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Haptoglobin is a plasma protein of mammals that plays a crucial role in vascular homeostasis by binding free haemoglobin released from ruptured red blood cells. Trypanosoma brucei can exploit this by internalising haptoglobin-haemoglobin complex to acquire host haem. Here, we investigated the impact of haptoglobin deficiency (Hp-/-) on T. brucei brucei infection and the parasite´s capacity to internalise haemoglobin in a Hp-/- mouse model. The infected Hp-/- mice exhibited normal disease progression, with minimal weight loss and no apparent organ pathology, similarly to control mice. While the proteomic profile of mouse sera significantly changed in response to T. b. brucei , no differences in the infection response markers of blood plasma between Hp-/- and control Black mice were observed. Similarly, very few quantitative differences were observed between the proteomes of parasites harvested from Hp-/- and Black mice, including both endogenous proteins and internalised host proteins. While haptoglobin was indeed absent from parasites isolated from Hp-/-mice, haemoglobin peptides were unexpectedly detected in parasites from both Hp-/- and Black mice. Combined, the data support the dispensability of haptoglobin for haemoglobin internalisation by T. b. brucei during infection in mice. Since the trypanosomes knock-outs for their haptoglobin-haemoglobin receptor (HpHbR) internalised significantly less haemoglobin from Hp-/- mice compared to those isolated from Black mice, it suggests that T. b. brucei employs also an HpHbR-independent haptoglobin-mediated mode for haemoglobin internalisation. Our study reveals a so-far hidden flexibility of haemoglobin acquisition by T. b. brucei and offers novel insights into alternative haemoglobin uptake pathways., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Horáková, Vrbacký, Tesařová, Stříbrná, Pilný, Vavrušková, Vancová, Sobotka, Lukeš and Perner.)

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 H 2 O 2 -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., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The fast and selective separation method of intact monoacylglycerol (MG) and diacylglycerol (DG) isomers using chiral supercritical fluid chromatography-mass spectrometry (SFC-MS) was developed and employed to study lipase selectivity in the hydrolysis of triacylglycerols (TGs). The synthesis of 28 enantiomerically pure MG and DG isomers was performed in the first stage using the most commonly occurring fatty acids in biological samples such as palmitic, stearic, oleic, linoleic, linolenic, arachidonic, and docosahexaenoic acids. To develop the SFC separation method, different chromatographic conditions such as column chemistry, mobile phase composition and gradient, flow rate, backpressure, and temperature were carefully assessed. Our SFC-MS method used a chiral column based on a tris(3,5-dimethylphenylcarbamate) derivative of amylose and neat methanol as a mobile phase modifier, which provides baseline separation of all the tested enantiomers in 5 min. This method was used to evaluate hydrolysis selectivity of lipases from porcine pancreas (PPL) and Pseudomonas fluorescens (PFL) using nine TGs differing in acyl chain length (14-22 carbon atoms) and number of double bonds (0-6) and three DG regioisomer/enantiomers as hydrolysis intermediate products. PFL exhibited preference of the fatty acyl hydrolysis from the sn -1 position of TG more pronounced for the substrates with long polyunsaturated acyls, while PPL did not show considerable stereoselectivity to TGs. Conversely, PPL preferred hydrolysis from the sn -1 position of prochiral sn -1,3-DG regioisomer, whereas PFL exhibited no preference. Both lipases showed selectivity for the hydrolysis of outer positions of DG enantiomers. The results show complex reaction kinetics of lipase-catalyzed hydrolysis given by different stereoselectivities for substrates.

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 principle 1-4 . 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 tRNAs Glu 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 tRNA Trp CCA from five to four base pairs (bp). The canonical 5-bp tRNA Trp 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., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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., (© 2022. The Author(s).)

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., (© 2021 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

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 (tRNA Trp ) 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 tRNA Trp , 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.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Catalase is a widespread heme-containing enzyme, which converts hydrogen peroxide (H 2 O 2 ) to water and molecular oxygen, thereby protecting cells from the toxic effects of H 2 O 2 . 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 H 2 O 2 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., (© 2019 Federation of European Biochemical Societies.)

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., Competing Interests: V.Y. is an Academic Editor of Pathogens. Other authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 IC 50 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Sulfhydryl oxidase Erv1 is a ubiquitous and conserved protein of the mitochondrial intermembrane space that plays a role in the transport of small sulfur-containing proteins. In higher eukaryotes, Erv1 interacts with the mitochondrial import protein Mia40. However, Trypanosoma brucei lacks an obvious Mia40 homologue in its genome. Here we show by tandem affinity purification and mass spectrometry that in this excavate protist, Erv1 functions without a Mia40 homologue and most likely any other interaction partner. Down-regulation of TbErv1 caused a reduction of the mitochondrial membrane potential already within 24h to less than 50% when compared with control cells. The depletion of TbErv1 was accompanied by accumulation of trCOIV precursor, with a concomitant reduction of aconitase activity both in the cytosol and mitochondrion. Overall, TbErv1 seems to have a role in the mitochondrial translocation and Fe-S cluster assembly in the organelle., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The catalase gene is a virtually ubiquitous component of the eukaryotic genomes. It is also present in the monoxenous (i.e. parasitizing solely insects) trypanosomatids of the subfamily Leishmaniinae, which have acquired the enzyme by horizontal gene transfer from a bacterium. However, as shown here, the catalase gene was secondarily lost from the genomes of all Leishmania sequenced so far. Due to the potentially key regulatory role of hydrogen peroxide in the inter-stagial transformation of Leishmania spp., this loss seems to be a necessary prerequisite for the emergence of a complex life cycle of these important human pathogens. Hence, in this group of protists, the advantages of keeping catalase were uniquely outweighed by its disadvantages., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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 Tb Hrg in the procyclic stage. RNAi-induced down-regulation of Tb Hrg 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 Tb Hrg KO developed normally in the tsetse flies at rates comparable with wild-type cells. T. brucei cells overexpressing Tb Hrg 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 Tb Hrg indicated localization to the flagellar membrane, and scanning electron microscopy revealed more intense Tb Hrg accumulation toward the flagellar pocket. Based on these findings, we postulate that T. brucei senses heme levels via the flagellar Tb Hrg 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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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 IC 50 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.

ABC transporter mitochondrial 1 (Atm1) and multidrug resistance-like 1 (Mdl) are mitochondrial ABC transporters. Although Atm1 was recently suggested to transport different forms of glutathione from the mitochondrion, which are used for iron-sulfur (Fe-S) cluster maturation in the cytosol, the function of Mdl remains elusive. In Trypanosoma brucei, we identified one homolog of each of these genes, TbAtm and TbMdl, which were downregulated either separately or simultaneously using RNA interference. Individual depletion of TbAtm and TbMdl led to limited growth defects. In cells downregulated for TbAtm, the enzymatic activities of the Fe-S cluster proteins aconitase and fumarase significantly decreased in the cytosol but not in the mitochondrion. Downregulation of TbMdl did not cause any change in activities of the Fe-S proteins. Unexpectedly, the simultaneous downregulation of TbAtm and TbMdl did not result in any growth defect, nor were the Fe-S cluster protein activities altered in either the cytosolic or mitochondrial compartments. Additionally, TbAtm and TbMdl were able to partially restore the growth of the Saccharomyces cerevisiae Δatm1 and Δmdl2 null mutants, respectively. Because T. brucei completely lost the heme b biosynthesis pathway, this cofactor has to be obtained from the host. Based on our results, TbMdl is a candidate for mitochondrial import of heme b, which was markedly decreased in both TbMdl and TbAtm + TbMdl knockdowns. Moreover, the levels of heme a were strongly decreased in the same knockdowns, suggesting that TbMdl plays a key role in heme a biosynthesis, thus affecting the overall heme homeostasis in T. brucei., (© 2015 FEBS.)

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., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The importance of mitochondria for a typical aerobic eukaryotic cell is undeniable, as the list of necessary mitochondrial processes is steadily growing. Here, we summarize the current knowledge of mitochondrial biology of an early-branching parasitic protist, Trypanosoma brucei, a causative agent of serious human and cattle diseases. We present a comprehensive survey of its mitochondrial pathways including kinetoplast DNA replication and maintenance, gene expression, protein and metabolite import, major metabolic pathways, Fe-S cluster synthesis, ion homeostasis, organellar dynamics, and other processes. As we describe in this chapter, the single mitochondrion of T. brucei is everything but simple and as such rivals mitochondria of multicellular organisms., (Copyright © 2015. Published by Elsevier Inc.)

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.

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., (© 2013 John Wiley & Sons Ltd.)

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.

Transformation of the metabolically down-regulated mitochondrion of the mammalian bloodstream stage of Trypanosoma brucei to the ATP-producing mitochondrion of the insect procyclic stage is accompanied by the de novo synthesis of citric acid cycle enzymes and components of the respiratory chain. Because these metabolic pathways contain multiple iron-sulfur (FeS) proteins, their synthesis, including the formation of FeS clusters, is required. However, nothing is known about FeS cluster biogenesis in trypanosomes, organisms that are evolutionarily distant from yeast and humans. Here we demonstrate that two mitochondrial proteins, the cysteine desulfurase TbiscS and the metallochaperone TbiscU, are functionally conserved in trypanosomes and essential for this parasite. Knock-downs of TbiscS and TbiscU in the procyclic stage by means of RNA interference resulted in reduced activity of the marker FeS enzyme aconitase in both the mitochondrion and cytosol because of the lack of FeS clusters. Moreover, down-regulation of TbiscS and TbiscU affected the metabolism of procyclic T. brucei so that their mitochondria resembled the organelle of the bloodstream stage; mitochondrial ATP production was impaired, the activity of the respiratory chain protein complex ubiquinol-cytochrome-c reductase was reduced, and the production of pyruvate as an end product of glucose metabolism was enhanced. These results indicate that mitochondrial FeS cluster assembly is indispensable for completion of the T. brucei life cycle.

MRP1 and MRP2 are multifunctional mitochondrial RNA-binding proteins with a regulatory role in RNA editing and putative role(s) in RNA processing in Trypanosoma brucei. Silencing of MRP1 and/or MRP2 by RNA interference affected the assembly and functionality of respiratory complexes. The absence of several subunits of complexes I, III and IV resulted in their disintegration and subsequent decrease of specific activities and also caused a significant decrease of membrane potential. The overall respiration in the interfered cells decreased by only about 20%, since the trypanosome alternative oxidase effectively replaced the missing cytochromes and became the principal terminal oxidase.

The function, stability and mutual interactions of selected nuclear-encoded subunits of respiratory complexes III and IV were studied in the Trypanosoma brucei procyclics using RNA interference (RNAi). The growth rates and oxygen consumption of clonal cell lines of knock-downs for apocytochrome c1 (apoc1) and the Rieske Fe-S protein (Rieske) of complex III, and cytochrome c oxidase subunit 6 (cox6) of complex IV were markedly decreased after RNAi induction. Western analysis of mitochondrial lysates using specific antibodies confirmed complete elimination of the targeted proteins 4-6 days after induction. The Rieske protein was reduced in the apoc1 knock-down and vice versa, indicating a mutual interdependence of these components of complex III. However, another subunit of complex IV remained at the wild-type level in the cox6 knock-down. As revealed by two-dimensional blue native/SDS-PAGE electrophoresis, silencing of a single subunit resulted in the disruption of the respective complex, while the other complex remained unaffected. Membrane potential was reproducibly decreased in the knock-downs and the activities of complex III and/or IV, but not complex I, were drastically reduced, as measured by activity assays and histochemical staining. Using specific inhibitors, we have shown that in procyclics with depleted subunits of the respiratory complexes the flow of electrons was partially re-directed to the alternative oxidase. The apparent absence in T. brucei procyclics of a supercomplex composed of complexes I and III may represent an ancestral state of the respiratory chain.

Objective: The aim of this study was to evaluate the influence of Group B streptococcus (GBS) screening and intrapartum antibiotic prophylaxis to the incidence of GBS disease in newborns., Design: Prospective non-randomised study., Setting: IInd Clinic of Obstetric and Gynaecology LFUK and FNsP Ruzinov, Bratislava, Slovakia., Methods: We enrolled 3023 newborns (754 in the study group, 2269 in the control group), which were born between 1.9.2000 and 31.3.2003. In both groups we compared following variables: total number of infectious diseases in newborns, number and forms of GBS neonatal disease, number of perinatal death due to GBS disease., Results: There was no GBS disease in the study group of 754 newborns. Mothers of these newborns had one screening culture in 35-36th week of gestation. One swab was taken from vagina and anus. GBS carriers (161-21.4%) were administered i.v. intrapartum antibiotic prophylaxis with Penicillin G i.v., or, when allergy to penicillin was in history, with Clindamycin i.v. In the control group of 2269 newborns, whose mothers had no prevention, the incidence of GBS neonatal disease reached 7.5/1000 newborns (17 cases). The incidence of invasive GBS neonatal disease was 2.6/1000 newborns., Conclusion: The authors have noticed a significant decrease in incidence of GBS neonatal disease after implication of GBS screening and intrapartum antibiotic prophylaxis.

The model of oxidative stress induced by Fe/ascorbate in rat brain in vitro was used to compare the antioxidant capacity of known antioxidants. Creatine kinase (CK) was selected as a marker of protein injury in such studies. Of the antioxidant enzymes (catalase, superoxide dismutase), oxygen radical scavengers (mannitol, glutathione), and the chelator (EDTA) tested in this work and this system, only catalase and glutathione prevented the injury induced by oxidative stress, indicating that H2O2 and the glutathione peroxidase reaction were involved in the preventive effect. Additionally, the preventive effect of glutathione may be caused also by the fact that glutathione easily reacts with 4-hydroxynonenal (HNE), generated in rat brain homogenate, thus protecting CK from inactivation by this aldehyde. To find out whether and if at which concentrations CK may be oxidatively modified by HNE, pure CK was incubated in the presence of 10 and 64 micromol/l HNE for 30 min at 37 degrees C. The activity of CK incubated with HNE decreased significantly. Simultaneously, the protein carbonyls, determined by electrophoresis and immunoblotting increased at 10 micromol/l HNE or disappeared probably due to crosslinking of CK at 64 micromol/l HNE. The concentration of HNE in rat brain homogenates after oxidative stress was determined by HPLC and was in the range of 10-16 nmol/mg prot., corresponding to a concentration of 10-16 micromol/l HNE. This indicates that CK of rat brain homogenates oxidized by Fe/ascorbate may be impaired not only directly by oxygen radicals but also secondarily by HNE.

The authors performed a serological observation of toxoplasmosis by using the methods of IFT, CFT and ELISA in 118 mothers and their children with orofacial clefts. The results were compared with those gained in the control group of healthy mothers and their children. Titre means of toxoplasma antibodies in inflicted mothers and their children were higher than in the control group. The results were evaluated statistically. The authors assume that a certain proportion of orofacial clefts may be induced by toxoplasmosis. (Tab. 6, Ref. 13.).