Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.

• Klíčová slova
• Trypanosoma, Cilia, Ciliogenesis, Distal appendages, Flagella, Transition fibres,
• MeSH
• bazální tělíska metabolismus   MeSH
• časové faktory MeSH
• cilie genetika   metabolismus   MeSH
• flagella genetika   metabolismus   MeSH
• konzervovaná sekvence MeSH
• protozoální proteiny * genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• transport proteinů MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protozoální proteiny * MeSH

Inhibition of hypoxanthine-guanine-xanthine phosphoribosyltransferase activity decreases the pool of 6-oxo and 6-amino purine nucleoside monophosphates required for DNA and RNA synthesis, resulting in a reduction in cell growth. Therefore, inhibitors of this enzyme have potential to control infections, caused by Plasmodium falciparum and Plasmodium vivax, Trypanosoma brucei, Mycobacterium tuberculosis, and Helicobacter pylori. Five compounds synthesized here that contain a purine base covalently linked by a prolinol group to one or two phosphonate groups have Ki values ranging from 3 nM to >10 μM, depending on the structure of the inhibitor and the biological origin of the enzyme. X-ray crystal structures show that, on binding, these prolinol-containing inhibitors stimulated the movement of active site loops in the enzyme. Against TBr in cell culture, a prodrug exhibited an EC50 of 10 μM. Thus, these compounds are excellent candidates for further development as drug leads against infectious diseases as well as being potential anticancer agents.

• MeSH
• inhibitory enzymů * farmakologie   chemie   chemická syntéza   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• pentosyltransferasy * antagonisté a inhibitory   metabolismus   MeSH
• racionální návrh léčiv * MeSH
• Trypanosoma brucei brucei účinky léků   enzymologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• hypoxanthine-guanine-xanthine phosphoribosyltransferase MeSH Prohlížeč
• inhibitory enzymů * MeSH
• pentosyltransferasy * MeSH

Studies have shown that depending on the substitution pattern, microtubule (MT)-targeting 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) can produce different cellular responses in mammalian cells that may be due to these compounds interacting with distinct binding sites within the MT structure. Selected TPDs are also potently bioactive against the causative agent of human African trypanosomiasis, Trypanosoma brucei, both in vitro and in vivo. So far, however, there has been no direct evidence of tubulin engagement by these TPDs in T. brucei. Therefore, to enable further investigation of anti-trypanosomal TPDs, a TPD derivative amenable to photoaffinity labeling (PAL) was designed, synthesized, and evaluated in PAL experiments using HEK293 cells and T. brucei. The data arising confirmed specific labeling of T. brucei tubulin. In addition, proteomic data revealed differences in the labeling profiles of tubulin between HEK293 and T. brucei, suggesting structural differences between the TPD binding site(s) in mammalian and trypanosomal tubulin.

• Klíčová slova
• African trypanosomiasis, Trypanosoma brucei, microtubules, photoaffinity labeling, seventh site, triazolopyrimidines, vinca site,
• MeSH
• HEK293 buňky MeSH
• lidé MeSH
• proteomika MeSH
• pyrimidiny chemie   MeSH
• savci metabolismus   MeSH
• trypanocidální látky * chemie   MeSH
• Trypanosoma brucei brucei * metabolismus   MeSH
• trypanozomóza africká * farmakoterapie   MeSH
• tubulin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• pyrimidiny MeSH
• trypanocidální látky * MeSH
• tubulin MeSH

BACKGROUND: Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. RESULTS: We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. CONCLUSIONS: In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.

• Klíčová slova
• Blastocrithidia, In silico, Metabolic predictions, Non-canonical genetic code, Trypanosomatid,
• MeSH
• Eukaryota genetika   MeSH
• genetický kód MeSH
• paraziti * genetika   MeSH
• terminační kodon MeSH
• Trypanosoma brucei brucei * genetika   MeSH
• Trypanosomatina * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• terminační kodon MeSH

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• fosfofruktokinasy metabolismus   MeSH
• glukoneogeneze * genetika   MeSH
• glukosa metabolismus   MeSH
• glycerol metabolismus   MeSH
• lidé MeSH
• savci MeSH
• transaldolasa metabolismus   MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• uhlík metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• fosfofruktokinasy MeSH
• glukosa MeSH
• glycerol MeSH
• transaldolasa MeSH
• uhlík MeSH

UPF1-like helicases play roles in telomeric heterochromatin formation and X-chromosome inactivation, and also in monogenic variant surface glycoprotein (VSG) expression via VSG exclusion-factor-2 (VEX2), a UPF1-related protein in the African trypanosome. We show that VEX2 associates with chromatin specifically at the single active VSG expression site on chromosome 6, forming an allele-selective connection, via VEX1, to the trans-splicing locus on chromosome 9, physically bridging two chromosomes and the VSG transcription and splicing compartments. We further show that the VEX-complex is multimeric and self-regulates turnover to tightly control its abundance. Using single cell transcriptomics following VEX2-depletion, we observed simultaneous derepression of many other telomeric VSGs and multi-allelic VSG expression in individual cells. Thus, an allele-selective, inter-chromosomal, and self-limiting VEX1-2 bridge supports monogenic VSG expression and multi-allelic VSG exclusion.

• MeSH
• alely MeSH
• membránové glykoproteiny genetika   MeSH
• telomery metabolismus   MeSH
• Trypanosoma brucei brucei * metabolismus   MeSH
• Trypanosoma * metabolismus   MeSH
• trypanosomové variantní povrchové glykoproteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• membránové glykoproteiny MeSH
• trypanosomové variantní povrchové glykoproteiny MeSH

Suramin is one of the oldest drugs in use today. It is still the treatment of choice for the hemolymphatic stage of African sleeping sickness caused by Trypanosoma brucei rhodesiense, and it is also used for surra in camels caused by Trypanosoma evansi. Yet despite one hundred years of use, suramin's mode of action is not fully understood. Suramin is a polypharmacological molecule that inhibits diverse proteins. Here we demonstrate that a DNA helicase of the pontin/ruvB-like 1 family, termed T. brucei RuvBL1, is involved in suramin resistance in African trypanosomes. Bloodstream-form T. b. rhodesiense under long-term selection for suramin resistance acquired a homozygous point mutation, isoleucine-312 to valine, close to the ATP binding site of T. brucei RuvBL1. The introduction of this missense mutation, by reverse genetics, into drug-sensitive trypanosomes significantly decreased their sensitivity to suramin. Intriguingly, the corresponding residue of T. evansi RuvBL1 was found mutated in a suramin-resistant field isolate, in that case to a leucine. RuvBL1 (Tb927.4.1270) is predicted to build a heterohexameric complex with RuvBL2 (Tb927.4.2000). RNAi-mediated silencing of gene expression of either T. brucei RuvBL1 or RuvBL2 caused cell death within 72 h. At 36 h after induction of RNAi, bloodstream-form trypanosomes exhibited a cytokinesis defect resulting in the accumulation of cells with two nuclei and two or more kinetoplasts. Taken together, these data indicate that RuvBL1 DNA helicase is involved in suramin action in African trypanosomes.

• Klíčová slova
• Drug target, Drug-resistance, RuvB-like 1 DNA helicase, Suramin, Trypanosoma brucei, Trypanosoma evansi,
• MeSH
• DNA-helikasy genetika   MeSH
• suramin farmakologie   terapeutické užití   MeSH
• Trypanosoma brucei brucei * genetika   MeSH
• Trypanosoma brucei rhodesiense genetika   MeSH
• Trypanosoma * genetika   MeSH
• trypanozomóza africká * farmakoterapie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA-helikasy MeSH
• suramin MeSH

The African trypanosome, Trypanosoma brucei, has developed into a flexible and robust experimental model for molecular and cellular parasitology, allowing us to better combat these and related parasites that cause worldwide suffering. Diminishing case numbers, due to efficient public health efforts, and recent development of new drug treatments have reduced the need for continued study of T. brucei in a disease context. However, we argue that this pathogen has been instrumental in revolutionary discoveries that have widely informed molecular and cellular biology and justifies continuing research as an experimental model. Ongoing work continues to contribute towards greater understanding of both diversified and conserved biological features. We discuss multiple examples where trypanosomes pushed the boundaries of cell biology and hope to inspire researchers to continue exploring these remarkable protists as tools for magnifying the inner workings of cells.

• Klíčová slova
• GPI-anchor, RNA editing, alternative oxidase, endocytosis, genetic code, glycosome, tRNA import,
• MeSH
• molekulární biologie MeSH
• Trypanosoma brucei brucei * genetika   MeSH
• Trypanosoma * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Tubulin and microtubules (MTs) are potential protein targets to treat parasitic infections and our previous studies have shown that the triazolopyrimidine (TPD) class of MT-active compounds hold promise as antitrypanosomal agents. MT-targeting TPDs include structurally related but functionally diverse congeners that interact with mammalian tubulin at either one or two distinct interfacial binding sites; namely, the seventh and vinca sites, which are found within or between α,β-tubulin heterodimers, respectively. Evaluation of the activity of 123 TPD congeners against cultured Trypanosoma brucei enabled a robust quantitative structure-activity relationship (QSAR) model and the prioritization of two congeners for in vivo pharmacokinetics (PK), tolerability and efficacy studies. Treatment of T. brucei-infected mice with tolerable doses of TPDs significantly decreased blood parasitemia within 24 h. Further, two once-weekly doses at 10 mg/kg of a candidate TPD significantly extended the survival of infected mice relative to infected animals treated with vehicle. Further optimization of dosing and/or the dosing schedule of these CNS-active TPDs may provide alternative treatments for human African trypanosomiasis.

• Klíčová slova
• Triazolopyrimidines, Trypanosoma brucei, drug discovery, human African trypanosomiasis, microtubules,
• MeSH
• lidé MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• pyrimidiny farmakologie   terapeutické užití   chemie   MeSH
• savci metabolismus   MeSH
• trypanocidální látky * farmakologie   terapeutické užití   chemie   MeSH
• Trypanosoma brucei brucei * metabolismus   MeSH
• trypanozomóza africká * farmakoterapie   MeSH
• tubulin metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• pyrimidiny MeSH
• trypanocidální látky * MeSH
• tubulin MeSH

The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the FoF1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the FoF1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨm. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨm. In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• fosforylace MeSH
• mitochondrie metabolismus   MeSH
• Trypanosoma brucei brucei * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH