Identification and Molecular Characterization of Superoxide Dismutases Isolated From A Scuticociliate Parasite: Physiological Role in Oxidative Stress.

Philasterides dicentrarchi is a free-living microaerophilic scuticociliate that can become a facultative parasite and cause a serious parasitic disease in farmed fish. Both the free-living and parasitic forms of this scuticociliate are exposed to oxidative stress associated with environmental factors and the host immune system. The reactive oxygen species (ROS) generated by the host are neutralized by the ciliate by means of antioxidant defences. In this study we aimed to identify metalloenzymes with superoxide dismutase (SOD) activity capable of inactivating the superoxide anion (•O2-) generated during induction of oxidative stress. P. dicentrarchi possesses the three characteristic types of SOD isoenzymes in eukaryotes: copper/zinc-SOD, manganese-SOD and iron-SOD. The Cu/Zn-SOD isoenzymes comprise three types of homodimeric proteins (CSD1-3) of molecular weight (MW) 34-44 kDa and with very different AA sequences. All Cu/Zn-SODs are sensitive to NaCN, located in the cytosol and in the alveolar sacs, and one of them (CSD2) is extracellular. Mn- and Fe-SOD transcripts encode homodimeric proteins (MSD and FSD, respectively) in their native state: a) MSD (MW 50 kDa) is insensitive to H2O2 and NaN3 and is located in the mitochondria; and b) FSD (MW 60 kDa) is sensitive to H2O2, NaN3 and the polyphenol trans-resveratrol and is located extracellularly. Expression of SOD isoenzymes increases when •O2- is induced by ultraviolet (UV) irradiation, and the increase is proportional to the dose of energy applied, indicating that these enzymes are actively involved in cellular protection against oxidative stress.

Transcriptome analysis on chlorpyrifos detoxification in Uronema marinum (Ciliophora, Oligohymenophorea).

Chlorpyrifos (CPF) pollution has drawn widespread concerns in aquatic environments due to its risks to ecologic system, however, the response mechanisms of ciliates to CPF pollution were poorly studied. In our current work, the degradation of CPF by ciliates and the morphological changes of ciliates after CPF exposure were investigated. In addition, the transcriptomic profiles of the ciliate Uronema marinum, with and without exposure with CPF, were detected using digital gene expression technologies. De novo transcriptome assembly 166,829,634 reads produced from three groups (untreated, CPF treatment at 12 h and 24 h) by whole transcriptome sequencing (RNA-Seq). Gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways were analyzed in all unigenes and different expression genes to identify their biological functions and processes. Furthermore, the results indicated that genes related to the stress response, cytoskeleton and cell structure proteins, and antioxidant systems might play an important role in the resistance mechanism of ciliates. The enzyme activities of SOD and GST after CPF stress were also analyzed, and the result showed the good antioxidant capacity of SOD and GST in ciliates inferred from the increase of the activities of the two enzymes. The ciliate Uronema marinum showed a resistance response to chlorpyrifos stress at the transcriptomic level in the present work, which indicates that ciliates can be considered as a potential bioremediation agent.

Exceptional in vivo catabolism of neurodegeneration-related aggregates.

Neurodegenerative diseases are linked to a systemic enzyme resistance of toxic aggregated molecules and their pathological consequences. This paper presents a unique phenomenon that Philodina acuticornis, a bdelloid rotifer, is able to catabolize different types of neurotoxic peptide and protein aggregates (such as beta-amyloids /Aß/, alpha-synuclein, and prion) without suffering any damage. P. acuticornis is capable of using these aggregates as an exclusive energy source (i.e., as 'food', identified in the digestive system and body) in a hermetically isolated microdrop environment, increasing their survival. As regards Aß1-42, five other bdelloid rotifer species were also found to be able to perform this phenomenon. Based on our experiments, the Aß1-42-treated bdelloid rotifers demonstrate significantly increased survival (e.g. mean lifespan = 51 ± 2.71 days) compared to their untreated controls (e.g. mean lifespan = 14 ± 2.29 days), with similar improvements in a variety of phenotypic characteristics. To our knowledge, no other animal species have so far been reported to have a similar capability. For all other microscopic species tested, including monogonant rotifers and non-rotifers, the treatment with Aß1-42 aggregates proved to be either toxic or simply ineffective. This paper describes and proves the existence of an unprecedented in vivo catabolic capability of neurotoxic aggregates by bdelloid rotifers, with special focus on P. acuticornis. Our results may provide the basis for a new preclinical perspective on therapeutic research in human neurodegenerative diseases.

Regulation of Fatty Acid Production and Release in Benthic Algae: Could Parallel Allelopathy Be Explained with Plant Defence Theories?

Many organisms produce chemical compounds, generally referred as secondary metabolites, to defend against predators and competitors (allelopathic compounds). Several hypotheses have been proposed to explain the interaction between environmental factors and secondary metabolites production. However, microalgae commonly use simple metabolites having a role in primary metabolism as allelopathic compounds. The aim of this study was to determine whether classical theories of plant chemical defences could be applied to microalgae producing allelochemicals derived from the primary metabolism. Our study was designed to investigate how growth phase, algal population density, nutrient limitation and carbon assimilation affect the production and release of allelopathic free fatty acids (FFAs) among other FFAs. The model species used was Uronema confervicolum, a benthic filamentous green alga that produces two allelopathic FFAs (linoleic and α-linolenic acids) inhibiting diatom growth. FFAs have been quantified in algal biomass and in culture medium. Our results were analysed according to two classical plant defence theories: the growth-differentiation balance hypothesis (GDBH) and the optimal defence theory (ODT), based on the metabolic capacities for defence production and on the need for defence, respectively. While a higher production of allelopathic compounds under increased light conditions supports the use of GDBH with this microalga, the observation of a negative feedback mechanism mostly supports ODT. Therefore, both theories were insufficient to explain all the observed effects of environmental factors on the production of these allelochemicals. This highlights the needs of new theories and models to better describe chemical interactions of microalgae.

Differential localization and functional specialization of centrin analogs in the parasitic ciliate Trichodina pediculus.

Trichodinids are ciliated protozoans that reversibly attach to the tegument of marine and freshwater host-organisms via an adhesive disc. In this study, we have used permeabilized cell models of Trichodina pediculus to examine the distribution of centrins, a Ca(2+)-binding protein associated with centrioles and/or contractile filamentous structures in a large number of protists. The previous finding that filamentous material of the adhesive disc comprised a 23-kDa centrin analog suggested that this protein might be a disc-specific isoform. This possibility was explored through immunolabeling methods using two distinct antibodies, anti-ecto-endoplasmic boundary (EEB) and anti-Hscen2 previously shown to react respectively with centrin-based filament networks and with centrioles. Immunofluorescence microscopy showed that anti-EEB reacts with filamentous material of the disc but not with basal bodies. Conversely, anti-Hscen2 cross-reacted with basal bodies but failed to label any type of structure occurring in the disc area. More detailed data on localization of this protein was obtained by immunoelectron microscopy showing gold particles deposits in the lumen of basal bodies. The different patterns revealed by this immunochemical approach suggest that the two protein antigens concerned by this study are distinct centrin isoforms that presumably perform organelle-specific function in the ciliate T. pediculus.

Evidence of an alternative oxidase pathway for mitochondrial respiration in the scuticociliate Philasterides dicentrarchi.

The presence of an alternative oxidase (AOX) in the mitochondria of the scuticociliate P. dicentrarchi was investigated. The mitochondrial oxygen consumption was measured in the presence of KCN, an inhibitor of cytochrome pathway (CP) respiration and salicylhydroxamic acid (SHAM), a specific inhibitor of alternative pathway (AP) respiration. AOX expression was monitored by western blotting with an AOX polyclonal antibody. The results showed that P. dicentrarchi possesses a branched mitochondrial electron transport chain with both cyanide-sensitive and -insensitive oxygen consumption. Mitochondrial respiration was partially inhibited by cyanide and completely inhibited by the combination of cyanide and SHAM, which is direct evidence for the existence of an AP in this ciliate. SHAM significantly inhibited in vitro growth of trophozoites both under normoxic and hypoxic conditions. AOX is a 42kD monomeric protein inducible by hypoxic conditions in experimental infections and by CP inhibitors such as cyanide and antimycin A, or by AP inhibitors such as SHAM. CP respiration was greatly stimulated during the exponential growth phase, while AP respiration increased during the stationary phase, in which AOX expression is induced. As the host does not possess AOX, and because during infection P. dicentrarchi respires via AP, it may be possible to develop inhibitors targeting the AP as a novel anti-scuticociliate therapy.

Effect of resveratrol on oxygen consumption by Philasterides dicentrarchi, a scuticociliate parasite of turbot.

The phytoalexin resveratrol (RESV) displays antiparasitic activity against Philasterides dicentrarchi, a scuticociliate pathogen of turbot, and causes oxidative stress, inhibition of antioxidant enzyme activity and morphological alterations in the parasite mitochondria. In this study, we analysed the mitochondrial biology of P. dicentrarchi and assessed the effect of RESV on mitochondrial metabolism. We found that RESV caused dose-dependent inhibition of mitochondrial electron transport and O2 consumption in ciliates permeabilized with digitonin. Although the RESV molecule has a high capacity for antiradical and antioxidant activity, it induced a high level of pro-oxidant activity against the ciliate, thus causing a significant increase in intracellular ROS production. The increased ROS production was accompanied by mitochondrial collapse and dysfunction of mitochondrial membrane potential (ΔΨm) and by a significant increase in intracellular Ca⁺² levels. RESV inhibited parasite growth in a similar way to antimycin A, an inhibitor of mitochondrial electron transport and ROS generator. The findings confirm the mitochondria as a target in the potential development of effective antiparasitic treatments.

Maximal force characteristics of the Ca(2+)-powered actuator of Vorticella convallaria.

The millisecond stalk contraction of the sessile ciliate Vorticella convallaria is powered by energy from Ca(2+) binding to generate contractile forces of ∼10 nN. Its contractile organelle, the spasmoneme, generates higher contractile force under increased stall resistances. By applying viscous drag force to contracting V. convallaria in a microfluidic channel, we observed that the mechanical force and work of the spasmoneme depended on the stalk length, i.e., the maximum tension (150-350 nN) and work linearly depended on the stalk length (∼2.5 nN and ∼30 fJ per 1 µm of the stalk). This stalk-length dependency suggests that motor units of the spasmoneme may be organized in such a way that the mechanical force and work of each unit cumulate in series along the spasmoneme.

Development of a fatty acid and RNA stable isotope probing-based method for tracking protist grazing on bacteria in wastewater.

Removal of potential pathogenic bacteria, for example, during wastewater treatment, is effected by sorption, filtration, natural die-off, lysis by viruses, and grazing by protists, but the actual contribution of grazing has never been assessed quantitatively. A methodical approach for analyzing the grazing of protists on (13)C-labeled prey bacteria was developed which enables mass balances of the carbon turnover to be drawn, including yield estimation. Model experiments for validating the approach were performed in closed microcosms with the ciliate Uronema sp. and (13)C-labeled Escherichia coli as model prey. The transfer of bacterial (13)C into grazing protist biomass was investigated by fatty acid (FA) and RNA stable isotope probing (SIP). Uronema sp. showed ingestion rates of ∼390 bacteria protist(-1) h(-1), and the temporal patterns of (13)C assimilation from the prey bacteria to the protist FA were identified. Nine fatty acids specific for Uronema sp. were found (20:0, i20:0, 22:0, 24:0, 20:1ω9c, 20:1ω9t, 22:1ω9c, 22:1ω9t, and 24:1). Four of these fatty acids (22:0, 20:1ω9t, 22:1ω9c, and 22:1ω9t) were enriched very rapidly after 3 h, indicating grazing on bacteria without concomitant cell division. Other fatty acids (20:0, i20:0, and 20:1ω9c) were found to be indicative of growth with cell division. The fatty acids were found to be labeled with a percentage of labeled carbon (atoms percent [atom%]) up to 50. Eighteen percent of the E. coli-derived (13)C was incorporated into Uronema biomass, whereas 11% was mineralized. Around 5 mol bacterial carbon was necessary in order to produce 1 mol protist carbon (y(x)(/)(s) ≈ 0.2), and the temporal pattern of (13)C labeling of protist rRNA was also shown. A consumption of around 1,000 prey bacteria (∼98 atom% (13)C) per protist cell appears to be sufficient to provide detectable amounts of label in the protist RNA. The large shift in the buoyant density fraction of (13)C-labeled protist RNA demonstrated a high incorporation of (13)C, and reverse transcription-real-time PCR (RT-qPCR) confirmed that protist rRNA increasingly dominated in the heavy RNA fraction.

Mechanics of Vorticella contraction.

Vorticella convallaria is one of a class of fast-moving organisms that can traverse its body size in less than a millisecond by rapidly coiling a slender stalk anchoring it to a nearby surface. The stalk houses a fiber called the spasmoneme, which winds helically within the stalk and rapidly contracts in response to calcium signaling. We have developed a coupled mechanical-chemical model of the coiling process, accounting for the coiling of the elastic stalk and the binding of calcium to the protein spasmin. Simulations of the model describe the contraction and recovery processes quantitatively. The stalk-spasmoneme system is shown to satisfy geometric constraints, which explains why the cell body sometimes rotates during contraction. The shape of the collapsing and recovering stalk bounds its effective bending stiffness. Simulations suggest that recovery from the contracted state is driven by the stalk at a rate controlled by dissociation of calcium from spasmin.

Sequestered organelles sustain aerobic microbial life in anoxic environments.

We report aerobic eukaryotic microbial life in the dimly lit anoxic water layer of a small freshwater lake. The microbial eukaryote is the ciliated protozoon Histiobalantium natans. Electron microscopy of thin sections shows that the cytoplasm of the ciliate harbours sequestered chloroplasts and sequestered mitochondria. The sequestered chloroplasts are attached or in very close proximity to the ciliate's own mitochondria. The sequestered mitochondria also seem to be associated with host-ciliate mitochondria. We suggest that the oxygenic photosynthetic activity of sequestered chloroplasts, perhaps enhanced by respiration in sequestered mitochondria, contributes to servicing the respiratory oxygen requirements of the ciliate host in its anoxic habitat. Our observations are novel, with the discovery of an aerobic microbial eukaryote capable of thriving and completing its life cycle in an anoxic environment, fuelled by oxygen generated by sequestered chloroplasts. The acknowledged flexibility and functional diversity within eukaryotic microbial communities still have many secrets to release.

Chemical modification of glycerinated stalks shows tyrosine residues essential for spasmoneme contraction of Vorticella sp.

Chemical modification of glycerinated stalks of Vorticella with TNM is used to investigate the role of tyrosine residues in the Ca(2+)-induced contraction of the spasmoneme. Tetranitromethane (TNM) is often employed as a specific reagent for the nitration of tyrosine residues in a protein at neutral and slightly alkaline pHs although TNM can also oxidize cysteine residues in the acidic and neutral pH range. Prior incubation with Ca(2+) of stalks to be treated with TNM can protect the spasmoneme from irreversible denaturation. On the other hand, TNM treatment in the absence of free Ca(2+) causes an irreversible denaturation of the spasmoneme. It was revealed by us that an isolated Ca(2+)-binding protein called spasmin could not bind with Ca(2+) after TNM treatment, even if the treatment was performed in the presence of Ca(2+). In an additional experiment, we confirmed that the chemical modification of cysteine residues in the spasmoneme with N-7-dimethyl-amino-4methyl- coumarinyl- maleimide (DACM) has no effect on the contractibility. These results suggest that tyrosine residues in spasmin are essential for spasmoneme contraction and are protected from TNM in the presence of Ca(2+) when spasmin binds with its receptor protein in the spasmoneme.

Cloning and expression of a cDNA encoding a Vorticella convallaria spasmin: an EF-hand calcium-binding protein.

The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.