Identification of a Tetrahymena species infecting guppies, pathology, and expression of beta-tubulin during infection.

Tetrahymenosis is caused by the ciliated protozoan Tetrahymena and is responsible for serious economic losses to the aquaculture industry worldwide. However, information regarding the molecular mechanism leading to tetrahymenosis is limited. In previous transcriptome sequencing work, it was found that one of the two ß-tubulin genes in T. pyriformis was significantly expressed in infected fish, we speculated that ß-tubulin is involved in T. pyriformis infecting fish. Herein, the potential biological function of the ß-tubulin gene in Tetrahymena species when establishing infection in guppies was investigated by cloning the full-length cDNA of this T. pyriformis ß-tubulin (BTU1) gene. The full-length cDNA of T. pyriformis BTU1 gene was 1873 bp, and the ORF occupied 1134 bp, whereas 5' UTR 434 bp, and 3' UTR 305 bp whose poly (A) tail contained 12 bases. The predicted protein encoded by T. pyriformis BTU1 gene had a calculated molecular weight of 42.26 kDa and pI of 4.48. Moreover, secondary structure analysis and tertiary structure prediction of BTU1 protein were also conducted. In addition, morphology, infraciliature, phylogeny, and histopathology of T. pyriformis isolated from guppies from a fish market in Harbin were also investigated. Furthermore, qRT-PCR analysis and experimental infection assays indicated that the expression of BTU1 gene resulted in efficient cell proliferation during infection. Collectively, our data revealed that BTU1 is a key gene involved in T. pyriformis infection in guppies, and the findings discussed herein provide valuable insights for future studies on tetrahymenosis.

Intracellular Hybrid Biosystem in a Protozoan to Trigger Visible-Light-Driven Photocatalysis.

Incorporating artificial photosensitizers with microorganisms has recently been recognized as an effective way to convert light energy into chemical energy. However, the incorporated biosystem is usually constructed in an extracellular manner and is vulnerable to the external environment. Here, we develop an intracellular hybrid biosystem in a higher organism protozoa Tetrahymena pyriformis, in which the in vivo synthesized CdS nanoparticles trigger photoreduction of nitrobenzene into aniline under visible-light irradiation. Integrating a photosensitizer CdS into T. pyriformis enables the photosensitizer CdS, inherent nitroreductase, and the cytoplasmic reductive substance in T. pyriformis to synergistically engage in the photocatalysis process, generating a greatly enhanced aniline yield with a 40-fold increment. Moreover, building an intracellular hybrid biosystem in mutant T. pyriformis could even grant it new capability of reducing nitrobenzene into aniline under visible-light irradiation. Such an intracellular hybrid biosystem paves a new way to functionalize higher organisms and diversify light energy conversion.

Sequencing and characterization of the macronuclear rDNA minichromosome of the protozoan Tetrahymena pyriformis.

Tetrahymena ribosomal DNA (rDNA) is an ideal system for studying eukaryotic DNA replication and gene transcription. In this study, we developed a new method to isolate rDNA from Tetrahymena cells and used it to sequence and annotate the complete 19,670 bp macronuclear rDNA minichromosome of Tetrahymena pyriformis, a species that lacks the germ-line micronucleus and is unable to undergo sexual reproduction. The key features of T. pyriformis and Tetrahymena thermophila rDNA sequences were then compared. Our results showed (i) the short inverted repeats (M repeats) essential for formation of rDNA minichromosome palindromic structure during sexual reproduction in Tetrahymena are highly conserved in T. pyriformis; (ii) in contrast to T. thermophila, which has two tandem domains that coordinately regulate rDNA replication, T. pyriformis has only a single domain; (iii) the 35S pre-rRNA precursor has 80.25% similarity between the two species; and (iv) the G + C content is higher in the transcribed region than the non-transcribed region in both species, but the GC-skew is more stable in T. pyriformis. The new isolation method and annotated information for the T. pyriformis rDNA minichromosome will provide a useful resource for studying DNA replication and chromosome copy number control in Tetrahymena.

Synthesis of CdS1-XSeX quantum dots in a protozoa Tetrahymena pyriformis.

Quantum dots (QDs) are recognized as the excellent fluorescence and photochemical materials to be applied in bioimaging, biomedical, and solar cell fields. Biosynthesized QDs (bio-QDs) have attracted attention due to their simple, eco-friendly, and excellent biocompatible traits. Moreover, bio-QDs could not be replaced by chemically fabricated QDs in many fields. Bio-QDs synthesized by different microorganisms have diverse characteristics. In this work, the biosynthesis of QDs by Tetrahymena pyriformis, a typical protozoa in aquatic environments, was achieved for the first time. The synthesized materials by T. pyriformis emitted yellow fluorescence and had an average diameter of 8.27 ± 0.77 nm. Spectral characterization results demonstrated that the synthesized QDs were CdS1-XSeX. Meanwhile, the fluorescence intensities of the synthesized bio-QDs showed a linear relationship with Cd2+ dosage ranging from 20 to 80 µM. The fluorescence enhancement of the synthesized QDs was highly selective to Cd2+ compared to other metal ions. The bio-QDs were demonstrated to have a great potential to be applied for Cd2+ detection. This work provides valuable information about the transformation of heavy metal ions in protozoan and is useful to accelerate the applications of the synthesized QDs.

In silico prediction of toxicity of phenols to Tetrahymena pyriformis by using genetic algorithm and decision tree-based modeling approach.

Risk assessment of chemicals is an important issue in environmental protection; however, there is a huge lack of experimental data for a large number of end-points. The experimental determination of toxicity of chemicals involves high costs and time-consuming process. In silico tools such as quantitative structure-toxicity relationship (QSTR) models, which are constructed on the basis of computational molecular descriptors, can predict missing data for toxic end-points for existing or even not yet synthesized chemicals. Phenol derivatives are known to be aquatic pollutants. With this background, we aimed to develop an accurate and reliable QSTR model for the prediction of toxicity of 206 phenols to Tetrahymena pyriformis. A multiple linear regression (MLR)-based QSTR was obtained using a powerful descriptor selection tool named Memorized_ACO algorithm. Statistical parameters of the model were 0.72 and 0.68 for Rtraining2 and Rtest2, respectively. To develop a high-quality QSTR model, classification and regression tree (CART) was employed. Two approaches were considered: (1) phenols were classified into different modes of action using CART and (2) the phenols in the training set were partitioned to several subsets by a tree in such a manner that in each subset, a high-quality MLR could be developed. For the first approach, the statistical parameters of the resultant QSTR model were improved to 0.83 and 0.75 for Rtraining2 and Rtest2, respectively. Genetic algorithm was employed in the second approach to obtain an optimal tree, and it was shown that the final QSTR model provided excellent prediction accuracy for the training and test sets (Rtraining2 and Rtest2 were 0.91 and 0.93, respectively). The mean absolute error for the test set was computed as 0.1615.

Cooperativity and evolution of Tetrahymena two-domain arginine kinase.

Tetrahymena pyriformis contains two arginine kinases, a 40-kDa enzyme (AK1) with a myristoylation signal sequence at the N-terminus and a two-domain 80-kDa enzyme (AK2). The former is localized mainly in cilia and the latter is in the cytoplasm. AK1 was successfully synthesized using an insect cell-free protein synthesis system and subjected to peptide mass fingerprinting (PMF) analysis. The masses corresponding to unmodified N-terminal tryptic peptide or N-terminal myristoylated peptide were not observed, suggesting that N-terminal peptides were not ionized in this analysis. We performed PMF analyses for two other phosphagen kinases (PKs) with myristoylation signals, an AK from Nematostella vectensis and a PK from Ectocarpus siliculosus. In both cases, the myristoylated, N-terminal peptides were clearly identified. The differences between the experimental and theoretical masses were within 0.0165-0.0583 Da, supporting the accuracy of the identification. Domains 1 and 2 of Tetrahymena two-domain AK2 were expressed separately in Escherichia coli and the extent of cooperativity was estimated on the basis of their kinetic constants. The results suggested that each of the domains functions independently, namely no cooperativity is displayed between the two domains. This is in sharp contrast to the two-domain AK from Anthopleura.

Amitotic chromosome loss predicts distinct patterns of senescence and non-senescence in ciliates.

Over time and repeated asexual divisions, many ciliate species display the characteristics of senescence, reduced fecundity and increased mortality. Their only path to recovery is sexual conjugation or autogamy. While more traditional models of cellular aging have been proposed, one of the most accepted explanations relies on the faulty mechanism by which ciliates duplicate their somatic nucleus, a process referred to as amitosis. Amitosis involves the random segregation of chromosomes with no consideration for homology. Over subsequent divisions, chromosome copy numbers will fluctuate until an entire chromosome is lost, resulting in death. Via simulations of this process, we find that senescence and death via chromosome loss is not the only possible result of amitosis. Random chromosome loss is less damaging to populations than previously thought, and strict adherence to the model predicts that Paramecium tetraurelia would not senesce. A combination of the reciprocal nature of amitosis and lethal selection against low-copy number chromosomes is responsible for this startling prediction. Additionally, our results provide an alternate explanation to recent evidence for selection on chromosome copy number in Tetrahymena thermophila and peculiar patterns of senescence in Tetrahymena pyriformis.

Quantitative Regression Models for the Prediction of Chemical Properties by an Efficient Workflow.

Rapid safety assessment is more and more needed for the increasing chemicals both in chemical industries and regulators around the world. The traditional experimental methods couldn't meet the current demand any more. With the development of the information technology and the growth of experimental data, in silico modeling has become a practical and rapid alternative for the assessment of chemical properties, especially for the toxicity prediction of organic chemicals. In this study, a quantitative regression workflow was built by KNIME to predict chemical properties. With this regression workflow, quantitative values of chemical properties can be obtained, which is different from the binary-classification model or multi-classification models that can only give qualitative results. To illustrate the usage of the workflow, two predictive models were constructed based on datasets of Tetrahymena pyriformis toxicity and Aqueous solubility. The qcv (2) and qtest (2) of 5-fold cross validation and external validation for both types of models were greater than 0.7, which implies that our models are robust and reliable, and the workflow is very convenient and efficient in prediction of various chemical properties.

Identification and characterization of the arsenite methyltransferase from a protozoan, Tetrahymena pyriformis.

Arsenic (As) methylation in aquatic microbes plays a major role in the biogeochemistry of As. Protozoa, especially the free-living freshwater species, are important players in aquatic ecological health. In this study, an arsenite (As(III)) methyltransferase, TpyArsM, was identified and characterized in a free-living protozoan, Tetrahymena pyriformis. In order to confirm its function, TpyarsM gene was knocked-out in Tetrahymena and was also heterologously expressed in hypersensitive E. coli; these events resulted in expected decreases in As tolerance and methylation ability, respectively. In-vitro tests revealed that purified TpyArsM protein methylated inorganic As to mono- and di- methylarsenate, and also had the novel property of producing trimethylarsenite (TMA(III)) and dimethylarsine (Me2AsH) gases. This new methyltransferase gene, identified in a species near the base of the food web, has enriched our knowledge of As methyltransferases and has great potential for bioremediation of As-contaminated environments.

Cell cycle-dependent modulations of fenestrin expression in Tetrahymena pyriformis.

In Tetrahymena, besides apparent cell polarity generated by specialized cortical structures, several proteins display a specific asymmetric distribution suggesting their involvement in the generation and the maintenance of cell polarization. One of these proteins, a membrane skeleton protein called fenestrin, forms an antero-posterior gradient, and is accepted as a marker of cell polarity during different cellular processes, such as cell division or oral replacement. In conjugating cells, fenestrin forms an intracytoplasmic net which participates in pronuclear exchange. The function of fenestrin is still unknown. To better understand the role of fenestrin we characterized this protein in an amicronuclear Tetrahymena pyriformis. We show that in this ciliate not only does fenestrin localization change in a cell division-dependent manner, but its mRNA and protein level is also cell cycle-regulated. We determine that the two available anti-fenestrin antibodies, 3A7 and 9A7, recognize different pools of fenestrin isoforms, and that 9A7 is the more general. In addition, our results indicate that fenestrin is a phosphoprotein. We also show that the level of fenestrin in the amicronuclear T. pyriformis and the amicronuclear BI3840 strain of T. thermophila is several times lower than in micronuclear T. thermophila.

DNA content alterations in Tetrahymena pyriformis macronucleus after exposure to food preservatives sodium nitrate and sodium benzoate.

The toxicity, in terms of changes in the DNA content, of two food preservatives, sodium nitrate and sodium benzoate was studied on the protozoan Tetrahymena pyriformis using DNA image analysis technology. For this purpose, selected doses of both food additives were administered for 2 h to protozoa cultures and DNA image analysis of T. pyriformis nuclei was performed. The analysis was based on the measurement of the Mean Optical Density which represents the cellular DNA content. The results have shown that after exposure of the protozoan cultures to doses equivalent to ADI, a statistically significant increase in the macronuclear DNA content compared to the unexposed control samples was observed. The observed increase in the macronuclear DNA content is indicative of the stimulation of the mitotic process and the observed increase in MOD, accompanied by a stimulation of the protozoan proliferation activity is in consistence with this assumption. Since alterations at the DNA level such as DNA content and uncontrolled mitogenic stimulation have been linked with chemical carcinogenesis, the results of the present study add information on the toxicogenomic profile of the selected chemicals and may potentially lead to reconsideration of the excessive use of nitrates aiming to protect public health.

[Influence of activator and inhibitors of Ca2+ channels on proliferative activity in Tetrahymena pyriformis infusoria].

It was determined that change in DNA content in macronuclei occurs in the T. pyriformis infusoria under the influence of an activator (caffeine) and inhibitors of Ca2+ channels (verapamil), NiCl2, and CdCl2. Caffeine (10 mM) stimulates DNA synthesis. Verapamil (5 microM), CdCl2 (125 microM), and NiCl2 (100 microM) decrease DNA content in macronuclei by 30 min after proliferative stimulation. By 4 h of incubation, there is, on average, 10% less DNA in macronuclei of Tetrahymena preprocessed with verapamil than in the control cells. The cells preprocessed with CdCl2 and NiCl2 differ from the control cells by lower DNA content almost at all studied periods, but they restore the level of nuclear DNA by 4 h. It is assumed that transmission of proliferative signals in the T. pyriformis has a Ca2+ -dependent character.

Verification of epigenetic inheritance in a unicellular model system: multigenerational effects of hormonal imprinting.

The unicellular Tetrahymena has receptors for hormones of higher vertebrates, produces these hormones, and their signal pathways are similar. The first encounter with a hormone in higher dose provokes the phenomenon of hormonal imprinting, by which the reaction of the cell is quantitatively modified. This modification is transmitted to the progeny generations. The duration of the single imprinter effect of two representative signal molecules, insulin and 5-HT (5-hydroxytryptamine), in two concentrations (10(-6) and 10(-15) M) were studied. The effects of imprinting were followed in 5 physiological indices: (i) insulin binding, (ii) 5-HT synthesis, (iii) swimming behaviour, (iv) cell growth and (v) chemotaxis in progeny generations 500 and 1000. The result of each index was different from the non-imprinted control functions, growth rate, swimming behaviour and chemotactic activity to insulin being enhanced, while others, e.g. synthesis and chemotactic responsiveness of 5-HT and the binding of insulin were reduced. This means that a function-specific heritable epigenetic change during imprinting occurs, and generally a single encounter with a femtomolar hormone concentration is enough for provoking durable and heritable imprinting in Tetrahymena. The experiments demonstrate the possibility of epigenetic effects at a unicellular level and call attention to the possibility that the character of unicellular organisms has changed through to the present day due to an enormous amount of non-physiological imprinter substances in their environment. The results - together with results obtained earlier in mammals - point to the validity of epigenetic imprinting effects throughout the animal world.

5-Fluorouracil accumulation in green microalgae and its biogenetic transfer into ciliate protozoan.

The study has demonstrated that anticancer drug 5-fluorouracil causes acute toxicity and interferes with the growth of green microalgae, Scenedesmus vacuolatus. It accumulates in microalgae biomass with bioaccumulation factor of 1.84 × 10(4) and further integrates into the DNA and RNA of microalgae. In addition, the labelled microalgae genome is transferred into protozoan Tetrahymena pyriformis on feeding and is retained in the food vacuoles of predator organisms. This biotransfer of labelled 5-fluorouracil via genomic material was evaluated using radioactivity in Tetrahymena cell pellets though radioactivity did not detect anticancer drug in the genome of the predator organism.

A hydrogen-bonding network formed by the B10-E7-E11 residues of a truncated hemoglobin from Tetrahymena pyriformis is critical for stability of bound oxygen and nitric oxide detoxification.

Truncated hemoglobins (trHbs) are distributed from bacteria to unicellular eukaryotes and have roles in oxygen transport and nitric oxide detoxification. It is known that trHbs exist in ciliates of the Tetrahymena group, but trHb structure and function remain poorly understood. To investigate trHb function with respect to stability of bound oxygen and protein structure, we measured the oxygen binding kinetics of Tetrahymena pyriformis trHb, and determined the crystal structure of the protein. The O(2) association and dissociation rate constants of T. pyriformis trHb were 5.5 µM(-1 )s(-1) and 0.18 s(-1), respectively. The autooxidation rate constant was 3.8 × 10(-3) h(-1). These values are similar to those of HbN from Mycobacterium tuberculosis. The three-dimensional structure of an Fe(II)-O(2) complex of T. pyriformis trHb was determined at 1.73-Å resolution. Tyr25 (B10) and Gln46 (E7) were hydrogen-bonded to a heme-bound O(2) molecule. Tyr25 donated a hydrogen bond to the terminal oxygen atom, whereas Gln46 hydrogen-bonded to the proximal oxygen atom. Furthermore, Tyr25 was hydrogen-bonded to the Gln46 and Gln50 (E11) residues. Mutations at Tyr25, Gln46, and Gln50 increased the O(2) dissociation and autooxidation rate constants. An Fe(III)-H(2)O complex of T. pyriformis trHb was formed following reaction of the Fe(II)-O(2) complex of T. pyriformis trHb, in a crystal state, with nitric oxide. This suggests that T. pyriformis trHb functions in nitric oxide detoxification.

[Functional characteristics of calcium-sensitive adenylyl cyclase of ciliate Tetrahymena pyriformis].

Calcium-sensitive forms of adenylyl cyclase (AC) were revealed in most vertebrates and invertebrates and also in some unicellular organisms, in particular ciliates. We have shown for the first time that calcium cations influence the AC activity of ciliate Tetrahymena pyriformis. These cations at the concentrations of 0.2-20 microM stimulated the enzyme activity, and maximum of catalytic effect was observed at 2 microM Ca2+. Calcium cations at a concentrations of 100 microM or higher inhibited the AC activity. Calmodulin antagonists W-5 and W-7 at the concentrations of 20-100 microM inhibited the catalytic effect induced by 5 microM Ca2+ and blocked the effect at higher concentrations of Ca2+. Chloropromazine, another calmodulin antagonist, reduced Ca2+-stimulated AC activity only at the concentrations of 200-1000 microM. AC stimulating effects of serotonin, EGF and cAMP increased in the presence of 5 microM Ca2+. AC stimulating effects of EGF, cAMP and insulin decreased in the presence of 100 microM Ca2+, and AC stimulating effect of cAMP decreased also in the presence of calmodulin antagonists (1 mM). At the same time, stimulating effect of D-glucose in the presence of Ca2+ and calmodulin antagonists did not change essentially. The data obtained speak in favor of the presence of calcium-sensitive forms of AC in ciliate T. pyriformis which mediate enzyme stimulation by EGF, cAMP, insulin, and serotonin.

TpMRK regulates cell division of Tetrahymena in response to oxidative stress.

TpMRK was identified as a stress-responsive mitogen activated protein kinase (MAPK)-related kinase and has been shown to play a critical role in the stress signalling in Tetrahymena cells. Here, we found that the mRNA expression of TpMRK was correlated with cell division of Tetrahymena with decreased expression occurring in cells prior to entering synchronous cell division induced by heat treatment. Notably, cell division was delayed with a lower division index of 40% after exposure to hydrogen peroxide while 85% of cells underwent cell division synchronously at 75 min after heat treatment without the oxidative exposure. Furthermore, inactivation of TpMRK signalling by p38 MAPK inhibitor SB203580 or MEK inhibitor PD 98059 partially derepressed cell division induced by hydrogen peroxide. Our data suggest that oxidative stimuli might cause aberration of synchronous cell division of Tetrahymena through activating the TpMRK cascade.

N-Methyl-D-aspartate receptor-mediated chemotaxis and Ca(2+) signaling in Tetrahymena pyriformis.

Although the ciliate Tetrahymena is a good model for the study of chemotaxis, its profound motility makes it difficult to monitor intracellular calcium (Ca(2+)) changes induced by chemotactic stimuli. In this study, we report a microfluidic-based chemotaxis system generating directional chemotactic gradients under highly viscous conditions, suppressing T. pyriformis motility, and allowing for the stable confocal imaging of changes in intracellular Ca(2+) in the ciliate. Once the viscous condition was achieved, directional chemical gradients were formed inside the center chamber via the release of N-methyl-d-aspartate (NMDA), a known chemoattractant, from the surrounding chemical reservoirs into the center chamber. As a result, intracellular Ca(2+) in the ciliate increased up to three-fold, and its distribution was skewed in the direction of NMDA stimulation. However, the Ca(2+) in ciliates pretreated with phospholipase C (PLC) or phosphatidylinositol-3-kinase (PI3K) blockers did not increase even after stimulation. Additionally, the PI3K blocker induced the secretion of granules, the size of which was dependent on the concentration of the blocker. Collectively, the results indicate that both PLC and PI3K perform pivotal roles in controlling the levels of intracellular Ca(2+) in T. pyriformis during chemotaxis.

Formation of categories from structure-activity relationships to allow read-across for risk assessment: toxicity of alpha,beta-unsaturated carbonyl compounds.

alpha,beta-Unsaturated carbonyl compounds are common environmental pollutants that are able to interact with proteins, enzymes, and DNA through various mechanisms. As such, they are able to stimulate a range of environmental toxicities and adverse health effects. In this study, a "category" of alpha,beta-unsaturated carbonyl compounds (aldehydes and ketones), assumed to act by a common mechanism of action (Michael type addition), was formed. This toxicologically and mechanistically important category was formed on the premise of structure-activity relationships. The acute aquatic toxicities to Tetrahymena pyriformis of compounds within the category were obtained in an effort to develop approaches for (qualitative) read-across. In addition, Salmonella typhimurium (strain TA100) mutagenicity data were analyzed to establish the structural differences between mutagenic and nonmutagenic compounds. These structural differences were compared with the structural characteristics of molecules associated with acute aquatic toxicity in excess of narcosis as well as other end points, for example, skin sensitization. The results indicate that a category can be formed that allows structural information and boundaries to be elucidated. This knowledge will guide future toxicity prediction within this category and assist in the development of category formation.

DNA content of Tetrahymena pyriformis as a biomarker for different toxic agents.

The toxicity of different substances was studied on the protozoan Tetrahymena pyriformis, using as an endpoint the DNA content of the macronucleus. Substances from various chemical classes were administered to the Tetrahymena cultures and then the DNA content of the protozoan macronuclei was measured by means of Image Analysis System. The increase in the DNA content of the nuclei is indicative of the stimulation of the mitotic process. Since mitogenic stimuli can substantially alter susceptibility to chemical carcinogenesis, the results of such experiments, which are cheap and easy to run, may contribute to the investigation of the toxic action of several substances on cellular level.