Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment.

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle1-4. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAsGlu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNATrpCCA from five to four base pairs (bp). The canonical 5-bp tRNATrp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.

A mitochondrial cytidine deaminase is responsible for C to U editing of tRNATrp to decode the UGA codon in Trypanosoma brucei.

In kinetoplastid protists, all mitochondrial tRNAs are encoded in the nucleus and imported from the cytoplasm to maintain organellar translation. This also applies to the tryptophanyl tRNA (tRNATrp) encoded by a single-copy nuclear gene, with a CCA anticodon to read UGG codon used in the cytosolic translation. Yet, in the mitochondrion it is unable to decode the UGA codon specifying tryptophan. Following mitochondrial import of tRNATrp, this problem is solved at the RNA level by a single C34 to U34 editing event that creates the UCA anticodon, recognizing UGA. To identify the enzyme responsible for this critical editing activity, we scrutinized the genome of Trypanosoma brucei for putative cytidine deaminases as the most likely candidates. Using RNAi silencing and poisoned primer extension, we have identified a novel deaminase enzyme, named here TbmCDAT for mitochondrial Cytidine Deaminase Acting on tRNA, which is responsible for this organelle-specific activity in T. brucei. The ablation of TbmCDAT led to the downregulation of mitochondrial protein synthesis, supporting its role in decoding the UGA tryptophan codon.

Effect of selected organic materials on soil humic acids chemical properties.

Environmental friendly agricultural management has an urgent need for finding a sustainable strategy for the usage of different by-products from bioenergy production. These are either used as soil amendments or fertilizers. This study is aimed at evaluating if and how soil organic matter changes after the application of biochar, compost, and digestate. A pot experiment was conducted with Haplic Cambisol (low range arable soil) in Phytotron CLF PlantMaster (Wertingen, Germany). The chemical composition of isolated humic acids (HA) was determined by an inductively coupled plasma-mass spectrometer (ICP-MS). FT-IR spectroscopy and CHNS analysis were used for detailed chemical and optical characterization. Soil magnetic properties - radical concentration, g-parameters of radicals, and iron ions were evaluated by EPR spectroscopy. The results showed that amending arable soil with biochar, digestate and compost results in chemical and structural changes of humic substances. The radicals originated in biochar and digestate are built-in to the structure of the humic acid, which was confirmed by EPR g-parameter values. Despite a relatively high concentration of paramagnetic metal ions Fe and Mn the effect of semiquinone radical quenching was not observed. That suggests a conclusion that metal ions of studied amendments are binding in HA structure and did not disturb natural radical processes in the soil. It was also concluded that the effect of applied material depends mainly on its chemical properties and the soil type.

The Trypanosoma brucei TbHrg protein is a heme transporter involved in the regulation of stage-specific morphological transitions.

The human parasite Trypanosoma brucei does not synthesize heme de novo and instead relies entirely on heme supplied by its vertebrate host or its insect vector, the tsetse fly. In the host bloodstream T. brucei scavenges heme via haptoglobin-hemoglobin (HpHb) receptor-mediated endocytosis occurring in the flagellar pocket. However, in the procyclic developmental stage, in which T. brucei is confined to the tsetse fly midgut, this receptor is apparently not expressed, suggesting that T. brucei takes up heme by a different, unknown route. To define this alternative route, we functionally characterized heme transporter TbHrg in the procyclic stage. RNAi-induced down-regulation of TbHrg in heme-limited culture conditions resulted in slower proliferation, decreased cellular heme, and marked changes in cellular morphology so that the cells resemble mesocyclic trypomastigotes. Nevertheless, the TbHrg KO developed normally in the tsetse flies at rates comparable with wild-type cells. T. brucei cells overexpressing TbHrg displayed up-regulation of the early procyclin GPEET and down-regulation of the late procyclin EP1, two proteins coating the T. brucei surface in the procyclic stage. Light microscopy of immunostained TbHrg indicated localization to the flagellar membrane, and scanning electron microscopy revealed more intense TbHrg accumulation toward the flagellar pocket. Based on these findings, we postulate that T. brucei senses heme levels via the flagellar TbHrg protein. Heme deprivation in the tsetse fly anterior midgut might represent an environmental stimulus involved in the transformation of this important human parasite, possibly through metabolic remodeling.

Synthesis and characterization of new inhibitors of cholinesterases based on N-phenylcarbamates: In vitro study of inhibitory effect, type of inhibition, lipophilicity and molecular docking.

Based on current treatment of Alzheimer's disease, where the carbamate inhibitor Rivastigmine is used, two series of carbamate derivatives were prepared: (i) N-phenylcarbamates with additional carbamate group (1-12) and (ii) N-phenylcarbamates with monosaccharide moiety (13-24). All compounds were tested for the inhibitory effect on both of the cholinesterases, electric eel acetylcholinesterase (eeAChE) and butyrylcholinesterase from equine serum (eqBChE) and the inhibitory activity (expressed as IC50 values) was compared with that of the established drugs Galanthamine and Rivastigmine. The compounds with two carbamate groups 1-12 revealed higher inhibitory efficiency on both cholinesterases in compared with monosaccharide derived carbamates 13-24 and with Rivastigmine. The significant decrease of inhibitory efficiency on eqBChE (also for eeAChE but in less manner) was observed after deacetalization of monosaccharide. Moreover, the type of inhibitory mechanism of five chosen compounds was studied. It was found, that compounds with two carbamate groups act presumably via a mixed inhibitory mechanism and the compounds with monosaccharide moiety act as non-competitive inhibitors. The lipophilicity of tested compounds was determined using partition coefficient. Specific positions of the inhibitors in the binding sites of cholinesterases were determined using molecular modeling and the results indicate the importance of phenylcarbamate orientation in the catalytic gorges of both enzymes.

Iron-associated biology of Trypanosoma brucei.

BACKGROUND: Every eukaryote requires iron, which is also true for the parasitic protist Trypanosoma brucei, the causative agent of sleeping sickness in humans and nagana in cattle. T. brucei undergoes a complex life cycle during which its single mitochondrion is subject to major metabolic and morphological changes. SCOPE OF REVIEW: This review covers what is known about processes associated with iron-sulfur clusters and heme metabolism in T. brucei. We discuss strategies by which iron and heme are acquired and utilized by this model parasite, emphasizing the differences between its two life cycle stages residing in the bloodstream of the mammalian host and gut of the insect vector. Finally, the role of iron in the host-parasite interactions is discussed along with their possible exploitation in fighting these deadly parasites. MAJOR CONCLUSIONS: The processes associated with acquisition and utilization of iron, distinct in the two life stages of T. brucei, are fine tuned for the dramatically different host environment occupied by them. Although the composition and compartmentalization of the iron-sulfur cluster assembly seem to be conserved, some unique features of the iron acquisition strategies may be exploited for medical interventions against these parasites. GENERAL SIGNIFICANCE: As early-branching protists, trypanosomes and related flagellates are known to harbor an array of unique features, with the acquisition of iron being another peculiarity. Thanks to intense research within the last decade, understanding of iron-sulfur cluster assembly and iron metabolism in T. brucei is among the most advanced of all eukaryotes.

A common tRNA modification at an unusual location: the discovery of wyosine biosynthesis in mitochondria.

Establishment of the early genetic code likely required strategies to ensure translational accuracy and inevitably involved tRNA post-transcriptional modifications. One such modification, wybutosine/wyosine is crucial for translational fidelity in Archaea and Eukarya; yet it does not occur in Bacteria and has never been described in mitochondria. Here, we present genetic, molecular and mass spectromery data demonstrating the first example of wyosine in mitochondria, a situation thus far unique to kinetoplastids. We also show that these modifications are important for mitochondrial function, underscoring their biological significance. This work focuses on TyW1, the enzyme required for the most critical step of wyosine biosynthesis. Based on molecular phylogeny, we suggest that the kinetoplastids pathways evolved via gene duplication and acquisition of an FMN-binding domain now prevalent in TyW1 of most eukaryotes. These findings are discussed in the context of the extensive U-insertion RNA editing in trypanosome mitochondria, which may have provided selective pressure for maintenance of mitochondrial wyosine in this lineage.

Synthesis, characterization and in vitro evaluation of substituted N-(2-phenylcyclopropyl)carbamates as acetyl- and butyrylcholinesterase inhibitors.

A serie of O-substituted N-2-phenylcyclopropylcarbamates was prepared and characterized. These carbamates were tested as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). It was found, that these compounds exhibit moderate inhibition activity with values of IC50 in the range of 54.8-94.4 µM (for AChE) and up to 5.8 µM (for BChE). The AChE/BChE selectivity for each carbamate was calculated. These values varied from 0.50 to 9.46, two carbamate derivatives inhibited only AChE selectively. The most promising derivative was prepared in all optically pure forms (four isomers). It was found that individual stereoisomers differed only slightly in the inhibition ability. The cytotoxicity of all carbamates was evaluated using the standard in vitro test with Jurkat cells. With regard to their inhibition activity and cytotoxicity as well as easy preparation, O-substituted N-2-phenylcyclopropylcarbamates can be considered as promising compounds for potential medicinal applications.

The T. brucei TRM5 methyltransferase plays an essential role in mitochondrial protein synthesis and function.

All tRNAs undergo post-transcriptional chemical modifications as part of their natural maturation pathway. Some modifications, especially those in the anticodon loop, play important functions in translational efficiency and fidelity. Among these, 1-methylguanosine, at position 37 (m(1)G37) of the anticodon loop in several tRNAs, is evolutionarily conserved and participates in translational reading frame maintenance. In eukaryotes, the tRNA methyltransferase TRM5 is responsible for m(1)G formation in nucleus-encoded as well as mitochondria-encoded tRNAs, reflecting the universal importance of this modification for protein synthesis. However, it is not clear what role, if any, mitochondrial TRM5 serves in organisms that do not encode tRNAs in their mitochondrial genomes. These organisms may easily satisfy the m(1)G37 requirement through their robust mitochondrial tRNA import mechanisms. We have explored this possibility in the parasitic protist Trypanosoma brucei and show that down-regulation of TRM5 by RNAi leads to the expected disappearance of m(1)G37, but with surprisingly little effect on cytoplasmic translation. On the contrary, lack of TRM5 causes a marked growth phenotype and a significant decrease in mitochondrial functions, including protein synthesis. These results suggest mitochondrial TRM5 may be needed to mature unmethylated tRNAs that reach the mitochondria and that could pose a problem for translational fidelity. This study also reveals an unexpected lack of import specificity between some fully matured and potentially defective tRNA species.

Mitochondrial and nucleolar localization of cysteine desulfurase Nfs and the scaffold protein Isu in Trypanosoma brucei.

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

Both human ferredoxins equally efficiently rescue ferredoxin deficiency in Trypanosoma brucei.

Ferredoxins are highly conserved proteins that function universally as electron transporters. They not only require Fe-S clusters for their own activity, but are also involved in Fe-S formation itself. We identified two homologues of ferredoxin in the genome of the parasitic protist Trypanosoma brucei and named them TbFdxA and TbFdxB. TbFdxA protein, which is homologous to other eukaryotic mitochondrial ferredoxins, is essential in both the procyclic (= insect-transmitted) and bloodstream (mammalian) stage, but is more abundant in the active mitochondrion of the former stage. Depletion of TbFdxA caused disruption of Fe-S cluster biogenesis and lowered the level of intracellular haem. However, TbFdxB, which is present exclusively within kinetoplastid flagellates, was non-essential for the procyclic stage, and double knock-down with TbFdxA showed this was not due to functional redundancy between the two homologues. Heterologous expressions of human orthologues HsFdx1 and HsFdx2 fully rescued the growth and Fe-S-dependent enzymatic activities of TbFdxA knock-down. In both cases, the genuine human import signals allowed efficient import into the T. brucei mitochondrion. Given the huge evolutionary distance between trypanosomes and humans, ferredoxins clearly have ancestral and highly conserved function in eukaryotes and both human orthologues have retained the capacity to participate in Fe-S cluster assembly.

Intricate balance of dually-localized catalase modulates infectivity of Leptomonas seymouri (Kinetoplastea: Trypanosomatidae).

Nearly all aerobic organisms are equipped with catalases, powerful enzymes scavenging hydrogen peroxide and facilitating defense against harmful reactive oxygen species. In trypanosomatids, this enzyme was not present in the common ancestor, yet it had been independently acquired by different lineages of monoxenous trypanosomatids from different bacteria at least three times. This observation posited an obvious question: why was catalase so "sought after" if many trypanosomatid groups do just fine without it? In this work, we analyzed subcellular localization and function of catalase in Leptomonas seymouri. We demonstrated that this enzyme is present in the cytoplasm and a subset of glycosomes, and that its cytoplasmic retention is H2O2-dependent. The ablation of catalase in this parasite is not detrimental in vivo, while its overexpression resulted in a substantially higher parasite load in the experimental infection of Dysdercus peruvianus. We propose that the capacity of studied flagellates to modulate the catalase activity in the midgut of its insect host facilitates their development and protects them from oxidative damage at elevated temperatures.

Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells.

We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ-producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100(-/-) L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100(-/-) leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100(-/-) exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.

The convoluted history of haem biosynthesis.

The capacity of haem to transfer electrons, bind diatomic gases, and catalyse various biochemical reactions makes it one of the essential biomolecules on Earth and one that was likely used by the earliest forms of cellular life. Since the description of haem biosynthesis, our understanding of this multi-step pathway has been almost exclusively derived from a handful of model organisms from narrow taxonomic contexts. Recent advances in genome sequencing and functional studies of diverse and previously neglected groups have led to discoveries of alternative routes of haem biosynthesis that deviate from the 'classical' pathway. In this review, we take an evolutionarily broad approach to illuminate the remarkable diversity and adaptability of haem synthesis, from prokaryotes to eukaryotes, showing the range of strategies that organisms employ to obtain and utilise haem. In particular, the complex evolutionary histories of eukaryotes that involve multiple endosymbioses and horizontal gene transfers are reflected in the mosaic origin of numerous metabolic pathways with haem biosynthesis being a striking case. We show how different evolutionary trajectories and distinct life strategies resulted in pronounced tensions and differences in the spatial organisation of the haem biosynthesis pathway, in some cases leading to a complete loss of a haem-synthesis capacity and, rarely, even loss of a requirement for haem altogether.

Heme-deficient metabolism and impaired cellular differentiation as an evolutionary trade-off for human infectivity in Trypanosoma brucei gambiense.

Resistance to African trypanosomes in humans relies in part on the high affinity targeting of a trypanosome lytic factor 1 (TLF1) to a trypanosome haptoglobin-hemoglobin receptor (HpHbR). While TLF1 avoidance by the inactivation of HpHbR contributes to Trypanosoma brucei gambiense human infectivity, the evolutionary trade-off of this adaptation is unknown, as the physiological function of the receptor remains to be elucidated. Here we show that uptake of hemoglobin via HpHbR constitutes the sole heme import pathway in the trypanosome bloodstream stage. T. b. gambiense strains carrying the inactivating mutation in HpHbR, as well as genetically engineered T. b. brucei HpHbR knock-out lines show only trace levels of intracellular heme and lack hemoprotein-based enzymatic activities, thereby providing an uncommon example of aerobic parasitic proliferation in the absence of heme. We further show that HpHbR facilitates the developmental progression from proliferating long slender forms to cell cycle-arrested stumpy forms in T. b. brucei. Accordingly, T. b. gambiense was found to be poorly competent for slender-to-stumpy differentiation unless a functional HpHbR receptor derived from T. b. brucei was genetically restored. Altogether, we identify heme-deficient metabolism and disrupted cellular differentiation as two distinct HpHbR-dependent evolutionary trade-offs for T. b. gambiense human infectivity.

Suicidal Leishmania.

Leishmania are obligate intracellular parasites known to have developed successful ways of efficient immunity evasion. Because of this, leishmaniasis, a disease caused by these flagellated protists, is ranked as one of the most serious tropical infections worldwide. Neither prophylactic medication, nor vaccination has been developed thus far, even though the infection has usually led to strong and long-lasting immunity. In this paper, we describe a "suicidal" system established in Leishmania mexicana, a human pathogen causing cutaneous leishmaniasis. This system is based on the expression and (de)stabilization of a basic phospholipase A2 toxin from the Bothrops pauloensis snake venom, which leads to the inducible cell death of the parasites in vitro. Furthermore, the suicidal strain was highly attenuated during macrophage infection, regardless of the toxin stabilization. Such a deliberately weakened parasite could be used to vaccinate the host, as its viability is regulated by the toxin stabilization, causing a profoundly reduced pathogenesis.

Catalase compromises the development of the insect and mammalian stages of Trypanosoma brucei.

Catalase is a widespread heme-containing enzyme, which converts hydrogen peroxide (H2 O2 ) to water and molecular oxygen, thereby protecting cells from the toxic effects of H2 O2 . Trypanosoma brucei is an aerobic protist, which conspicuously lacks this potent enzyme, present in virtually all organisms exposed to oxidative stress. To uncover the reasons for its absence in T. brucei, we overexpressed different catalases in procyclic and bloodstream stages of the parasite. The heterologous enzymes originated from the related insect-confined trypanosomatid Crithidia fasciculata and the human. While the trypanosomatid enzyme (cCAT) operates at low temperatures, its human homolog (hCAT) is adapted to the warm-blooded environment. Despite the presence of peroxisomal targeting signal in hCAT, both human and C. fasciculata catalases localized to the cytosol of T. brucei. Even though cCAT was efficiently expressed in both life cycle stages, the enzyme was active in the procyclic stage, increasing cell's resistance to the H2 O2 stress, yet its activity was suppressed in the cultured bloodstream stage. Surprisingly, following the expression of hCAT, the ability to establish the T. brucei infection in the tsetse fly midgut was compromised. In the mouse model, hCAT attenuated parasitemia and, consequently, increased the host's survival. Hence, we suggest that the activity of catalase in T. brucei is beneficial in vitro, yet it becomes detrimental for parasite's proliferation in both invertebrate and vertebrate hosts, leading to an inability to carry this, otherwise omnipresent, enzyme.

An enigmatic catalase of Blastocrithidia.

Here we report that trypanosomatid flagellates of the genus Blastocrithidia possess catalase. This enzyme is not phylogenetically related to the previously characterized catalases in other monoxenous trypanosomatids, suggesting that their genes have been acquired independently. Surprisingly, Blastocrithidia catalase is less enzymatically active, compared to its counterpart from Leptomonas pyrrhocoris, posing an intriguing biological question why this gene has been retained in the evolution of trypanosomatids.