effect on viral viability, which has never been tested. To evaluate such activity, we chose human adenovirus (HAdV), which is representative of waterborne viruses. We conducted experiments using HAdV serotype 5, whose titer was determined by infecting HeLa cell cultures. HAdV5 was shown to be sensitive to climacostol at a concentration of 0.0002 mg/mL, with an approximate 3 Log10 reduction when the initial titer of HAdV5 was approximately 104 and 103 TCID50/mL. These preliminary results could be an important starting point for further research aimed at improving the characterization of climacostol activity under different experimental conditions and against various viruses, including enveloped ones (i.e., the coronavirus). The production of climacostol by a protist living in fresh water also suggests a possible application in the activated sludge of wastewater treatment plants.

Natural Function and Structural Modification of Climacostol, a Ciliate Secondary Metabolite.

The review highlights the main results of two decades of research on climacostol (5-[(2Z)-non-2-en-1-yl]benzene-1,3-diol), the resorcinolic lipid produced and used by the ciliated protozoan Climacostomum virens for chemical defense against a wide range of predators, and to assist its carnivorous feeding. After the first studies on the physiological function of climacostol, the compound and some analogues were chemically synthesized, thus allowing us to explore both its effect on different prokaryotic and eukaryotic biological systems, and the role of its relevant structural traits. In particular, the results obtained in the last 10 years indicate climacostol is an effective antimicrobial and anticancer agent, bringing new clues to the attempt to design and synthesize additional novel analogues that can increase or optimize its pharmacological properties.

Distinguishing Activities in the Photodynamic Arsenals of the Pigmented Ciliates Blepharisma sinuosum Sawaya, 1940 and Blepharisma japonicum Suzuki, 1954 (Ciliophora: Heterotrichea).

Blepharismins are photodynamic hypericin-like dianthrones produced as a variable pigment blend in Blepharisma ciliates and mostly studied in the Afro-Asiatic Blepharisma japonicum. The present work describes the bioactivity of pigments from the Brazilian Blepharisma sinuosum. Comparative analyses showed that the pigments from both species can trigger photo-induced modifications in phospholipids, but different redox properties and biological activities were assigned for each pigment blend. Stronger activities were detected for B. sinuosum pigments, with the lethal concentration LC50 10 × lower than B. japonicum pigments in light-irradiated tests against Bacillus cereus and less than half for treatments on the human HeLa tumor cells. HPLC showed B. sinuosum producing a simpler pigment blend, mostly with the blepharismin-C (~ 70%) and blepharismin-E (~ 30%) types. Each blepharismin engaged a specific dose-response profile on sensitive cells. The blepharismin-B and blepharismin-C were the most toxic pigments, showing LC50  ~ 2.5-3.0 µm and ~ 100 µm on B. cereus and HeLa cells, respectively, after illumination. Similarity clustering analysis compiling the bioactivity data revealed two groups of blepharismins: the most active, B and C, and the less active, A, D and E. The B. sinuosum pigment blend includes one representative of each clade. Functional and medical implications are discussed.

The Natural Compound Climacostol as a Prodrug Strategy Based on pH Activation for Efficient Delivery of Cytotoxic Small Agents.

We synthesized and characterized MOMO as a new small molecule analog of the cytotoxic natural product climacostol efficiently activated in mild extracellular acidosis. The synthesis of MOMO had a key step in the Wittig olefination for the construction of the carbon-carbon double bond in the alkenyl moiety of climacostol. The possibility of obtaining the target (Z)-alkenyl MOMO derivative in very good yield and without presence of the less active (E)-diastereomer was favored from the methoxymethyl ether (MOM)-protecting group of hydroxyl functions in aromatic ring of climacostol aldehyde intermediate. Of interest, the easy removal of MOM-protecting group in a weakly acidic environment allowed us to obtain a great quantity of climacostol in biologically active (Z)-configuration. Results obtained in free-living ciliates that share the same micro-environment of the climacostol natural producer Climacostomum virens demonstrated that MOMO is well-tolerated in a physiological environment, while its cytotoxicity is rapidly and efficiently triggered at pH 6.3. In addition, the cytostatic vs. cytotoxic effects of acidified-MOMO can be modulated in a dose-dependent manner. In mouse melanoma cells, MOMO displayed a marked pH-sensitivity since its cytotoxic and apoptotic effects become evident only in mild extracellular acidosis. Data also suggested MOMO being preferentially activated in the unique extra-acidic microenvironment that characterizes tumoural cells. Finally, the use of the model organism Drosophila melanogaster fed with an acidic diet supported the efficient activity and oral delivery of MOMO molecule in vivo. MOMO affected oviposition of mating adults and larvae eclosion. Reduced survival of flies was due to lethality during the larval stages while emerging larvae retained their ability to develop into adults. Interestingly, the gut of eclosed larvae exhibited an extended damage (cell death by apoptosis) and the brain tissue was also affected (reduced mitosis), demonstrating that orally activated MOMO efficiently targets different tissues of the developing fly. These results provided a proof-of-concept study on the pH-dependence of MOMO effects. In this respect, MOM-protection emerges as a potential prodrug strategy which deserves to be further investigated for the generation of efficient pH-sensitive small organic molecules as pharmacologically active cytotoxic compounds.

Bioactivity and Structural Properties of Novel Synthetic Analogues of the Protozoan Toxin Climacostol.

Climacostol (5-[(2Z)-non-2-en-1-yl]benzene-1,3-diol) is a resorcinol produced by the protozoan Climacostomum virens for defence against predators. It exerts a potent antimicrobial activity against bacterial and fungal pathogens, inhibits the growth of several human and rodent tumour cells, and is now available by chemical synthesis. In this study, we chemically synthesized two novel analogues of climacostol, namely, 2-methyl-5 [(2Z)-non-2-en-1-yl]benzene-1,3-diol (AN1) and 5-[(2Z)-non-2-en-1-yl]benzene-1,2,3-triol (AN2), with the aim to increase the activity of the native toxin, evaluating their effects on prokaryotic and free-living protists and on mammalian tumour cells. The results demonstrated that the analogue bearing a methyl group (AN1) in the aromatic ring exhibited appreciably higher toxicity against pathogen microbes and protists than climacostol. On the other hand, the analogue bearing an additional hydroxyl group (AN2) in the aromatic ring revealed its ability to induce programmed cell death in protistan cells. Overall, the data collected demonstrate that the introduction of a methyl or a hydroxyl moiety to the aromatic ring of climacostol can effectively modulate its potency and its mechanism of action.

Dysfunctional autophagy induced by the pro-apoptotic natural compound climacostol in tumour cells.

Autophagy occurs at a basal level in all eukaryotic cells and may support cell survival or activate death pathways. Due to its pathophysiologic significance, the autophagic machinery is a promising target for the development of multiple approaches for anti-neoplastic agents. We have recently described the cytotoxic and pro-apoptotic mechanisms, targeting the tumour suppressor p53, of climacostol, a natural product of the ciliated protozoan Climacostomum virens. We report here on how climacostol regulates autophagy and the involvement of p53-dependent mechanisms. Using both in vitro and in vivo techniques, we show that climacostol potently and selectively impairs autophagy in multiple tumour cells that are committed to die by apoptosis. In particular, in B16-F10 mouse melanomas climacostol exerts a marked and sustained accumulation of autophagosomes as the result of dysfunctional autophagic degradation. We also provide mechanistic insights showing that climacostol affects autophagosome turnover via p53-AMPK axis, although the mTOR pathway unrelated to p53 levels plays a role. In particular, climacostol activated p53 inducing the upregulation of p53 protein levels in the nuclei through effects on p53 stability at translational level, as for instance the phosphorylation at Ser15 site. Noteworthy, AMPKα activation was the major responsible of climacostol-induced autophagy disruption in the absence of a key role regulating cell death, thus indicating that climacostol effects on autophagy and apoptosis are two separate events, which may act independently on life/death decisions of the cell. Since the activation of p53 system is at the molecular crossroad regulating both the anti-autophagic action of climacostol and its role in the apoptosis induction, it might be important to explore the dual targeting of autophagy and apoptosis with agents acting on p53 for the selective killing of tumours. These findings also suggest the efficacy of ciliate bioactive molecules to identify novel lead compounds in drug discovery and development.

Chemical Defense by Erythrolactones in the Euryhaline Ciliated Protist, Pseudokeronopsis erythrina.

Pseudokeronopsis erythrina produces three new secondary metabolites, erythrolactones A2, B2 and C2, and their respective sulfate esters (A1, B1, C1), the structures of which have been recently elucidated on the basis of NMR spectroscopic data coupled to high resolution mass measurements (HR-MALDI-TOF). An analysis of the discharge of the protozoan pigment granules revealed that the non-sulfonated erythrolactones are exclusively stored in these cortical organelles, which are commonly used by a number of ciliates as chemical weapons in offense/defense interactions with prey and predators. We evaluated the toxic activity of pigment granule discharge on a panel of free-living ciliates and micro-invertebrates, and the activity of each single purified erythrolactone on three ciliate species. We also observed predator-prey interactions of P. erythrina with unicellular and multicellular predators. Experimental results confirm that only P. erythrina cells with discharged pigment granules were preferentially or exclusively hunted and eaten by at least some of its predators, whereas almost all intact (fully pigmented) cells remained alive. Our results indicate that erythrolactones are very effective as a chemical defense in P. erythrina.

Natural products from aquatic eukaryotic microorganisms for cancer therapy: Perspectives on anti-tumour properties of ciliate bioactive molecules.

Several modern drugs, including those for cancer therapy, have been isolated from natural sources, are based on natural products and its derivatives, or mime natural products. Some of them are in clinical use, others in clinical trials. The success of natural products in drug discovery is related to their biochemical characteristics and to the technologic methods used to study their feature. Natural compounds may acts as chemo-preventive agents and as factors that increase therapeutic efficacy of existing drugs, thus overcoming cancer cell drug resistance that is the main factor determining the failure in conventional chemotherapy. Water environment, because of its physical and chemical conditions, shows an extraordinary collection of natural biological substances with an extensive structural and functional diversity. The isolation of bioactive molecules has been reported from a great variety of aquatic organisms; however, the therapeutic application of molecules from eukaryotic microorganisms remains inadequately investigated and underexploited on a systematic basis. Herein we describe the biological activities in mammalian cells of selected substances isolated from ciliates, free-living protozoa common almost everywhere there is water, focusing on their anti-tumour actions and their possible therapeutic activity. In particular, we unveil the cellular and molecular machine mediating the effects of cell type-specific signalling protein pheromone Er-1 and secondary metabolites, i.e. euplotin C and climacostol, in cancer cells. To support the feasibility of climacostol-based approaches, we also present novel findings and report additional mechanisms of action using both in vitro and in vivo models of mouse melanomas, with the scope of highlighting new frontiers that can be explored also in a therapeutic perspective. The high skeletal chemical difference of ciliate compounds, their sustainability and availability, also through the use of new organic synthesis/modifications processes, and the results obtained so far in biological studies provide a rationale to consider some of them a potential resource for the design of new anti-cancer drugs.

Climacostol reduces tumour progression in a mouse model of melanoma via the p53-dependent intrinsic apoptotic programme.

Climacostol, a compound produced by the ciliated protozoan Climacostomum virens, displayed cytotoxic properties in vitro. This study demonstrates that it has anti-tumour potential. Climacostol caused a reduction of viability/proliferation of B16-F10 mouse melanoma cells, a rapidly occurring DNA damage, and induced the intrinsic apoptotic pathway characterised by the dissipation of the mitochondrial membrane potential, the translocation of Bax to the mitochondria, the release of Cytochrome c from the mitochondria, and the activation of Caspase 9-dependent cleavage of Caspase 3. The apoptotic mechanism of climacostol was found to rely on the up-regulation of p53 and its targets Noxa and Puma. In vivo analysis of B16-F10 allografts revealed a persistent inhibition of tumour growth rate when melanomas were treated with intra-tumoural injections of climacostol. In addition, it significantly improved the survival of transplanted mice, decreased tumour weight, induced a remarkable reduction of viable cells inside the tumour, activated apoptosis and up-regulated the p53 signalling network. Importantly, climacostol toxicity was more selective against tumour than non-tumour cells. The anti-tumour properties of climacostol and the molecular events associated with its action indicate that it is a powerful agent that may be considered for the design of pro-apoptotic drugs for melanoma therapy.

Cold-shock based method to induce the discharge of extrusomes in ciliated protists and its efficiency.

Extrusomes are ejectable organelles in protists, which are able to discharge their contents to the outside of the cell in response to external stimuli. It is known that a large number of extrusomes functions as organelles for offense or defense in predator-prey interactions among protists and/or microinvertebrates. To date, the main approach to study these interactions was to compare artificially-induced extrusome-deficient cells with normal cells as prey for predators. Commonly applied methods to obtain extrusome-deficient cells use external chemicals, which could alter the viability of cells and/or interfere with the subsequent analysis of the substances (secondary metabolites) contained in the extrusomes. The cold-shock based method here presented has proven to be effective to remove different kinds of extrusomes from several protist species without harming the treated cells and without adding external reagents. This method could be also useful to simplify the related analysis of the chemical nature of the secreted secondary metabolites.

Chemical offense by means of toxicysts in the freshwater ciliate, Coleps hirtus.

Coleps hirtus is a small common freshwater ciliate belonging to the protostomatid group, its body covered by calcified plates assembled to form an armor. Coleps feeds on bacteria, algae, flagellates, living and dead ciliates, animal and plant tissues. To assist its carnivorous feeding the ciliate is equipped with offensive extrusomes (toxicysts), clustering mainly in and around its oral aperture. In this study, we isolated the discharge of the toxicysts from living cells, evaluating its cytotoxic effects against various ciliate species, and demonstrating that it is essential for the effectiveness of Coleps' predatory behavior. The analysis of the toxicyst discharge performed by liquid chromatography-electrospray-mass spectrometry and gas chromatography-mass spectrometry, revealed the presence of a mixture of 19 saturated, monounsaturated and polyunsaturated free fatty acids with the addition of a minor amount of a diterpenoid (phytanic acid).

DNA binding and oxidative DNA damage induced by climacostol-copper(II) complexes: implications for anticancer properties.

Climacostol is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to the group of resorcinolic lipids. Climacostol exerts a potent antimicrobial activity against a panel of bacterial and fungal pathogens. In addition it inhibits the growth of tumor cell lines in a dose-dependent manner by inducing programmed cell death via intrinsic pathway. In this work, we investigated the possibility that climacostol exerts a prooxidant effect, inducing plasmid DNA strand breakage and eukaryotic DNA damage in presence of Cu(II) ions. Inhibition of DNA breakage using SOD, catalase and neocuproine confirmed the involvement of reactive oxygen species and Cu(I) ions in the DNA damage. UV-visible absorption changes and mass spectrometric analysis identified a product of reaction as a deprotonated form of climacostol. Study of the interaction with DNA, using fluorescence spectroscopic techniques, showed that climacostol binds with DNA. Given the structure-activity relationship of this compound and the mechanism of its prooxidant effect, we propose that the Cu(II)-mediated oxidative DNA damage by climacostol could explain its antimicrobial and antiproliferative activity.

The defensive function of trichocysts in Paramecium tetraurelia against metazoan predators compared with the chemical defense of two species of toxin-containing ciliates.

The time-honored assumption about the defensive function of trichocysts in Paramecium against predators was recently verified experimentally against different species of unicellular predators. In the present study, we examined the defensive function of trichocysts against three metazoan predators, Cephalodella sp. (Rotifera), Eucypris sp. (Arthropoda), and Stenostomum sphagnetorum (Platyhelminthes). The results confirmed the defensive function of trichocysts against two of these metazoan predators (Cephalodella sp. and Eucypris sp.), while they seem ineffective against S. sphagnetorum. We also compared the defensive efficiency of the trichocysts of P. tetraurelia with that of toxin-containing extrusomes of two ciliates.

Cell aging-induced methionine oxidation causes an autocrine to paracrine shift of the pheromone activity in the protozoan ciliate, Euplotes raikovi.

Ciliates of the genus Euplotes rely on the autocrine (self) and paracrine (non-self) activities of their water-borne protein pheromones to control the two fundamental phenomena of their life cycle, i.e. vegetative (mitotic) growth and sex manifested as cell union in mating pairs. We observed that cell aging determines the synthesis of increasing concentrations of pheromones that are oxidized at the level of methionine residues which are more exposed on the molecular surface. The oxidized form of the E. raikovi pheromone Er-1 was purified and its interactions with its source cells were shown no longer to be of autocrine type directed to promote cell growth, but changed to interactions of the paracrine type directed to induce cell unions in mating pairs of the selfing type (i.e. involving genetically identical cells). These pairs generate viable offspring, like pairs formed between genetically different cells. It was therefore concluded that aging cells may paradoxically gain beneficial effects from the synthesis of oxidized forms of their pheromones. By undergoing mating in response to the interactions with these forms, they can re-initiate a new life cycle and, in fact, rejuvenate.