Molecular Cloning, Expression and Enzymatic Characterization of Tetrahymena thermophila Glutathione-S-Transferase Mu 34.

Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079 mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190 µmol/min/mg) as substrate. Enzyme kinetic analysis revealed Km values of 0.68 mM with GSH and 0.40 mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25 °C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage.

Use of E-64 cysteine protease inhibitor for the recombinant protein production in Tetrahymena thermophila.

Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 °C and 38 °C. In vitro protease activity analysis using secretome as protease enzyme source from E-64-supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 µM concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila. This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation.

Genome-wide identification and in-silico analysis of papain-family cysteine protease encoding genes in Tetrahymena thermophila.

Tetrahymena thermophila is a promising host for recombinant protein production, but its utilization in biotechnology is mostly limited due to the presence of intracellular and extracellular papain-family cysteine proteases (PFCPs). In this study, we employed bioinformatics approaches to investigate the T. thermophila PFCP genes and their encoded proteases (TtPFCPs), the most prominent protease family in the genome. Results from the multiple sequence alignment, protein modeling, and conserved motif analyses revealed that all TtPFCPs showed considerably high homology with mammalian cysteine cathepsins and contained conserved amino acid motifs. The total of 121 TtPFCP-encoding genes, 14 of which were classified as non-peptidase homologs, were found. Remaining 107 true TtPFCPs were divided into four distinct subgroups depending on their homology with mammalian lysosomal cathepsins: cathepsin L-like (TtCATLs), cathepsin B-like (TtCATBs), cathepsin C-like (TtCATCs), and cathepsin X-like (TtCATXs) PFCPs. The majority of true TtPFCPs (96 out of the total) were in TtCATL-like peptidase subgroup. Both phylogenetic and chromosomal localization analyses of TtPFCPs supported the hypothesis that TtPFCPs likely evolved through tandem gene duplication events and predominantly accumulated on micronuclear chromosome 5. Additionally, more than half of the identified TtPFCP genes are expressed in considerably low quantities compared to the rest of the TtPFCP genes, which are expressed at a higher level. However, their expression patterns fluctuate based on the stage of the life cycle. In conclusion, this study provides the first comprehensive in-silico analysis of TtPFCP genes and encoded proteases. The results would help designing an effective strategy for protease knockout mutant cell lines to discover biological function and to improve the recombinant protein production in T. thermophila.

Recombinant production of hormonally active human insulin from pre-proinsulin by Tetrahymena thermophila.

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.

Discovery of deoxyribonuclease II-like proteins in bacteria.

Deoxyribonuclease II (DNase II) is one of the earliest enzymes discovered in the history of biochemistry. Its role in apoptosis and development has been documented with great detail in eukaryotes. Prior in silico analyses showed its complete absence in bacterial genomes, with the exception of single bacterial genus: Burkholderia. It is therefore considered to be a eukaryotic enzyme. Here we show that the presence of DNase II is not limited to Burkholderia, as we find over one hundred DNase II-like sequences spanning 90 bacteria species belonging to 54 different genera and seven phyla. The majority of the significant hits (85%) come from Bacteroidetes and Proteobacteria phyla. Sequence analyses reveal that bacterial DNase II-like proteins possess a signature catalytic motif of eukaryotic DNase II. In phylogenetic analyses, we find that bacterial DNase II-like proteins are divided into two distinct clades. Our structural analyses reveal high levels of similarity between experimentally determined crystal structures of recombinant Burkholderia thailandensis DNase II and candidate bacterial DNase II-like proteins. We also biochemically show that Chromobacterium violaceum cell lysate possesses acidic DNase II-like activities. Collectively, our results indicate that DNase II has deeper evolutionary roots than previously thought. We argue that either some prokaryotic lineages have undergone losses of DNase II genes, resulting in rare conservation, or some lineages have acquired DNase II genes from eukaryotes through lateral gene transfer. We also discuss the possible involvement of DNase II as a part of an anti-phage defense system in bacteria.

Identification of glutathione-S-transferase m19 and m34 among responsive GST genes against 1-chloro-2,4-dinitrobenzene treatment of Tetrahymena thermophila.

Industrial xenobiotic pollutants have toxic effects on diverse organisms in their natural environments. This study aims to identify the Glutathione-S-transferases (GST) from Tetrahymena thermophila that are highly responsive to the treatment of synthetic substrate 1-chloro-2,4-dinitrobenzene (CDNB). The LD50 value of CDNB was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test as 0.079 mM at 9 h exposure. The glutathione affinity-purified 22 kDa and 23 kDa GSTs from CDNB-treated cells were identified as GSTm19 and GSTm34 with 2D-gel electrophoresis coupled MALDI-Tof MS/MS analysis. The specific activitiy of the affinity-purified GSTs was upregulated upon the treatment of 0.072 mM CDNB with the decreased cell survival. GSTm19 and GSTm34 had also upregulated the mRNA expression under the highest dose treatment. The high cell survival and elevated total GST enzyme activity at 9 h under CDNB doses could be the result of both transcriptional upregulations as well as post-translational modifications. As a result, the cell survival of Tetrahymena thermophila was significantly affected by CDNB exposure in a concentration-dependent manner with the effect of low-dose stimulation and high-dose inhibition.

The in vivo Tetrahymena thermophila extracellular glucose drop assay for characterization of mammalian insulin activity.

Insulin activity is generally determined by an in vivo rabbit blood glucose drop assay in research and industriel laboratories. The humane experimental techniques imply the use of alternative invertebrate organisms in place of animals, known as replacement rule of the 3Rs. In this study, we report an alternative in vivo extracellular glucose drop assay using unicellular invertebrate Tetrahymena thermophila to replace the use of rabbit and mouse. This assay has four major steps; growing cells, starving cells, treatment of cells and measurement of glucose drop. In this assay, 0.2 mg/ml of human, porcine and bovine insulins dropped extracellular glucose level to 16%, 14% and 12%, respectively in ten minutes. In addition, mammalian insulins respectively increased the cell area about 19%, 15%, and 16% at 6th hour with statistically significant effect on the cell growth, but not in the cell viability. The results showed that the in vivo Tetrahymena thermophila extracellular glucose drop assay could be used as an alternative assay to replace the mouse or the rabbit insulin blood glucose drop assay.

Characterization and use of Tetrahymena thermophila artificial chromosome 2 (TtAC2) constructed by biomimetic of macronuclear rDNA minichromosome.

Efficient expression vectors for unicellular ciliate eukaryotic Tetrahymena thermophila are still needed in recombinant biology and biotechnology applications. Previously, the construction of the T. thermophila Macronuclear Artificial Chromosome 1 (TtAC1) vector revealed additional needs for structural improvements such as better in vivo stability and maintenance as a recombinant protein expression platform. In this study, we designed an efficiently maintained artificial chromosome by biomimetic of the native macronuclear rDNA minichromosome. TtAC2 was constructed by sequential cloning of subtelomeric 3'NTS region (1.8 kb), an antibiotic resistance gene cassette (2 kb neo4), a gene expression cassette (2 kb TtsfGFP), rDNA coding regions plus a dominant C3 origin sequence (10.3 kb), and telomeres (2.4 kb) in a pUC19 backbone plasmid (2.6 kb). The 21 kb TtAC2 was characterized using fluorescence microscopy, qPCR, western blot and Southern blot after its transformation to vegetative T. thermophila CU428.2 strain, which has a recessive B origin allele. All experimental data show that circular or linear forms of novel TtAC2 were maintained as free replicons in T. thermophila macronucleus with or without antibiotic treatment. Notably, TtAC2 carrying strains expressed a TtsfGFP marker protein, demonstrating the efficacy and functionality of the protein expression platform. We show that TtAC2 is functionally maintained for more than two months, and can be efficiently used in recombinant DNA, and protein production applications.

Construction and dynamic characterization of a Tetrahymena thermophila macronuclear artificial chromosome.

Artificial chromosomes were previously generated for use in bacteria, protists, yeast and human cells. A Tetrahymena thermophila artificial chromosome could serve as a versatile platform to study diverse aspects of Tetrahymena biology and beyond. Here, we placed a C3-type rDNA replication origin and telomere sequences from T. thermophila into a pNeo4 vector, producing the first T. thermophila macronuclear artificial chromosome (TtAC1). Circular or linear forms of TtAC1 can be stably transformed into both vegetative and conjugative T. thermophila cells. Linear TtAC1 was stably double in copy number under antibiotic selection, but its copy number was dropping without antibiotic selection pressure. Southern blot, Real-Time PCR and E. coli retransformation analyses together showed that TtAC1 vector did not integrate into the macronuclear genome, and was maintained as a linear or a circular chromosome in T. thermophila macronucleus under antibiotic selection. The use of TtAC1 for recombinant protein production was demonstrated by western blot analysis of a secreted 27 kDa TtsfGFP-12XHis protein. We present the first macronuclear artificial chromosome with species-specific chromosomal elements for use in T. thermophila studies and to aid broad recombinant biotechnology applications.

Genome-wide analysis of the Tetrahymena thermophila glutathione S-transferase gene superfamily.

The ciliate Tetrahymena thermophila has a rapid response to detoxify xenobiotics, which presents opportunity to study the diversification of Glutathione S-Transferase superfamily. In-silico identification of putative GST genes were resulted with 70 GST genes; 49 TtGSTmu, 7 TtGSTomega, 5 TtGSTtheta, 2 TtGSTzeta, 4 TtMAPEG and 3 TtEF1G. TtGST superfamily has short intron carrying or intronless genes. The most expressed mRNAs of TtGST are limited to 4 members at all life stages. TtGST genes are widely distributed to all five micronuclear chromosomes with the highest diversified members from different classes in chromosome 4. The clustering and the orientation of some TtGSTs in the T. thermophila genome give clues about the recent gene duplication. Analysis of GSH affinity-purified GST proteins with Western blot and activity assay showed GST activity carrying purified TtGST populations. In conclusion, the enhanced genome capacity of TtGST superfamily may have evolved through improved GST enzymatic activity.

A comparative in-silico analysis of autophagy proteins in ciliates.

Autophagy serves as a turnover mechanism for the recycling of redundant and/or damaged macromolecules present in eukaryotic cells to re-use them under starvation conditions via a double-membrane structure known as autophagosome. A set of eukaryotic genes called autophagy-related genes (ATGs) orchestrate this highly elaborative process. The existence of these genes and the role they play in different eukaryotes are well-characterized. However, little is known of their role in some eukaryotes such as ciliates. Here, we report the computational analyses of ATG genes in five ciliate genomes to understand their diversity. Our results show that Oxytricha trifallax is the sole ciliate which has a conserved Atg12 conjugation system (Atg5-Atg12-Atg16). Interestingly, Oxytricha Atg16 protein includes WD repeats in addition to its N-terminal Atg16 domain as is the case in multicellular organisms. Additionally, phylogenetic analyses revealed that E2-like conjugating protein Atg10 is only present in Tetrahymena thermophila. We fail to find critical autophagy components Atg5, Atg7 and Atg8 in the parasitic ciliate Ichthyophthirius multifiliis. Contrary to previous reports, we also find that ciliate genomes do not encode typical Atg1 since all the candidate sequences lack an Atg1-specific C-terminal domain which is essential for Atg1 complex formation. Consistent with the absence of Atg1, ciliates also lack other members of the Atg1 complex. However, the presence of Atg6 in all ciliates examined here may rise the possibility that autophagosome formation could be operated through Atg6 in ciliates, since Atg6 has been shown as an alternative autophagy inducer. In conclusion, our results highlight that Atg proteins are partially conserved in ciliates. This may provide a better understanding for the autophagic destruction of the parental macronucleus, a developmental process also known as programmed nuclear death in ciliates.

Efficient expression of codon-adapted affinity tagged super folder green fluorescent protein for synchronous protein localization and affinity purification studies in Tetrahymena thermophila.

BACKGROUND: A superior Green Fluorescent Protein (GFP) mutant, known as superfolder GFP (sfGFP), is more soluble, faster folding, and is the brightest of the known GFP mutants. This study aimed to create a codon-adapted sfGFP tag (TtsfGFP) for simultaneous protein localization and affinity purification in Tetrahymena thermophila. RESULTS: In vivo fluorescence spectroscopic analyses of clones carrying a codon-adapted and 6 × His tagged TtsfGFP cassette showed approximately 2-4-fold increased fluorescence emission compared with the control groups at 3 h. Fluorescence microscopy also revealed that TtsfGFP reached its emission maxima at 100 min, which was much earlier than controls expressing EGFP and sfGFP (240 min). A T. thermophila ATP-dependent DNA ligase domain containing hypothetical gene (H) was cloned into the 3' end of 6 × His-TtsfGFP to assess the affinity/localization dual tag feature. Fluorescence microscopy of the 6 × His-TtsfGFP-H clone confirmed its localization in the macro- and micronucleus of vegetative T. thermophila. Simultaneous affinity purification of TtsfGFP and TtsfGFP-H with Ni-NTA beads was feasible, as shown by Ni-NTA purified proteins analysis by SDS-PAGE and western blotting. CONCLUSIONS: We successfully codon adapted the N-terminal 6 × His-TtsfGFP tag and showed that it could be used for protein localization and affinity purification simultaneously in T. thermophila. We believe that this dual tag will advance T. thermophila studies by providing strong visual traceability of the target protein in vivo and in vitro during recombinant production of heterologous and homologous proteins.

Identification of neutral and acidic deoxyribonuclease activities in Tetrahymena thermophila life stages.

Deoxyribonucleases (DNases) play a major role in apoptotic DNA fragmentation/degradation, and apoptotic-like DNA degradation is also observed during conjugation of the ciliate Tetrahymena thermophila; however, the characteristics of neutral and acidic DNases are still undefined in its life stages. Here, we report the biochemical characterization of DNase activities displayed in three different Tetrahymena life stages in a comparative manner. Maximum DNase activity of Tetrahymena was observed under acidic conditions, indicating that Tetrahymena has strong DNase II-like activities. Zymography revealed that Tetrahymena has at least five distinct DNase activity bands at 28, 32, 33.8, 35.5, and 69-kDa, and that the activities at 32 and 33.8-kDa were also secreted into starvation buffer. Cofactor analysis demonstrated that Mg(2+) exerted inhibitory effects on neutral DNase activities. Unexpectedly, Mg(2+) and Ca(2+) had favorable effects on acidic DNase activities. The DNase activity profile of conjugating Tetrahymena cells revealed that the 32 and 33.8-kDa activities at pH 5.0 increased from 14 to 18 h of conjugation, corresponding to the final resorption of the old macronucleus by lysosomal enzymes during programmed nuclear death (PND). Overall, we found that Tetrahymena DNases exhibit different biochemical properties and a possible involvement of DNase II-like activities in PND.

In silico identification and characterization of the MAPK family members of unicellular model eukaryote Tetrahymena thermophila.

The biological function and evolutionary diversity of the mitogen-activated protein kinase (MAPK) family have mostly been studied in fungi, animals and plants, with very limited information from lower eukaryotes. This study aimed to describe the MAPKs of unicellular Tetrahymena thermophila. Eight members of the T. thermophila MAPK (TtMPK) gene family, in addition to previously reported TtMPK1, TtMPK2 and TtMPK3, were identified bioinformatically using a T. thermophila genome database. Phylogenetic analysis assigned the TtMPKs into two major groups, ERK1/2-like (TtMPK1, 2, 3, 5, 6, 7, 8, and 9) as stress-responsive MAPKs for biotic and abiotic stresses, and ERK7/8-like (TtMPK4, 10, and 11) as cell-cycle-associated protein kinases for biotic factors. Semi-quantitative RT-PCR analysis of the TtMPKs showed high mRNA expression at 30°C; however, only TtMPK5 and TtMPK6 showed high expression at 37°C. Osmotic shock by 100mM NaCl only increased the expression of TtMPK2, whereas 20mM NaCl reduced the expression of all MPKs to almost zero. The results suggested that T. thermophila MAPKs are among the closest representatives of the ancestors of the eukaryotic MAPK family. Although no functional characterization of MPKs was performed, this study is the first report of the genome-wide MAPK family in T. thermophila.

Cloning, expression and characterization of a gene encoding mitogen activated protein kinase 2 (MPK2) from Tetrahymena thermophila.

Environmental effects and mitogens determine cell phenotype in eukaryotes mainly through MAPK pathways. However, MAPK signaling pathways in T. thermophila have not been studied comprehensively. This study aims to express recombinant MPK2, a MAPK from T. thermophila, in E. coli to characterize its kinase activity. MPK2 was cloned by RT-PCR using degenerate oligonucleotide primers and RACE method. The full-length cDNA of the MPK2 gene is 1705bp that includes 1281bp ORF coding for a putative protein of 426 amino acids having a mass of 50.2kDa. The putative MPK2 protein contains all eleven conserved subdomains that are characteristics of serine/threonine protein kinases, and a TDY motif, which is a putative dual phosphorylation site common in Protista. MPK2 displays highest 48% overall identity to human ERK5 (MAPK7). The expression vector pGEX4T-1-MPK2 was constructed by inserting the coding region of MPK2 cDNA into pGEX4T-1 after introducing the nine point mutations, and then transformed into E. coli BL21(DE3). Autophosphorylation of 76kDa GST-MPK2 at tyrosine residues was confirmed not only by Western blot using anti-phosphotyrosine monoclonal antibody but also by in vitro kinase assay. GST-MPK2 was also able to phosphorylate the artificial substrate myelin basic protein. This study concludes that the free-living unicellular protist T. thermophila MPK2 has commonly conserved MAPK enzyme features, possibly involved in the regulation of cell survival responding to abiotic or biotic stressors, and the production and movement of haploid gametic nuclei between pairs during conjugation.

Synthesis and antibacterial activity of tert-butyl [1-benzyl-2[(4-aryl-2-thiazolyl)hydrazono]ethyl]carbamate derivatives.

The increasing clinical importance of drug-resistant fungal and bacterial pathogens has lent additional urgency to microbiological research and new antibacterial compound development. For this purpose, new tert-butyl[1-benzyl-2[(4-aryl-2-thiazolyl)hydrazono]ethyl]carbamate derivatives were synthesized and evaluated for antibacterial activity. The reaction of Boc-L-phenylalaninal with thiosemicarbazide gave the thiosemicarbazone which furnished the title compounds by reaction with phenacyl bromides. The newly synthesized compounds were screened for antibacterial activity and toxicity. While microdilution broth susceptibility assay was used for the antibacterial activity evaluation of the compounds against the strains E. coli (NRRL B-3704), M. luteus (NRRL B-4375), B. cereus (NRRL B-3711), P. aeruginosa (NRRL B-23), and S. fecalis (NRRL B-14617), the Artemia salina 96-well assay was used to determine cytotoxicities of the compounds. Observations obtained from the bioassays showed that some of the compounds are highly active against E. coli, M. luteus, and B. cereus when compared with the control agent and showed low toxicity.