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Abstract A transmission-blocking vaccine targeting the sexual stages of Plasmodium species could play a key role in eradicating malaria. Multiple studies have identified the P. falciparum proteins Pfs25 and Pfs48/45 as prime targets for transmission-blocking vaccines. Although significant advances have been made in recombinant expression of these antigens, they remain difficult to produce at large scale and lack strong immunogenicity as subunit antigens. We linked a self-assembling protein, granule lattice protein 1 (Grl1p), from the ciliated protozoan, Tetrahymena thermophila , to regions of the ectodomains of either Pfs25 or Pfs48/45. We found that resulting protein chimera could be produced in E. coli as nanoparticles that could be readily purified in soluble form. When produced in the E. coli SHuffle strain, fusion to Grl1p dramatically increased solubility of target antigens while at the same time directing the formation of particles with diameters centering on 38 and 25 nm depending on the antigen. In a number of instances, co-expression with chaperone proteins and induction at a lower temperature further increased expression and solubility. Based on Western blotting and ELISA analysis, Pfs25 and Pfs48/45 retained their transmission-blocking epitopes within E. coli -derived particles, and the particles themselves elicited strong antibody responses in rabbits when given with an aluminum-based adjuvant. Antibodies against Pfs25-containing nanoparticles blocked parasite transmission in standard membrane-feeding assays. In conclusion, fusion to Grl1p can act as a solubility enhancer for proteins with limited solubility while retaining correct folding, which may be useful for applications such as the production of vaccines and other biologics. Highlights • Malaria antigens were fused with a self-assembling protein from T. thermophila. • Fusion dramatically increased the solubility of Pfs25 and Pfs48/45 in E. coli. • The fusion proteins formed particles and retained correct folding. • Antibodies raised against the fusion proteins blocked malaria transmission. [ABSTRACT FROM AUTHOR]

Tetrahymena is a unicellular microbial eukaryotic organism that has been used extensively in toxicology and environmental research. This work attempts to model for the first time the wiring of proteins involved in cellular mechanisms of Cd toxicity in Tetrahymena thermophila. 1975 high-confidence PPIs between 68 Cd-binding proteins and 422 partners were inferred through a novel structural systems biology approach that utilizes comparative analysis between Tetrahymena and other eukaryotes for which experimentally supported protein interactomes exist. The PPIs of the potential network were confirmed by known domain interactions in the Protein Data Bank and its topological characteristics were compared with publicly available experimental information for T. thermophila. To experimentally validate the robustness of the proposed PPI network, the interaction between the two most interconnected hub proteins was detected through GST pull-down assay. Potential effects on Tetrahymena's cellular and metabolic processes by PPIs involving Cd-binding proteins were uncovered. Furthermore, 244 PPIs in which Cd-binding proteins or/and their partners are encoded by orthologs of human disease genes in T. thermophila, but not in yeast, were identified and analyzed. The findings suggest that Tetrahymena could be possibly a useful model for an improved understanding of molecular mechanisms of Cd toxicity in human diseases. [ABSTRACT FROM AUTHOR]

Spatially separated populations of the same species often exhibit correlated fluctuations in abundance, a phenomenon known as spatial synchrony. Dispersal can generate spatial synchrony. In nature, most individuals disperse short distances with a minority dispersing long distances. The effect of occasional long distance dispersal on synchrony is untested, and theoretical predictions are contradictory. Occasional long distance dispersal might either increase both overall synchrony and the spatial scale of synchrony, or reduce them.We conducted a protist microcosm experiment to test whether occasional long distance dispersal increases or decreases overall synchrony and the spatial scale of synchrony.We assembled replicate 15‐patch ring metapopulations of the protist predator Euplotes patella and its protist prey Tetrahymena pyriformis. All metapopulations experienced the same dispersal rate, but differed in dispersal distance. Some metapopulations experienced strictly short distance (nearest neighbour) dispersal, others experienced a mixture of short‐ and long distance dispersal.Occasional long distance dispersal increased overall spatial synchrony and the spatial scale of synchrony for both prey and predators, though the effects were not statistically significant for predators. As predicted by theory, dispersal generated spatial synchrony by entraining the phases of the predator–prey cycles in different patches, a phenomenon known as phase locking.Our results are consistent with theoretical models predicting that occasional long distance dispersal increases spatial synchrony. However, our results also illustrate that the spatial scale of synchrony need not match the spatial scale of the processes generating synchrony. Even strictly short distance dispersal maintained high spatial synchrony for many generations at spatial scales much longer than the dispersal distance, thanks to phase locking. The authors show experimentally that even occasional long distance dispersal can synchronize population cycles on large spatial scales. This helps explain why population cycles in nature often are synchronized over distances much longer than the typical dispersal distance. Pictured is the main study organism, the ciliate protist Euplotes patella (photo by Jeremy Fox). [ABSTRACT FROM AUTHOR]

Tetrahymena mitochondrial cox1 barcodes and nuclear SSUrRNA sequences are particularly effective at distinguishing among its many cryptic species. In a project to learn more about Tetrahymena natural history, the majority of >1,000 Tetrahymena‐like fresh water isolates were assigned to established Tetrahymena species with the remaining assigned to 37 new species of Tetrahymena, nine new species of Dexiostoma and 12 new species of Glaucoma. Phylogenetically, all but three Tetrahymena species belong to the well‐established "australis" or "borealis" clades; the minority forms a divergent "paravorax" clade. Most Tetrahymena species are micronucleate, but others are exclusively amicronucleate. The self‐splicing intron of the LSUrRNA precursor is absent in Dexiostoma and Glaucoma and was likely acquired subsequent to the "australis/borealis" split; in some instances, its sequence is diagnostic of species. Tetrahymena americanis, T. elliotti, T. gruchyi n. sp., and T. borealis, together accounted for >50% of isolates, consistent with previous findings for established species. The biogeographic range of species found previously in Austria, China, and Pakistan was extended to the Nearctic; some species show evidence of population structure consistent with endemism. Most species were most frequently collected from ponds or lakes, while others, particularly Dexiostoma species, were collected most often from streams or rivers. The results suggest that perhaps hundreds of species remain to be discovered, particularly if collecting is global and includes hosts of parasitic forms. [ABSTRACT FROM AUTHOR]

Paramecium tetraurelia expresses four types of arginine kinase (AK1-AK4). In a previous study, we showed that AK3 is characterized by typical arginine substrate inhibition, where enzymatic activity markedly decreases near a concentration of 1 mM of arginine substrate. This is in sharp contrast to the three other AK types, which obey the Michaelis-Menten reaction curve. Since cellular arginine concentration in another ciliate Tetrahymena is estimated to be 3-15 mM in vivo, Paramecium AK3 likely functions in conditions that are strongly affected by substrate inhibition. The purpose of this work is to find some novel aspect on the kinetic mechanism of the substrate inhibition of Paramecium AK3 enzyme. Substrate inhibition kinetics for AK3 were analyzed using three models and their validity were evaluated with three static parameters (R2, AICc, and Sy.x). The most accurate model indicated that not only ES but also the SES complex reacts to form products, the latter being the complex with two substrates in the active center. The maximum reaction rate for the SES complex, VmaxSES = 30.4 µmol Pi/min/mg protein, was one-eighth of the ES complex, VmaxES = 241.7. The dissociation constant for the SES complex (KiSES: 0.34 mM) was two times smaller than that of the ES complex (KsES: 0.61 mM), suggesting that after the primary binding of the arginine substrate (ES complex formation), the binding of a second arginine to the secondarily induced inhibitory site is accelerated to form an SES complex with a lower VmaxSES. The same kinetics were used for the S79A, S80A, and V81A mutants. The results indicate that the S79 residue is significantly involved in the process of binding the second arginine substrate. Herein, the KiSES value was ten times (3.62 mM) the value for the wild-type (0.34 mM), weakening substrate inhibition. In contrast, VmaxES and VmaxSES values for the mutants decreased by one-third, except for the VmaxSES of the S79A mutant, which had a value that was comparable with the value for the wild-type. [ABSTRACT FROM AUTHOR]

Abstract During sexual reproduction or conjugation, ciliates form a specialized cell adhesion zone for the purpose of exchanging gametic pronuclei. Hundreds of individual membrane fusion events transform the adhesion zone into a perforated membrane curtain, the mating junction. Pronuclei from each mating partner are propelled through this fenestrated membrane junction by a web of short, cris-crossing microtubules. Pronuclear passage results in the formation of two breaches in the membrane junction. Following pronuclear exchange and karyogamy (fertilization), cells seal these twin membrane breaches thereby re-establishing cellular independence. This would seem like a straightforward problem: simply grow membrane in from the edges of each breach in a fashion similar to how animal cells "grow" their cytokinetic furrows or how plant cells construct a cell wall during mitosis. Serial section electron microscopy and 3-D electron tomography reveal that the actual mechanism is less straightforward. Each of the two membrane breaches transforms into a bowed membrane assembly platform. The resulting membrane protrusions continue to grow into the cytoplasm of the mating partner, traverse the cytoplasm in anti-parallel directions and make contact with the plasma membrane that flanks the mating junction. This investigation reveals the details of a novel, developmentally-induced mechanism of membrane disruption and restoration associated with pronuclear exchange and fertilization in the ciliate, Tetrahymena thermophila. Highlights • Tetrahymena thermophila , a ciliate, undergoes complex membrane dynamics during sexual reproduction and pronuclear exchange. • Mating cells initially form a specialized mating junction, where the two plasma membranes adhere. • The adhesion zone undergoes membrane fusion events creating 100s of membrane pores that expand, creating a membrane curtain. • Gamete pronuclei are propelled through this membrane curtain by a meshwork of cris-crossing short microtubules. • Following bilateral nuclear exchange, the junction is restored by an elaborate process of membrane folding and growth. [ABSTRACT FROM AUTHOR]

Gelsemine is an important toxic substance extracted from Gelsemium elegans, which has a lot of biological functions in cells and organisms, but its toxicity has been rarely reported in Tetrahymena thermophila. In this study, we used the protozoan T. thermophila as an experimental model to investigate the potential toxicity-induced mechanism of gelsemine in the unicellular eukaryote. Our results clearly showed gelsemine inhibited T. thermophila growth in a dose-dependent manner. This exposure also resulted in oxidative stress on T. thermophila cells and antioxidant enzyme levels were significantly altered at high gelsemine levels (p < 0.05). Gelsemine produced a slight apoptotic effect at the highest (0.8 mg/mL) gelsemine level used here (p < 0.05). Furthermore, the toxin-induced DNA damage in a dose-dependent manner. The ultrastructural analysis also revealed mitophagic vacuoles at 0.4 and 0.8 mg/mL levels of gelsemine exposure. Moreover, expressions of oxidative stress-related and MAP kinase genes were significantly changed after exposure to 0.8 mg/mL level of gelsemine (p < 0.05). Altogether, our results clearly show that gelsemine from G. elegans can inhibit the growth via inducing oxidative stress and DNA damage in T. thermophila cells. [ABSTRACT FROM AUTHOR]

The biotoxicity of perfluorooctane sulfonate (PFOS) has been a concern. However, the effects of PFOS on Tetrahymena thermophila , a unicellular model organism, remain unclear. This study aimed to investigate the toxicity and detoxification mechanism of PFOS in this protozoan. PFOS did not show prominent toxic effects on T. thermophila. Cell viability of T. thermophila can be concentration-dependently increased by PFOS. PFOS also increased the stability of cell membranes and the activity of lysosomes. However, PFOS inhibited efflux transporter activities. Most of the PFOS amount remained in the culture medium during the culture periods. Only a low amount of PFOS was absorbed by cells, where PFOS molecules were mainly combined with membrane proteins. The expressions of four membrane protein genes involved in transporting xenobiotics were analyzed by real time-PCR. The gene abcg25 was significantly up-regulated. The growth of abcg25 gene knockout protozoans under PFOS treatment was slightly inhibited. However, the amount of PFOS adsorbed by the knockout protozoans showed no significant difference from the Wild-type protozoans. We concluded that the ABCG25 protein might play a key role in preventing PFOS from entering the cell or being exported from the cells to protect T. thermophila against PFOS. However, ABCG25 was not the only membrane protein able to bind with PFOS. [Display omitted] • Adsorbed PFOS in Tetrahymena thermophila mainly combined with membrane proteins. • Gene abcg25 was upregulated under PFOS exposure. • Growth of abcg25 knockout protozoa (abcg25 KO) was inhibited under PFOS exposure. • No difference was found between abcg25 KO and normal cells on adsorbed PFOS amount. • ABCG25 was vital, but not the only protein in preventing PFOS from entering cells. [ABSTRACT FROM AUTHOR]

Alternative cell factories, such as the unicellular ciliate eukaryotic Tetrahymena thermophila, may be required for the production of protein therapeutics that are challenging to produce in conventional expression systems. T. thermophila (Tt) can secrete proteins with the post-translational modifications necessary for their function in humans. In this study, we tested if T. thermophila could process the human pre-proinsulin to produce hormonally active human insulin (hINS) with correct modifications. Flask and bioreactor culture of T. thermophila were used to produce the recombinant Tt-hINS either with or without an affinity tag from a codon-adapted pre-proinsulin sequence. Our results indicate that T. thermophila can produce a 6 kDa Tt-hINS monomer with the appropriate disulfide bonds after removal of the human insulin signal sequence or endogenous phospholipase A signal sequence, and the C-peptide of the human insulin. Additionally, Tt-hINS can form 12 kDa dimeric, 24 kDa tetrameric, and 36 kDa hexameric complexes. Tt-hINS-sfGFP fusion protein was localized to the vesicles within the cytoplasm and was secreted extracellularly. Assessing the affinity-purified Tt-hINS activity using the in vivo T. thermophila extracellular glucose drop assay, we observed that Tt-hINS induced a significant reduction (approximately 21 %) in extracellular glucose levels, indicative of its functional insulin activity. Our results demonstrate that T. thermophila is a promising candidate for the pharmaceutical and biotechnology industries as a host organism for the production of human protein drugs. • T. thermophila produces a 6 kDa human insulin (Tt-hINS) from pre-proinsulin. • The purified Tt-hINS has insulin activity based on results from the glucose drop assay. • The extracellular secretion of Tt-hINS reveals the removal of signal sequences. • Multimeric insulin Tt-hINS forms are 12 kDa, 24 kDa, and 36 kDa. • T. thermophila promises an alternative host for pharmaceutical industries. [ABSTRACT FROM AUTHOR]

The catalytic core of Group I self-splicing introns has been proposed to consist of two structural domains, P4-P6 and P3-P9. Each contains helical segments and conserved unpaired nucleotides, and the isolated P4-P6 domain has been shown to have substantial native tertiary structure. The proposed tertiary structure domains of the Tetrahymena intron were synthesized separately and shown to self-assemble into a catalytically active complex. Surprisingly, the concentration dependence of these reactions revealed that the domains interact with nanomolar apparent dissociation constants, even though there is no known base pairing between P4-P6 and P3-P9. This suggests that the domains interact through multiple tertiary contacts, the nature of which can now be explored in this system. For example, a circularly permuted version of the P4-P6 domain, which folds similarly to the native P4-P6 molecule, formed a stable but inactive complex. Interestingly, activity was demonstrated with the permuted molecule when nucleotides proposed to form a triple-strand interaction with P4 and P6 were restored as part of the P1-P3 substrate or as part of the P3-P9 RNA. Thus, beyond stabilization of the P4-P6 domain, the triple-strand region may facilitate correct orientation of the RNA domains or participate more directly in catalysis.

Multi-ciliated cells (MCCs) use polarized fields of undulating cilia (ciliary array) to produce fluid flow that is essential for many biological processes. Cilia are positioned by microtubule scaffolds called basal bodies (BBs) that are arranged within a spatially complex 3-dimensional geometry (3D). Here, we develop a robust and automated computational image analysis routine to quantify 3D BB organization in the ciliate, Tetrahymena thermophila. Using this routine, we generate the first morphologically constrained 3D reconstructions of Tetrahymena cells and elucidate rules that govern the kinetics of MCC organization. We demonstrate the interplay between BB duplication and cell size expansion through the cell cycle. In mutant cells, we identify a potential BB surveillance mechanism that balances large gaps in BB spacing by increasing the frequency of closely spaced BBs in other regions of the cell. Finally, by taking advantage of a mutant predisposed to BB disorganization, we locate the spatial domains that are most prone to disorganization by environmental stimuli. Collectively, our analyses reveal the importance of quantitative image analysis to understand the principles that guide the 3D organization of MCCs.

Protozoa are natural predators of bacteria, but some bacteria can evade digestion once phagocytosed. Some of these resistant bacteria can be packaged in the fecal pellets produced by protozoa, protecting them from physical stresses and biocides. Depending on the bacteria and protozoa involved in the packaging process, pellets can have different morphologies. In the present descriptive study, we evaluated the packaging process with 20 bacteria that have never been tested before for packaging by ciliates. These bacteria have various characteristics (shape, size, Gram staining). All of them appear to be included in pellets produced by the ciliates Tetrahymena pyriformis and/or T. thermophila in at least one condition tested. We then focused on the packaging morphology of four of these bacteria. Our results demonstrated that, as shown previously for Mycobacterium smegmatis, the packaging of Microbacterium oxydans, Micrococcus luteus, and Cupriavidus sp. was formed of a single layer of material. The packaging of Cellulosimicrobiumfunkei was made of indistinguishable material. A different pellet morphology was obtained for each of the four bacterial strains studied. The ingestion of small bacteria resulted in rounder, denser, and more regular pellets. These results support the idea that bacteria packaging is a relatively widespread phenomenon.

In the present work, an extensive analysis of the putative glutathione peroxidases (GPx) of the eukaryotic microorganism model Tetrahymena thermophila is carried out. A comparative analysis with GPx present in other Tetrahymena species and other very taxonomically diverse ciliates is also performed. A majority of ciliate GPx have replaced the selenocysteine (Sec) by Cys in its catalytic center, so they can be considered as phospholipid hydroperoxide glutathione peroxidases (PHGPx). Selenocysteine insertion sequence (SECIS) elements have been detected in several ciliate GPx that do not incorporate Sec in their amino acid sequences, and conversely, in other ciliate GPx with Sec, no SECIS elements are detected. These anomalies are analyzed and discussed. From the phylogenetic analysis using the ciliate GPx amino acid sequences, the existence of extensive intra- and interspecific gene duplications that produced multiple GPx isoforms in each species is inferred. The ancestral character of the selenoproteins is also corroborated. The analysis by qRT-PCR of six selected T. thermophila GPx genes has shown a quantitative differential expression between them, depending on the stressor (oxidizing agents, apoptotic inducer or metals) and the time of exposure.

Endosymbiosis between coccoid green algae and ciliates are widely distributed and occur in various phylogenetic lineages among the Ciliophora. Most mixotrophic ciliates live in symbiosis with different species and genera of the so-called Chlorella clade (Trebouxiophyceae). The mixotrophic ciliates can be differentiated into two groups: (i) obligate, which always live in symbiosis with such green algae and are rarely algae-free and (ii) facultative, which formed under certain circumstances such as in anoxic environments an association with algae. A case of the facultative endosymbiosis is found in the recently described species of Tetrahymena, T. utriculariae, which lives in the bladder traps of the carnivorous aquatic plant Utricularia reflexa. The green endosymbiont of this ciliate belonged to the genus Micractinium. We characterized the isolated algal strain using an integrative approach and compared it to all described species of this genus. The phylogenetic analyses using complex evolutionary secondary structure-based models revealed that this endosymbiont represents a new species of Micractinium, M. tetrahymenae sp. nov., which was further confirmed by the ITS2/CBC approach.

Katanin-like 2 protein (Katnal2) orthologs have a tripartite domain organization. Two highly conserved regions, an N-terminal LisH (Lis-homology) domain and a C-terminal AAA catalytic domain, are separated by a less conserved linker. The AAA domain of Katnal2 shares the highest amino acid sequence homology with the AAA domain of the canonical katanin p60. Katnal2 orthologs are present in a wide range of eukaryotes, from protists to humans. In the ciliate Tetrahymena thermophila, a Katnal2 ortholog, Kat2, co-localizes with the microtubular structures, including basal bodies and ciliary outer doublets, and this co-localization is sensitive to levels of microtubule glutamylation. The functional analysis of Kat2 domains suggests that an N-terminal fragment containing a LisH domain plays a role in the subcellular localization, dimerization, and stability of Kat2.

Persistence of Aeromonas hydrophila in aquatic environments is the principle cause of fish hemorrhagic septicemia. Protistan predation has been considered to be a strong driving force for the evolution of bacterial defense strategies. In this study, we investigated the adaptive traits of A. hydrophila NJ-35, a carp pathogenic strain, in response to Tetrahymena thermophila predation. After subculturing with Tetrahymena, over 70% of A. hydrophila colonies were small colony variants (SCVs). The SCVs displayed enhanced biofilm formation, adhesion, fitness, and resistance to bacteriophage infection and oxidative stress as compared to the non-Tetrahymena-exposed strains. In contrast, the SCVs exhibited decreased intracellular bacterial number in RAW264.7 macrophages and were highly attenuated for virulence in zebrafish. Considering the outer membrane proteins (OMPs) are directly involved in bacterial interaction with the external surroundings, we investigated the roles of OMPs in the antipredator fitness behaviors of A. hydrophila. A total of 38 differentially expressed proteins were identified in the SCVs by quantitative proteomics. Among them, three lipoproteins including SurA, Slp, and LpoB, and a serine/threonine protein kinase (Stpk) were evidenced to be associated with environmental adaptation of the SCVs. Also, the three lipoproteins were involved in attenuated virulence of SCVs through the proinflammatory immune response mediated by TLR2. This study provides an important contribution to the understanding of the defensive traits of A. hydrophila against protistan predators.

Emerging models of telomere and associated proteins in Tetrahymena have broadened our understanding of telomeric processes. In this issue of Structure , Ma et al. pinpoint a region in p50 that associates with the CST complex. The interaction mediated by p50 is crucial for the optimal positioning of TERT to maintain homeostasis at the chromosome ends. [ABSTRACT FROM AUTHOR]

The Tetrahymena ribozyme has been shown to catalyze an RNA polymerase-like reaction in which an RNA primer is extended by the sequential addition of pN nucleotides derived from GpN dinucleotides, where N = A, C, or U. Here, we show that this reaction is influenced by the presence of a template; bases that can form Watson-Crick base pairs with a template add as much as 25-fold more efficiently than mismatched bases. A mutant enzyme with an altered guanosine binding site can catalyze template-directed primer extension with all four bases when supplied with dinucleotides of the form 2-aminopurine-pN.

The substrate specificities of dynein, kinesin, and myosin substrate turnover activity and cytoskeletal filament-driven translocation were examined using 15 ATP analogues. The dyneins were more selective in their substrate utilization than bovine brain kinesin or muscle heavy meromyosin, and even different types of dyneins, such as 14S and 22S dynein from Tetrahymena cilia and the beta-heavy chain-containing particle from the outer-arm dynein of sea urchin flagella, could be distinguished by their substrate specificities. Although bovine brain kinesin and muscle heavy meromyosin both exhibited broad substrate specificities, kinesin-induced microtubule translocation varied over a 50-fold range in speed among the various substrates, whereas heavy meromyosin-induced actin translocation varied only by fourfold. With both kinesin and heavy meromyosin, the relative velocities of filament translocation did not correlate well with the relative filament-activated substrate turnover rates. Furthermore, some ATP analogues that did not support the filament translocation exhibited filament-activated substrate turnover rates. Filament-activated substrate turnover and power production, therefore, appear to become uncoupled with certain substrates. In conclusion, the substrate specificities and coupling to motility are distinct for different types of molecular motor proteins. Such nucleotide "fingerprints" of enzymatic activities of motor proteins may prove useful as a tool for identifying what type of motor is involved in powering a motility-related event that can be reconstituted in vitro.

Espinoza-Vergara et al. unveiled a novel transmission mode of Vibrio cholerae based on environmental protozoan predation, which the bacterial pathogen evades via its release in 'expelled food vacuoles.' Vacuole-enclosed bacteria are not only fairly protected against environmental stressors, but also show enhanced intestinal colonization fitness upon oral ingestion. [ABSTRACT FROM AUTHOR]

The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprising a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying their structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, whereas the lipid scramblase Scr7p colocalizes with Vps8Dp. The H+-ATPase subunit Vma4 is spongiome specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools versus lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. Although the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome might be isolated from bulk endocytic input., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Bacterivorous ciliates and lytic bacteriophages are two major predators in aquatic environments, competing for the same type of prey. This study investigated the possible interaction of these different microorganisms and their influence on the activity of each other. Therefore, two bacterivorous ciliates, Paramecium sp. RB1 and Tetrahymena sp. RB2, were used as representative ciliates; a T4-like Escherichia coli targeting lytic bacteriophage as a model virus; and E. coli ATCC 25922 as a susceptible bacterial host and prey. The growth of the two ciliates with E. coli ATCC 25922 as prey was affected by the presence of phage particles. The grazing activity of the two ciliates resulted in more than a 99% reduction of the phage titer and bacterial cell numbers. However, viable phage particles were recovered from individual washed cells of the two ciliates after membrane filtration. Therefore, ciliates such as Paramecium sp. RB1 and Tetrahymena sp. RB2 can remove bacteriophages present in natural and artificial waters by ingesting the viral particles and eliminating bacterial host cells required for viral replication. The ingestion of phage particles may marginally contribute to the nutrient supply of the ciliates. However, the interaction of phage particles with ciliate cells may contribute to the transmission of bacteriophages in aquatic environments., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The multimolecular assembly of three-dimensionally structured proteins forms their quaternary structures, some of which have high geometric symmetry. The size and complexity of protein quaternary structures often increase in a hierarchical manner, with simpler, smaller structures serving as units for larger quaternary structures. In this study, we exploited oligomerization of a ribozyme cyclic trimer to achieve larger ribozyme-based RNA assembly. By installing kissing loop (KL) interacting units to one-, two-, or three-unit RNA molecules in the ribozyme trimer, we constructed dimers, open-chain oligomers, and branched oligomers of ribozyme trimer units. One type of open-chain oligomer preferentially formed a closed tetramer containing 12 component RNAs to provide 12 ribozyme units. We also observed large assembly of ribozyme trimers, which reached 1000 nm in size.

Limited dispersal is classically considered as a prerequisite for ecological specialization to evolve, such that generalists are expected to show greater dispersal propensity compared with specialists. However, when individuals choose habitats that maximize their performance instead of dispersing randomly, theory predicts dispersal with habitat choice to evolve in specialists, while generalists should disperse more randomly. We tested whether habitat choice is associated with thermal niche specialization using microcosms of the ciliate Tetrahymena thermophila, a species that performs active dispersal. We found that thermal specialists preferred optimal habitats as predicted by theory, a link that should make specialists more likely to track suitable conditions under environmental changes than expected under the random dispersal assumption. Surprisingly, generalists also performed habitat choice but with a preference for suboptimal habitats. Since this result challenges current theory, we developed a metapopulation model to understand under which circumstances such a preference for suboptimal habitats should evolve. We showed that competition between generalists and specialists may favor a preference for niche margins in generalists under environmental variability. Our results demonstrate that the behavioral dimension of dispersal--here, habitat choice--fundamentally alters our predictions of how dispersal evolve with niche specialization, making dispersal behaviors crucial for ecological forecasting facing environmental changes. [ABSTRACT FROM AUTHOR]

The mechanisms that regulate γ‐tubulin, including its post‐translational modifications, are poorly understood. γ‐Tubulin is important for the duplication of centrioles and structurally similar basal bodies (BBs), organelles which contain a ring of nine triplet microtubules. The ciliate Tetrahymena thermophila carries hundreds of cilia in a single cell and provides an excellent model to specifically address the role of γ‐tubulin in the BBs assembly and maintenance. The genome of Tetrahymena contains a single γ‐tubulin gene. We show here that there are multiple isoforms of γ‐tubulin that are likely generated by post‐translational modifications. We identified evolutionarily conserved serine and threonine residues as potential phosphosites of γ‐tubulin, including S80, S129, S131, T283, and S360. Several mutations that either prevent (S80A, S131A, T283A, S360A) or mimic (T283D) phosphorylation were conditionally lethal and at a higher temperature phenocopied a loss of γ‐tubulin. Cells that overproduced S360D γ‐tubulin displayed phenotypes consistent with defects in the microtubule‐dependent functions, including an asymmetric division of the macronucleus and abnormalities in the pattern of BB rows, including gaps, fragmentation, and misalignment. In contrast, overexpression of S129D γ‐tubulin affected the orientation, docking, and structure of the BBs, including a loss of either the B‐ or C‐subfibers or the entire triplets. We conclude that conserved potentially phosphorylated amino acids of γ‐tubulin are important for either the assembly or stability of BBs. [ABSTRACT FROM AUTHOR]

Tetrahymena can facilitate plasmid transfer among Escherichia coli or from E. coli to Salmonella Enteritidis via vesicle accumulation. In this study, whether ciliates promote the interactive transfer of plasmids encoding blaIMP‐1 between fecal E. coli and environmental Aeromonas caviae was investigated. Both bacteria were mixed with or without ciliates and incubated overnight at 30°C. The frequency of plasmid‐acquired bacteria was estimated by colony counts using an agar plate containing ceftazidim (CAZ) followed by determination of the minimum inhibitory concentration (MIC). Cultures containing ciliates interactively transferred the plasmid between E. coli and Aeromonas with a frequency of 10−4 to 10−5. All plasmid‐acquired bacteria showed a MIC against CAZ of >128 μg/mL and the plasmid transfer was confirmed by PCR amplification of the blaIMP‐1 gene. Fluorescent observation showed that both bacteria accumulated in the same vesicle and that transwell sequestering significantly decreased the transfer frequency. Although ciliates preferentially ingested E. coli rather than A. caviae, both bacteria were co‐localized into the same vesicles of ciliates, indicating that their meeting is associated with the gene transfer. Thus, ciliates interactively promote plasmid transfer between E. coli and A. caviae. The results of this study will facilitate control of the spread of multiple‐antibiotic resistant bacteria. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Six multi-target compounds with potential anti-AD properties have been studied. • BACE 1 inhibitory assay shows moderate to good activity for some of them. • Theoretical docking studies provide insights on the compound-protein interaction. • The compounds bind Cu2+ selectively over other biologically relevant metal ions. • The multi-target compounds are excellent antioxidant agents. • ThT and turbidity assays reveal promising anti-amyloidogenic properties. Abstract The lack of any effective therapy along with the aging world population anticipates a growth of the worldwide incidence of Alzheimer’s disease (AD) to more than 100 million cases by 2050. Accumulation of extracellular amyloid-β (Aβ) plaques, intracellular tangles in the brain, and formation of reactive oxygen species (ROS) are the major hallmarks of the disease. In the amyloidogenic process, a β-secretase, known as BACE 1, plays a fundamental role in the production of Aβ fragments, and therefore, inhibition of such enzymes represents a major strategy for the rational design of anti-AD drugs. In this work, a series of four multi-target compounds (1 – 4), inspired by previously described ionophoric polyphenols, have been synthesized and studied. These compounds have been designed to target important aspects of AD, including BACE 1 enzymatic activity, Aβ aggregation, toxic concentrations of Cu2+ metal ions and/or ROS production. Two other compounds (5 and 6), previously reported by some of us as antimalarial agents, have also been studied because of their potential as multi-target species against AD. Interestingly, compounds 3 and 5 showed moderate to good ability to inhibit BACE 1 enzymatic activity in a FRET assay, with IC 50 ′s in the low micromolar range (4.4 ± 0.3 and 1.7 ± 0.3 μM, respectively), comparable to other multi-target species, and showing that the observed activity was in part due to a competitive binding of the compounds at the active site of the enzyme. Theoretical docking calculations overall agreed with FRET assay results, displaying the strongest binding affinities for 3 and 5 at the active site of the enzyme. In addition, all compounds selectively interacted with Cu2+ metal ions forming 2:1 complexes, inhibited the production of Aβ-Cu2+ catalyzed hydroxyl radicals up to a ∼100% extent, and scavenged AAPH-induced peroxyl radical species comparably to resveratrol, a compound used as reference in this work. Our results also show good anti-amyloidogenic ability: compounds 1 – 6 inhibited both the Cu2+-induced and self-induced Aβ(1–40) fibril aggregation to an extent that ranged from 31% to 77%, while they disaggregated pre-formed Aβ(1–40) mature fibrils up to a 37% and a 69% extent in absence and presence of Cu2+, respectively. Cytotoxicity was additionally studied in Tetrahymena thermophila and HEK293 cells, and compared to that of resveratrol, showing that compounds 1 – 6 display lower toxicity than that of resveratrol, a well-known non-toxic polyphenol. [ABSTRACT FROM AUTHOR]

Abstract: Endosymbiotic interactions are frequently found in nature, especially in the group of protists. Even though many endosymbioses have been studied in detail, little is known about the mechanistic origins and physiological prerequisites of endosymbiont establishment. A logical step towards the development of endocytobiotic associations is evading digestion and escaping from the host's food vacuoles. Surface properties of bacteria are probably involved in these processes. Therefore, we chemically modified the surface of a transformant strain of Escherichia coli prior to feeding to Tetrahymena pyriformis. N‐(3‐dimethylaminopropyl)‐N’‐ethylcarbodiimide allows any substance carrying amino‐ or carboxyl groups to be bound covalently to the bacterial surface by forming a peptide bond, thus, altering its properties biochemically and biophysically in a predictable manner. The effect of different traits on digestion of T. pyriformis was examined by fluorescence and transmission electron microscopy. The efficiency of digestion differs considerably depending on the coupled substances. Alkaline substances inhibit digestion partially, resulting in incomplete digestion and slightly enhanced escape rates. Increasing hydrophobicity leads to much higher escape frequencies. Both results point to possible mechanisms employed by pathogenic bacteria or potential endosymbionts in evading digestion and transmission to the host's cytoplasm. [ABSTRACT FROM AUTHOR]

In Tetrahymena , K antigens associate only with mature basal bodies and are expected to play important roles in the morphogenesis and function of the membrane skeleton around basal bodies, but these proteins have not been identified and their functions are unknown. Commercially available anti-human Rho GDP-dissociation inhibitor α (RhoGDIα) antibody (sc-33201) was accidentally found to show very similar immunofluorescence staining patterns to those of anti-K antigen antibodies, such as 424A8 and 10D12 mouse monoclonal antibodies, in Tetrahymena . A 40 kDa protein recognized by this antibody was partially purified and identified as granule lattice protein 1 (Grl1p) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. In immunoblotting experiments this antibody was suggested to recognize endogenous Grl1p. The three-dimensional structure of proGrl1p protein predicted by I-TASSER was similar to a spectrin family protein. Grl1 may be a K antigen and a spectrin-like protein in Tetrahymena . [ABSTRACT FROM AUTHOR]

Selenium (Se) is commonly recognized as a protective element with an antagonistic effect against mercury (Hg) toxicity. However, the mechanisms of this Hg–Se antagonism are complex and remain controversial. To gain insight into the Hg–Se antagonism, a type of unicellular eukaryotic protozoa ( Tetrahymena malaccensis , T. malaccensis ) was selected and individually or jointly exposed to two Hg and three Se species. We found that Se species showed different toxic effects on the proliferation of T. malaccensis with the toxicity following the order: selenite (Se(IV)) > selenomethionine (SeMeth) > selenate (Se(VI)). The Hg–Se antagonism in Tetrahymena was observed because the joint toxicity significantly decreased under co-exposure to highly toxic dosages of Hg and Se versus individual toxicity. Unlike Se(IV) and Se(VI), non-toxic dosage of SeMeth significantly decreased the Hg toxicity, revealing the influence of the Se species and dosages on the Hg–Se antagonism. Unexpectedly, inorganic divalent Hg (Hg 2 + ) and monomethylmercury (MeHg) also displayed detoxification towards extremely highly toxic dosages of Se, although their detoxifying efficiency was discrepant. These results suggested mutual Hg–Se detoxification in T. malaccensis , which was highly dependent on the dosages and species of both elements. As compared to other species, SeMeth and MeHg promoted the Hg–Se joint effects to a higher degree. Additionally, the Hg contents decreased for all the Hg–Se co-exposed groups, revealing a sequestering effect of Se towards Hg in T. malaccensis . [ABSTRACT FROM AUTHOR]

Analysis of drugs and pharmaceuticals in the environment is typically performed with non-chiral chromatographic techniques. The environmental risks posed by chiral compounds analysed in this way must therefore be assumed to be independent of chirality, meaning that each enantiomer is equally potent in toxicity and long-lived in stability. This manuscript examines the degradation of each of the four isomers of ephedrine in river simulating microcosms and links this to toxicity data obtained by exposing three different organisms ( D. magna, P. subcapitata and T. thermophila ) to each of the isomers individually. Microcosms showed that significant degradation only occurred in biotic conditions and that only two isomers ( 1R,2S- (-)-ephedrine, 1S,2S- (+)-pseudoephedrine) degraded significantly over a period of fourteen days. This is concerning because at least one of the non-degraded isomers ( 1S,2R- (+)-ephedrine) has been observed in wastewater effluent, which discharges directly into rivers, meaning these isomers could be persistent in the environment. We also observed formation of 1S,2R- (+)-ephedrine in single isomer 1R,2S- (-)-ephedrine river simulating microcosms. Human liver microsome assays and mass spectrometry based data mining revealed that 1S,2R- (+)-ephedrine is not human derived but it could be formed as a results of microbial metabolic processes. Across all three organisms tested the persistent isomers ( 1S,2R- (+)-ephedrine and 1R,2R -(-)-pseudoephedrine) were more toxic than those that undergo degradation; meaning that if these isomers are entering or formed in the environment they might represent a potentially hazardous contaminant. [ABSTRACT FROM AUTHOR]

In educational and research settings, Tetrahymena is an excellent model organism for engaging students to investigate function, morphology, structure, phagocytosis, and ciliary motion. Here, we present applications of Wright stain and Sytox green as useful low-cost tools for phenotypic analysis. We used heatfixed Tetrahymena followed by Wright-stain-labeled organelles at different stages of its life cycle. In addition, a low concentration of Wright stain, at 1 percent (vol/vol), enabled visualization of filled vacuoles with stain in live Tetrahymena. Furthermore, we identified that Sytox green fluorescence labels not only nuclei of pre-incubated cultures of Tetrahymena, but also nuclei and some notable cytoplasmic staining after heat fixation. These applications can be used alongside inverted, battery-operated, bright field, fluorescence microscopes (Miller et al., 2010), as well as Cellcams (Martin & Shin, 2016) for acquiring images and time-lapse movies. In the future, these useful approaches can be applied broadly in many lab inquiry settings, such as toxicology and molecular genetics. [ABSTRACT FROM AUTHOR]

Persistence of Aeromonas hydrophila in aquatic environments is the principle cause of fish hemorrhagic septicemia. Protistan predation has been considered to be a strong driving force for the evolution of bacterial defense strategies. In this study, we investigated the adaptive traits of A. hydrophila NJ-35, a carp pathogenic strain, in response to Tetrahymena thermophila predation. After subculturing with Tetrahymena, over 70% of A. hydrophila colonies were small colony variants (SCVs). The SCVs displayed enhanced biofilm formation, adhesion, fitness, and resistance to bacteriophage infection and oxidative stress as compared to the non-Tetrahymena-exposed strains. In contrast, the SCVs exhibited decreased intracellular bacterial number in RAW264.7 macrophages and were highly attenuated for virulence in zebrafish. Considering the outer membrane proteins (OMPs) are directly involved in bacterial interaction with the external surroundings, we investigated the roles of OMPs in the antipredator fitness behaviors of A. hydrophila. A total of 38 differentially expressed proteins were identified in the SCVs by quantitative proteomics. Among them, three lipoproteins including SurA, Slp, and LpoB, and a serine/threonine protein kinase (Stpk) were evidenced to be associated with environmental adaptation of the SCVs. Also, the three lipoproteins were involved in attenuated virulence of SCVs through the proinflammatory immune response mediated by TLR2. This study provides an important contribution to the understanding of the defensive traits of A. hydrophila against protistan predators. [ABSTRACT FROM AUTHOR]

Metallothioneins (MTs) are low-molecular-weight proteins with high Cys content and high metal-chelating ability. CdMT and CuMT subfamilies present different characteristics in Tetrahymena. To explore the effect of the cysteine arrangement and sequence length of MTs for binding different metal ions, MTT1, truncated MTT1 (TM1), MTT2, and truncated MTT2 (TM2) were expressed in E. coli. The half-maximal inhibiting concentrations (IC50) of Cd2+ and Cu+ for the recombinant strains were different. Furthermore, E. coli cells expressing MTT1 and TM1 exhibited higher accumulating ability for Cd2+ than cells expressing MTT2 and TM2. However, the opposite is true for Cu+. The binding ability of the different recombinant proteins to Cd2+ and Cu+ were also different. MTT1 and truncated mutant TM1 were the preference for Cd2+, whereas MTT2 and truncated mutant TM2 were the preference for Cu+ coordination. These results showed that metal ion tolerance and accumulation ability not only depended on cysteine arrangement pattern but also on sequence length of MT in Tetrahymena. [ABSTRACT FROM AUTHOR]

Self-assembly on target membranes is one of the important properties of all dynamin family proteins. Drp6, a dynaminrelated protein in Tetrahymena, controls nuclear remodelling and undergoes cycles of assembly/disassembly on the nuclear envelope. To elucidate the mechanism of Drp6 function, we have characterized its biochemical and biophysical properties using size exclusion chromatography, chemical cross-linking and electron microscopy. The results demonstrate that Drp6 readily forms high-molecular-weight self-assembled structures as determined by size exclusion chromatography and chemical cross-linking. Negative stain electron microscopy revealed that Drp6 assembles into rings and spirals at physiological ionic strength. We have also shown that the recombinant Drp6 expressed in bacteria is catalytically active and its GTPase activity is not enhanced by low salt. These results suggest that, in contrast to dynamins but similar to MxA, Drp6 self-assembles in the absence of membrane templates, and its GTPase activity is not affected by ionic strength of the buffer. We discuss the self-assembly structure of Drp6 and explain the basis for lack of membrane-stimulated GTPase activity. [ABSTRACT FROM AUTHOR]

Mutational analyses of axonemal dyneins are useful for elucidating the molecular mechanism of ciliary motility. This study demonstrates a mutation system for characterizing lethal P-loop mutations in Tetrahymena outer arm dynein (Dyh3p). The viable DYH3 -knockout (vKO-DYH3) cells isolated in this study enabled the examination of lethal mutations in P-loops 1 and 2. The P1 mutant dynein localized in the oral apparatus and the proximal region of the cilia, and the P2 mutant dynein localized only in the oral apparatus. Both results are different from the localization of wild-type Dyh3p. In addition, a co-precipitation assay showed that the P1 and P2 mutant dyneins did not dissociate from microtubules in ATP plus vanadate or in no-ATP conditions, in contrast to wild-type Dyh3p. This mutation system is useful for further molecular studies of axonemal dyneins and ciliary motility. [ABSTRACT FROM AUTHOR]

Abstract Group I intron ribozymes share common core elements that form a three-dimensional structure responsible for their catalytic activity. This core structure is unstable without assistance from additional factors that stabilize its tertiary structure. We examined biogenic triamine and tetraamine and also their fragments for their abilities to stabilize a structurally unstable group I ribozyme, ΔP5 ribozyme, derived from the Tetrahymena group I intron ribozyme by deleting its large activator module. Biogenic triamine (spermidine) and tetraamine (spermine) efficiently activated the ΔP5 ribozyme under conditions where the ribozyme was virtually inactive. These observations suggested that polyamines are promising small molecule modulators to activate and possibly inhibit the core catalytic ability of group I ribozymes. Highlights • Biogenic triamine and tetraamine efficiently activated the ∆P5 ribozyme. • Spermine (tetraamine) modestly inhibited the full-length Tetrahymena ribozyme. • Polyamines are promising small molecule modulators to activate and possibly inhibit the core catalytic ability of group I ribozymes. [ABSTRACT FROM AUTHOR]